Effectiveness and Evaluation of Wood Protection Against ...

DOCTORA L T H E S I S

Effectiveness and Evaluationof Wood Protection Against

Biological Deterioration Causedby Filamentous Fungi

Olena Myronycheva

Wood Science and Engineering

Doctoral Thesis

Division of Wood Science and Engineering

Department of Engineering Sciences and Mathematics

Luleå University of Technology

EFFECTIVENESS AND EVALUATION OF WOOD PROTECTION

AGAINST BIOLOGICAL DETERIORATION CAUSED BY FILAMENTOUS FUNGI

Olena Myronycheva

Supervisors:

Olov Karlsson

Margot Sehlstedt-Persson

Dennis Jones

Dick Sandberg

Skellefteå, Sweden

2021

Doctoral Thesis


Luleå University of Technology,

Skellefteå, Sweden

All rights reserved

Copyright © Olena Myronycheva, 2021

Division of Wood Science and Engineering


Luleå University of Technology,

Skellefteå, Sweden

931 87 Skellefteå, Sweden

Phone: +46 (0) 910 58 5312

Author e-mail: [email protected]

Picture for Cover page taken by Tomáš Kytka

ISBN: 978-91-7790-960-6 (print)

ISBN: 978-91-7790-961-3 (electronic)

Printed by Luleå University of Technology, Graphic Production 2021

mailto:[email protected]

"It is characteristic of science and of progress that they

continuously open new fields to our vision.

When moving forward toward the discovery of the unknown, the

scientist is like a traveller who reaches higher and higher

summits from which he sees in the distance new countries to

explore.

Take interest, I implore you, in those sacred dwellings which are

designated by the expression term, laboratories.

Demand that they be multiplied and advanced. These are the

temples of the future, temples of well-being and happiness ...

where humanity grows greater, stronger, better.”

Louis Pasteur (Dubos 1950)

https://www.forbes.com/quotes/author/louis-pasteur

ABSTRACT Developing a holistic understanding of the biological deterioration of wooden material by fungi

in the laboratory and outdoor conditions requires the development of new assessment procedures

and tools that allow describing the process with maximum precision and accuracy. Environmental

biodeterioration is a complex process including a combination of physical, chemical and

biological changes, with many uncertainties limiting the predictability and effectiveness of

selected preservatives after laboratory tests. Therefore, in the current thesis, the investigation of

the effectiveness of selected wood process parameters and protection systems against fungal

growth and evaluation of the applicability of near-infrared spectroscopy for wooden surfaces

assessment under fungal attack were accomplished.

The mould attack on copper impregnated Scots pine sapwood regulated to a greater extent by

planing depth than by the infection method. Air-borne contaminants can heavily occupy the

unplaned surfaces, but the extent of such occupation could be reduced with planing and

impregnation solutions. Despite the vulnerability of the differently planed and copper-

impregnated wood towards mould fungi, mass loss of that wood degraded by white-rot Trametes

versicolor was less than 5%. The distribution, quantity, and nature of lipophilic substances

beneath the surface in the air- and kiln-dried Scots pine sapwood boards significantly influenced

mould fungi attack. It was found that the concentration of total extractives was significantly higher

in kiln-dried than in air-dried samples and was higher close to the surface than in the layers

beneath. During kiln-drying, a migration front is created at a depth of 0.25 mm with a thickness

of about 0.5 mm. The evidence from the previous study is committed to understanding the

influence of extractives and other migrating compounds on the unplanned surface and,

consequently, on mould growth on that surface of Scots pine sapwood subjected to air and kiln

drying. Therefore, a multivariate regression model was developed.

The thermal modification at different temperatures of exotic African wood influenced the

chemistry. Iroko wood demonstrated stabilisation of pH and different patterns of chemical

changes compared to padouk.

The open process of wood treatment like heating-and-cooling (i.e. fully soaking heated wood in

cold liquor allowing the liquor to penetrate wood partially) can improve wood performance by

developing a protective layer beneath the surface on heat-induced curing. However, the applied

methacrylic resin demonstrated effectiveness during laboratory testing for biodeterioration but did

not perform efficiently during outdoor tests.

The test of available commercially of generally recognised as safe (GRAS) compounds and

biocidal treatment in laboratory conditions revealed a moderate inhibition effect on protection

against biodeterioration.

Hyperspectral imaging in the NIR region could be applied to classify thermally modified wood

but not for air/kiln-dried Scots pine wood. The use of a portable microNIR spectrometer

efficiently demonstrated the separation of no mould and mould specimens in laboratory tests of

Scots pine and allowed classifying boards treated with commercial biocides after outdoor

weathering.

SAMMANFATTNING För att uppnå en holistisk förståelse av svampars försämring av trämaterials egenskaper under

laboratorie- och utomhusförhållanden krävs utveckling av nya bedömningsförfaranden och

verktyg som gör det möjligt att beskriva processen med maximal precision och noggrannhet.

Biologisk påverkan i omgivande miljö utgörs av komplexa processer som inkluderar en

kombination av fysiska, kemiska och biologiska förändringar, och inkluderar många osäkerheter

som begränsar förutsägbarheten och effektiviteten hos utvalda träskyddsmedel vid specifika

laboratorietester. Avhandlingen baseras på undersökning av effektiviteten hos utvalda

träprocessparametrar och skyddssystem mot svamptillväxt samt utvärdering av tillämpligheten av

nära infraröd spektroskopi för bedömning av träytor vid svampattack.

Attack av mögelsvampar på kopparimpregnerad furusplint (Scots Pine) reglerades i större

utsträckning av hyveldjupet än av infektionsmetoden. Luftburna mikrober kan lättare fastna och

kolonisera de ohyvlade ytorna, men dess omfattning skulle kunna minskas genom hyvling och

användandet av lämpliga impregneringslösningar. Trots sårbarheten hos det ohyvlade virket mot

mögelsvampar var massförlusten av det kopparimpregnerade virket som bryts ned av vitrött-

svampen Trametes versicolor mindre än 5%. Fördelningen, kvantiteten och typ av lipofila ämnen

under ytan hos luft- och kammartorkade furuvirket samt inverkan på tillväxt av mögelsvampar i

mögelutsatt virke studerades. Mängden extraktivämnen var betydligt högre i kammartorkade än i

lufttorkade prover och högre i skikt nära virkesytan än i underliggande skikt. Det konstaterades

att vid kammartorkningen bildades en migrationsfront i virket med ett djup av 0,25 mm och med

en tjocklek av ca 0,5 mm. Resultat från studierna pekade på extraktivers och andra migrerande

föreningars inverkan på ohyvlade ytans kemi i det luft- och kammartorkade virket och

mögeltillväxten.

Kemiska egenskaper hos afrikanska träslag (Padouk and Iroko) som värmebehandlats vid olika

behandlingstemperaturer (20-210°C) studerades. Aciditet (pH) vid 20°C var olika för träslagen

men påverkades ej av en ökad behandlingstemperatur medan extraktivämneshalt och fenolhalt

skiftade mer.

Öppna och ej trycksatta processer för träbehandling som uppsugning av vätska genom

nedsänkning av uppvärmt virke i kall vätska har potential att förbättra träegenskaper genom att

utveckla ett skyddande skikt under ytan efter värmeinducerad fixering. Applicerat metakrylatharts

visade sig effektivt mot biologisk påverkan vid laboratoriebetingelser, men presterade inte

effektivt vid utomhus tester.

Behandlingar med kemiska föreningar och substrat som kan klassas som allmänt erkända som

säkra (GRAS) samt biocidinnehållande ytbehandlingsprodukter visade på en måttligt hämmande

effekt mot biologisk påverkan vid laboratorieförhållanden.

Erhållna resultat visar på att hyperspektral avbildning i NIR-regionen skulle kunna tillämpas för

att klassificera termiskt modifierat trä men inte för luft/kammartorkad furu. Användning av bärbar

mikroNIR-spektrometer visade sig vara ett effektivt hjälpmedel för separation av furuytor med

eller utan mögel. Tekniken tillät även klassificering av brädor som behandlats med kommersiella

biocidinnehållande produkter efter utomhusexponering.

АБСТРАКТ Розвиток всебічного розуміння біологічного руйнування дерев’яного матеріалу грибами в

лабораторних і зовнішніх умовах вимагає розробки нових процедур оцінки та інструментів,

які дозволяють описати процес з максимальною точністю і точністю. Біопсування у

навколишньому середовищі – це складний процес, що включає поєднання фізичних,

хімічних та біологічних змін, з багатьма невизначеними факторами, що обмежує

передбачуваність та ефективність обраних консервантів після лабораторних досліджень.

Тому в даній науковій роботі виконано дослідження ефективності вибраних параметрів

обробки деревини та систем захисту від грибкового росту та оцінка застосовності

спектроскопії ближнього інфрачервоного випромінювання для оцінки дерев’яних

поверхонь з наявністю грибка. Напад цвілі на просочену міддю заболонь сосни звичайної

регулюється більшою мірою глибиною стругання, ніж методом зараження. Забруднювачі,

що переносяться повітрям, можуть сильно забруднювати нестругані поверхні, але ступінь

такого забруднення можна зменшити за допомогою стругання та просочення різними

сполуками. Незважаючи на вразливість деревини, яка по-різному стругана та просочена

міддю, до цвілевих грибів, втрата маси цієї деревини, деградованої за допомогою білої

гнилі Trametes versicolor, становила менше 5%. Розподіл, кількість та природа ліпофільних

речовин під поверхнею в висушених на повітрі та в печі дошках із заболоні сосни звичайної

значно впливали на напад цвілевих грибів. Було виявлено, що концентрація загальних

екстрактивних речовин була значно вищою у висушених у печі зразках, ніж у висушених

на повітрі зразках, і була вищою біля поверхні, ніж у шарах під ними. Під час сушіння в

печі створюється фронт міграції на глибині 0,25 мм при товщині близько 0,5 мм. Дані з

попереднього дослідження спрямовані на розуміння впливу екстрактивних речовин та

інших мігруючих сполук на нестругану поверхню і, отже, на ріст цвілі на цій поверхні

заболоні звичайної, підданої сушці на повітрі та в печі. Тому була розроблена

багатовимірна регресійна модель.

Термічна модифікація при різних температурах екзотичної африканського дерева вплинула

на хімію деревени. Деревина іроко продемонструвала стабілізацію рН та різні напрямки

хімічних змін порівняно з падуком.

Відкритий процес обробки деревини, такий як нагрівання та подальше охолодження (тобто

повне замочування нагрітої деревини в холодному розчині, дозволяючи розчину частково

проникати в деревину), має потенціал для покращення характеристик деревини шляхом

створення захисного шару під поверхнею під час затвердіння під впливом тепла. Проте

застосована метакрилова смола продемонструвала ефективність під час лабораторних

випробувань на біопсування, але не ефективна під час випробувань на відкритому повітрі.

Випробування комерційних загальновизнаних безпечних (GRAS) сполук та біоцидної

обробки в лабораторних умовах виявило помірний інгібуючий ефект при захисті від

біопсування.

Гіперспектральне зображення в області NIR можна застосувати для класифікації термічно

модифікованої деревини, але не для деревини сосни звичайної, висушеної на повітрі або

при сушці в печі. Використання портативного спектрометра microNIR ефективно

продемонструвало поділ без плісняви та зразків з пліснявою в лабораторних дослідженнях

сосни звичайної та дозволило класифікувати дошки, оброблені комерційними біоцидами

після впливу зовніщнього середовища.

PREFACE After finishing my PhD journey and looking back, I feel happy with exciting opportunities

resulting from acquired knowledge. Coming to Sweden and being hungry for new things to

explore, I definitely could say that curiosity opens all opportunities and doors. The unique

connection between academia and industry I found in Sweden inspired me to work hard to develop

new applications because you can immediately see the results of your research in the practice and

how it influences the minds of people involved.

Firstly, I am grateful to the companies Martinsons (now Holmen) and particularly Peter

Jacobsson, NORRA Timber, Derome providing the test material and ideas for research. Support

from The European Regional Development Fund, the County Administration of Västerbotten, the

municipality of Skellefteå, and Wood Centre North is also greatly appreciated and acknowledged.

Furthermore, financial support from the Swedish Research Council for the Environment,

Agricultural Sciences and Spatial Planning (FORMAS) project “Experimental studies of capillary

phenomena in bio-based materials” 942-2016-64, “Fungal growth on modified wood-based

products under subarctic conditions” 2017-00419, is also gratefully acknowledged. Moreover,

support from the COST Action FP1407 “ModWoodLife” project “Understanding wood

modification through an integrated scientific and environmental impact approach” is gratefully

acknowledged. Finally, to equip laboratories would not be possible without support from Labfund

of LTU, Stiftelsen Rönnbäret and Kempe.

Secondly, my recognition to Ingrid and Elisabeth from Byske local community, Ayna Rönnblon

and her husband Lasse, Anna-Klara Granstrand with family, and Gunnar Landsell for compassion

during my hard time I will never forget.

My deep gratitude I express to my family Volodymyr and Agrypyna, mama, Maryna and Andriy,

for endless love and help. My Swedish friends Isabel, Symon, Anya and Solomon are being

around and supporting those years.

“Sist men inte minst” my most profound bow to supervisors for being all way supportive with

the commitment, diligence and patience not only with scientific content but also with a human

being, and for my joy to grow.

Finally, I am blessed to work with such excellent and intelligent professionals at the Wood Science

and Engineering Division at Luleå University of Technology, Skellefteå.

Sincerely,

Olena Myronycheva

LIST OF APPENDED PAPERS AND AUTHOR’S CONTRIBUTION Paper I

Myronycheva, Olena, Margot Sehlstedt-Persson, Olov Karlsson, and Dick Sandberg. 2018.

‘Growth of Mold and Rot Fungi on Copper-Impregnated Scots Pine Sapwood: Influence of

Planing Depth and Inoculation Pattern’. BioResources 13 (4): 8787-8801.

https://doi.org/10.15376/biores.13.4.8787-8801.

Author contribution: literature research, problem statement, planning and performing the

biodeterioration experiments, data analysis and draft of manuscript preparation.

Paper II

Myronycheva, Olena, Olov Karlsson, Margot Sehlstedt-Persson, Micael Öhman, and Dick

Sandberg. 2018. ‘Distribution of Low-Molecular Lipophilic Extractives beneath the Surface of

Air- and Kiln-Dried Scots Pine Sapwood Boards’. Edited by Martina Lahmann. PLOS ONE 13

(10): e0204212. https://doi.org/10.1371/journal.pone.0204212.

Author contribution: literature research, planning and performing the analytical experiments,

data analysis and draft of manuscript preparation, communication with reviewers.

Paper III

Karlsson, Olov, Olena Myronycheva, Margot Sehlstedt-Persson, Micael Öhman, and Dick

Sandberg. 2017. ‘Multivariate Modeling of Mould Growth in Relation to Extractives in Dried

Scots Pine Sapwood’. In 48th Conference of the International Research Group on Wood

Protection, IRG48. Ghent, Belgium. http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-63985.


data analysis and draft preparation.

Paper IV

Kroupa, Michal, Milan Gaff, Olov Karlsson, Olena Myronycheva, and Dick Sandberg. 2018.

‘Effects of Thermal Modification on Bending Properties and Chemical Structure of Iroko and

Padauk’. Proceedings of the 9th European Conference on Wood Modification 2018, Arnhem, the

Netherlands. 155–161. http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-72585.


data analysis and draft preparation regarding chemical analysis of extractives of African wood.

https://doi.org/10.15376/biores.13.4.8787-8801

https://doi.org/10.1371/journal.pone.0204212

http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-72585

.

Paper V

Elustondo, Diego, Olena Myronycheva, Bror Sundqvist, and Olov Karlsson. 2016. ‘Localized

Wood Surface Modification, Part I: Method Characterization’. BioResources 12 (1): 283-295–

295. https://doi.org/10.15376/biores.12.1.283-295.

Author contribution: contribution to literature research, experiment planning, writing, editing

and further planning of the part II experiment for the biodeterioration resistance check.

Paper VI

Karlsson, Olov, Olena Myronycheva, Dennis Jones, Diego Elustondo, and Dick Sandberg. 2018.

‘Thermally Modified Wood Treated with Methacrylate’. In IRG49 Scientific Conference on

Wood Protection, Johannesburg, South Africa. http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-

68771.

Author contribution: planning and performing the biodeterioration experiments, data analysis

and draft preparation.

Paper VII

Kim, I., Karlsson, O., Myronycheva, O., Jones, D., and Sandberg, D. (2020). "Methacrylic resin

for protection of wood from discoloration by mould growth and weathering," BioResources.

15(3), 7018-7033.

Author contribution: planning and performing the biodeterioration experiments, data analysis

and manuscript draft preparation.

Paper VIII

Myronycheva, Olena, Faksawat Poohphajai, Margot Sehlstedt-Persson, Tommy Vikberg, Olov

Karlsson, Helmut Junge, and Dick Sandberg. 2019. ‘Application of GRAS Compounds for the

Control of Mould Growth on Scots Pine Sapwood Surfaces: Multivariate Modelling of Mould

Grade’. Forests 10 (9): 714. https://doi.org/10.3390/f10090714.

Author contribution: literature research, problem statement, planning of performing the

biodeterioration experiments, data analysis and the draft of manuscript preparation,

communication with reviewers.

https://doi.org/10.15376/biores.12.1.283-295



https://doi.org/10.3390/f10090714

Paper IX

Myronycheva, Olena, Injeong Kim, Peter Jacobsson, Olov Karlsson, Margot Sehlstedt-Persson,

and Dick Sandberg. 2020. ‘The Influence of Four Commercial Wood-Surface Treatments on

Mould-Fungi Growth in a Pure Culture’. In , 19–19. InnoRenew CoE.

http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-81365.

Author contribution: project methodology planning, performed experimental work with data

analysis, prepared the manuscript, and presented in the conference.

Paper X

Myronycheva O., Sidorova E., Hagman O., Sehlstedt-Persson M., Karlsson O., Sandberg D.

(2018). Hyperspectral imaging surface analysis for dried and thermally modified wood: an

exploratory study. Journal of Spectroscopy. https://doi.org/10.1155/2018/7423501

Author contribution: planning and performance of the experiment, data collection, analysis and

modelling, manuscript writing, communication with editor and reviewers.

Paper XI

Myronycheva O, Karlsson O, Sehlstedt-Persson M, Öhman M, Sandberg D. (2019). Portable

microNIR sensor for the evaluation of mould contaminated boards. In: The Proceeding from the

62nd SWST International Convention. Yosemite National Park, California, USA.

Author contribution: methodology planning, data collection, analysis, validation and

visualization, writing – original draft and presenting on the conference.

Paper XII

Olena Myronycheva, Alona Sekan, Injeong Kim, Olov Karlsson, Peter Jacobsson, Margot

Sehlstedt-Persson, Dick Sandberg (2020). ‘Spectroscopic Characterisation of Wood Treated by

Different Coatings after Weathering in Subarctic Conditions’. In Proceedings of the 2020 Society

of Wood Science and Technology International Convention. Portoroz, Slovenia: Society of Wood

Science and Technology.

Author contribution: methodology planning, data collection, analysis, validation and

visualization, writing – original draft and presenting on the conference.

https://doi.org/10.1155/2018/7423501

Additional scientific work not included in the thesis:

1. Myronycheva O, Karlsson O. Extraction of extractives and analysis of low-molecular

lipophilic extractives beneath the surface of air- and kiln-dried scots pine sapwood boards

v1 (protocols.io.phqdj5w). doi:10.17504/protocols.io.phqdj5w

2. Wei M, Xiong S, Chen F, Geladi P, Eilertsen L, Myronycheva O, et al. Energy smart hot-

air pasteurisation as effective as energy intense autoclaving for fungal preprocessing of

lignocellulose feedstock for bioethanol fuel production. Renewable energy. 2020;155:

237–247. Available: http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-78347

3. Xiong S, Martín C, Eilertsen L, Wei M, Myronycheva O, Larsson SH, et al. Energy-

efficient substrate pasteurisation for combined production of shiitake mushroom

(Lentinula edodes) and bioethanol. Bioresource Technology. 2019;274: 65–72.

doi:10.1016/j.biortech.2018.11.071

4. Sekan AS, Myronycheva OS, Karlsson O, Gryganskyi AP, Blume Y. Green potential of

Pleurotus spp. in biotechnology. PeerJ. 2019;7: e6664. doi:10.7717/peerj.6664

5. Venditti T, Ladu G, Cubaiu L, Myronycheva O, D’hallewin G. Repeated treatments with

acetic acid vapors during storage preserve table grapes fruit quality. Postharvest biology

and technology. 2017;125: 91–98. Available:


6. Myronycheva O, Bandura I, Bisko N, Grygansky AP, Karlsson O. Assessment of the

Growth and Fruiting of 19 Oyster Mushroom Strains for Indoor Cultivation on

Lignocellulosic Wastes. BioResources. 2017;12: 4606–4626. Available:


7. Bandura I, Myronycheva O, Karlsson O, Anike FN, Isikhuemhen OS. Pretreatment of

wheat straw and solid state fermentation improves yield and biological efficiency in

Pleurotus ostreatus Jacq) P. Kumm. mushroom production. University of Palermo; 2017.

pp. 41–43. Available: http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-65995

8. Sehlstedt-Persson M, Öhman M, Marklund B, Myronycheva O. Hyveldjupets inverkan på

mögelbenägenhet hos råspont av furu och gran. TräCentrum Norr; 2016. Available:


9. Bandura I, Myronycheva O, Karlsson O. Assessment of Raw Plant material and Substrate

for Efficient production of Oyster Mushrooms (Pleurotusostreatus (Jacq.) P. Kumm.).

Technická univerzita vo Zvolene; 2016. pp. 27–33. Available:








TABLE OF CONTENTS

1. Introduction .............................................................................................................................. 19

2. Problem description ................................................................................................................. 21

2.1 Background ........................................................................................................................ 21

2.2 Vision ................................................................................................................................. 22

2.3 Objective ............................................................................................................................ 22

2.4 Research questions ............................................................................................................. 22

2.5 Limitations ......................................................................................................................... 22

3. State-of-the-art ......................................................................................................................... 25

3.1 Wood as a material with complex chemistry ..................................................................... 25

3.2 Fungi as decomposers of the wooden material .................................................................. 28

3.3 Fungi in the built environment ........................................................................................... 31

3.4 Wood protection methods against biodeterioration ........................................................... 35

3.5 Methods and evaluations describing fungal growth on wood ............................................ 38

4. Materials and methods ............................................................................................................. 45

4.1 Wood planing depth, impregnation and extractives influence on the fungal attack .......... 45 4.1.1 Wood specimens preparation for copper impregnation ............................................................ 45

4.1.2 Impregnation and evaluation of impregnation results .............................................................. 45

4.1.3 Wood specimens preparation for drying procedures evaluation .............................................. 46

4.1.4 Wood specimens preparation after thermal modification ......................................................... 46

4.1.5 Wood extractives chemical analysis ......................................................................................... 47

4.1.6 Test design for wood material biodeterioration ........................................................................ 47

4.2 Wood thermal modification, protection by GRAS and biocides under fungal attack ....... 49

4.3. Spectroscopic characterisation of wood under fungal attack ............................................ 51

4.4 Statistical analysis used in the thesis .................................................................................. 52

5. Results and Discussion ............................................................................................................. 53

5.1 Wood planing depth, impregnation and extractives influence on fungal attack ................ 53

5.2 Wood thermal modification, protection by GRAS and biocides under fungal attack ....... 61

5.3 Spectroscopic characterisation of wood under fungal attack ............................................. 66 5.3.1 Hyperspectral analysis of the surface ....................................................................................... 66

5.3.2 NIR modelling of kiln/air-dried wood under fungal attack ...................................................... 68

6. Conclusions .............................................................................................................................. 75

7. Future work .............................................................................................................................. 77

REFERENCES ............................................................................................................................. 79

19

1. INTRODUCTION

The desire of European leadership to transition towards a bio-based economy raises demand for

bio-based industrial solutions in all industries. Moreover, the European Union is aligned to the

United Nations 2030 Agenda and Sustainable Developmental Goals framework, where

sustainable raw materials are commonplace in use and crucial for efficient economic development

and prosperity (European Commission 2019).

Wood products are considered as a prominent “green” solution in terms of substitution of abiotic

and energy-intensive materials in construction (European Commission 2019) via processing of

raw forest products to value-added wood products, 80% of which are used in construction and

other civil-engineering projects (Swedish Wood 2021). That has resulted in a growth in the global

production rate of sawn timber of almost 17% since the decision was made in 2012, according to

the Food and Agriculture Organisation of the United Nations (FAO UN 2021). Notably, the

Swedish production volume of sawn timber reached the amount of 18.4 million m3 in 2020, of

which about 75% was exported, as reported by the Swedish Forest Industries (Skogsindustrierna

2021).

As a building and construction material, wood is still nearly 100% organic, renewable engineering

material of natural origin, forming a storage pool of wood-based carbon (UNECE 2008). Derived

from terrestrial vegetation, wood material is involved in the terrestrial carbon cycle and subjected

to decomposition caused by various abiotic and biotic factors (Carvalhais et al. 2014). After

manufactured processing and in moist conditions, sawn timber is highly susceptible to

biodeterioration and biodegradation by different microbes in the biosphere (Zabel and Morrell

1992). Therefore, different technologies of wood preservation have been developed since the

phenomenon was noticed (Feist and Hon 1984) to prolong material shelf life and extend the

maintenance period.

According to requirements within the Public Health Agency FoHMFS 2014:14 “The Swedish

Public Health Agency’s general advice on moisture and microorganisms” and The Swedish

National Board of Housing, Building and Planning rules (Boverket 2021) regulating moisture

security for materials and products included in the building section, wooden products are

considered to be more vulnerable compared to products from steel and concrete coming from non-

renewable sources. Since the hygroscopic nature of wood is known, and control of the living

environment is an obligation of property owners, some players in the building and construction

sector generate nebulous or “wishy-washy” type of knowledge (Olsson 2019). The considerable

maintenance cost of products and wood that has a heterogeneous origin that can impact the life

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cycle credibility. When national and local governments spend enormous amounts of money

supporting global initiatives (for example, https://www.bioinnovation.se/) and the domestic

wooden industry (Skelleftea Commune 2021). The current trends in designing innovative

environmentally-friendly protecting systems, with a known end-life scenario with minimal toxic

preservative release and bioaccumulation, are significant challenges for wood-based material

development (Salminen et al. 2014). This is not very easy achievable only by strict control of

preservatives usage and implicates knowelege gap in terms of wood preservatives efficiencyto

reduce different fungi growth and avialability of proper evaluation procedures.

https://www.bioinnovation.se/

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2. PROBLEM DESCRIPTION

2.1 BACKGROUND So far, the current EU standards are out of date and need to be revisited according to research

done in the last 30 years regarding material resistance and fungal biology (Kutnik, Suttie, and

Brischke 2014). At the same time, a little research has been conducted in the area of

biodegradation no shift from “wood preservation” that means to preserve by toxic chemicals

towards “wood protection”—aiming to develop flexible complex solutions considering the natural

properties of the wood-based material and its heterogeneity, interactions with water, biological

and organic contamination on the surface after process performance and during maintenance to

satisfy stakeholders’ preferences along the whole forest value chain is observed.

To achieve that state of wood protection the detailed knowledge about wood chemistry, its

interaction with different fungi and their physiological conditions in various wood material

applications should be produced using modern analytical equipment. That allows the development

of a comprehensive model based on the process parameters influence on wood chemistry and

understanding the influence of those parameters on the fungal attack. Moreover, a need for fast

assessing tools was stated from the industrial point of view.

Current work is dedicated to evaluating wood protection methods regarding the resulting wood

materials susceptibility towards fungal growth. The wooden species evaluated in our studies was

Scots pine, Norway spruce and two exotic African wood iroko and padouk, since pine and spruce

wood are the most abundant tree species used in the industrial production of wooden construction

material. Scots pine sapwood is quite vulnerable to fungal contaminations and growth. Several

surface treatments, modifications and impregnation were assessed for the material resistance

improvement. To compare the two exotic African wood species, padouk and iroko, were evaluated

regarding extractives enrichment and their chemical properties. The moisture influence was not

mainly focused since laboratory measurements were done in a highly saturated moisture

environment to promote fungal growth. The fungal cultures isolated from wood material with

native virulence were used throughout the tests. Special attention was paid to the distribution of

the extractives and conditions during the previous drying. Pattern recognition approach based on

multivariate statistical analysis seems to be a way to explore the data and draw relevant

conclusions about tests performances.

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2.2 VISION Development of a holistic understanding of biological deterioration of wooden material by

filamentous fungi in laboratory and outdoor conditions.

2.3 OBJECTIVE Investigation of the effectiveness of selected wood process parameter and protection systems to

reduce fungal growth and evaluation of the applicability of near-infrared spectroscopy for wooden

surfaces assessment under fungal attack.

2.4 RESEARCH QUESTIONS 1. How wood planing depth, copper impregnation and extractives influence fungal attack.

2. How wood thermal modification, protection by GRAS and biocidal compounds influence the

degree of fungal growth in different testing conditions.

3. Which tested near infrared spectroscopic method is more relevant for characterising

conventionally dried and thermally modified timber under fungal attack?

Figure 2.1 presents the connection of appended research paper to research questions.

2.5 LIMITATIONS There are several ways to evaluate biological deterioration. However, in current work, the

assessment for the mould growth was done in laboratory conditions without direct connection to

individual fungi, and community interactions existing in nature. The assessment of nutrients and

full extractives profile and link to individual fungi growth were not undertaken due to the

analytical complexity of the system. The sampling and analytical assessment is a dilemma as a

tiny layer about 100 micrometres below the surface is critical to moulding fungi residence.

Furthermore, the developed indexes used for fungal growth assessment was also a critical point

representing generalisation of the process. The question is still open regarding how to link the

surface assessment at a macro/micro level to environmental processes wood undergoes during

construction as well as exploitation to find decisive parameters in the description of fungi-wood

heterogeneity and move towards modelling, prediction of that system in the natural environment.

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3. STATE OF THE ART 3.1 WOOD AS A MATERIAL WITH COMPLEX CHEMISTRY

Wood as a raw construction material is a product from perennial woody plants prevailing in

land ecosystems in the form of trees. The formation of trees goes via complex biosynthetic

mechanisms of growth. Conifers or gymnosperm trees are a significant source of raw wood

material supply in Northern Europe (Ek et al. 2009). The stem has gymnosperm xylem

consisting of mostly tracheids arranged in 3D high-order macromolecular structures of lignified

cell walls of cellulose, hemicellulose and pectic substances that define the physical, chemical

and biological properties of softwoods that is a wood material from conifers (Terashima 2012).

At the macroscale, the roundwood before sawing consist of bark, heartwood and sapwood. Pine

heartwood is a dead tissue highly enriched by extractives. The detailed scheme of heartwood

formation and deposition of lipophilic and hydrophilic extractives are presented in Figure 3.1.

Figure 3.1. Model of heartwood formation in conifers: (a) Cross-section view of green (freshly

felled) pine with the sapwood (SW) darker due to the higher moisture content (MC) than the

heartwood (HW). (b) Sketch of the same cross-section view illustrating three main areas of

heartwood formation: (i) sapwood with a higher MC and living parenchyma cells; (ii) transition

zone where the radial oriented rays undergo programmed cell death and the tracheids dry out

the free water; and (iii) heartwood tracheids impregnated with heartwood constituents and with

a lower MC (around fibre saturation point (FSP). Figure inspired by Nakada and Fukatsu (2012).

Retrieved from (Felhofer et al. 2018).1

1 © The Author(s) 2018. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited

26

Mechanical strength of stem as a primary structural element of gymnosperm trees is formed at

a cellular level by the tracheid alignment in the trunk (Ek et al. 2009). The rigid cell wall of the

softwood tracheid on Figure 3.2 consists of the middle lamella, primary wall, outer, middle,

and inner secondary walls (S1, S2, S3) and warty layer. Rigidity to the structure added by

deposited lignin located in the middle lamella acted as the glue by bonding the cell walls

together, as represented in Figure 3.2 (Rowell 2005).

Figure 3.2. The structure of softwood tracheid cell wall (Rowell 2005).

The primary chemical constituents of the wooden cell wall are carbohydrates. Mainly, the

highest amount of cellulose is about 40-45% structurally formed from D-glucose molecules,

which are connected by β-1,4 glucosidic linkages. Hemicelluloses are presented in the range of

15-30% in the form of polysaccharides and divided into mannans, constituted by D-glucose, D-

mannose, D-galactose, and xylans (constituted of D-xylose, L-arabinose, D-(4-O-

methyl)glucuronic acid), and lesser amounts of other sugars such as L-rhamnose and D-fucose.

Polysaccharides are pectins, starches, xyloglucan, and monosugars, mainly saccharose,

fructose, and glucose (Kretz 1973). The majority of mono sugars are found in sapwood,

typically close to the cambium zone. Polymerised lignin is also present in the cell wall at lower

concentrations and is believed to form the matrix together with hemicellulose in between

cellulose fibrils contributing to (wet) strength, microbial resistance and stability of the wood

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fibre. Lignin is a branched and irregular macropolymer formed by the phenolic coupling of the

phenylpropane derivatives (coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol), resulting

in a solid structure. Pine heartwood is a dead tissue that possesses high durability due to the

natural deposition of phenolic compounds such as terpenes, flavonoids, lignans, stilbenes,

among others (Hillis 1987). However, the lipophilic (resin acids, long-chain fatty acids and

triglycerides), less hydrophobic (stilbenes, lignans) and non-structural sugars extractives are

also deposited in sapwood, but usually to lower content (Figure 3.1). The hydrophilic

extractives are polar compounds, mainly mono- and disaccharides that are food reserves. The

composition of these sugars is mainly of saccharose and other low-molecular sugars such as

glucose and fructose. The concentrations of those sugars vary during the winter and summer

seasons, respectively (Terziev et al. 1997). Regarding water-soluble polysaccharides in the

sapwood of Scots pine, they are primarily from neutral acetylated glucomannans with an

average monosaccharide ratio of Man:Glu:Gal of 4:1:0.5 (Willför et al. 2004). The extensive

amount of hydroxyl groups in those carbohydrates named polyoses or hemicellulose are mainly

responsible for hydrogen bonding of water molecules and hygroscopicity of wood material.

Acetyl side groups in hemicelluloses contribute to the acid formation during thermal treatment

(Sundqvist et al. 2006; Rowell 2005; Sundqvist 2004; Sundqvist 2002; Fengel et al 1989).

The surface of a wooden board could be described as a chemically heterogeneous and porous

structure affected by the anisotropy of wood. There is an unambiguous influence on the surface

of the wooden material of the complex natural processes during wood formation followed by

primary industrial processing such as harvesting, sawing and drying. Diffusion of extractives

after sawing and drying (Nussbaum 1999), and organic and inorganic contaminant

sedimentation adds further complexity to the wood surface and could change its hydrophilicity

and wettability. Wettability is a complex phenomenon that influences the adhesion of different

substances to the surface. It is found that wood itself has a acidic character (Mantanis and Young

1997), but infiltration by extractives turn it relatively more acidic according to Lewis acid-base

theory due to the redirection of functional groups and oxidation at the wood/extractives-air

interface (Wålinder 2000).

On the other hand, during weathering, the degradation of wood polymers and the leaching of

extractives notably accelerated on the surface makes the wood more hydrophylic. Different

opinions exist regarding the role of extractives on the wood surface, and their degradation

chemistry is not evident (Rowell 2005). The reduction in hydrophobicity and water exclusion

efficacy of Scots pine sapwood was reported during artificially accelerated weathering, but at

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outdoor ageing conditions, it was found that abiotic and biotic factors were acting

interdependently (Žlahtič et al. 2016). The durable species kept their initial hydrophobicity, but

it is not clear whether this was due to their photodegradation resistance or the wood's natural

durability. Almost no information about chemical changes and composition of wood’s non-

polar (hydrophobic) extractives during weathering could be found in the scientific literature. It

is a fact that, after harvesting, the wood material undergoes chemical changes that reduce the

initial durability of the grown tree (Ahmed et al. 2013). Natural ageing is mainly related to

surface inactivation caused by migration of extractives from bulk to the surface and changes in

the chemistry owing to the oxidation of the extractives at the surface (Ahmed et al. 2013). To

add complexity to this, one can say that the natural design and evolution of plant vascular

development defines the complicated natural polydispersity of wood material in terms of

chemical properties of its constituents and is not yet fully clarified from a natural biosynthesis

perspective, nor the impact of wood processing on it (Terashima 2013; Terashima et al. 2016).

Another challenge faced is diversity of structural components in the hemicellulose-cellulose-

lignin matrix and non-structural wood extractive compounds in terms of chemical bonds

polarity. Moreover, the mono/oligo/polymers degradation and volatility within wood during

drying/heat/thermal/preservative treatment, combined with the complexity of reaction ongoing

during processing, add additional questions about the existing variability of wood material.

3.2 FUNGI AS DECOMPOSERS OF THE WOODEN MATERIAL

There is a accepted theory about the genetic coevolution of the fungal biome with the evolution

of forests horizons in the Earth (Eastwood et al. 2011). Consequently, the fungal ability to

decompose lignocellulosic biomass was driven by nutritional adaptation and the occupation of

free ecological niches (Floudas et al. 2012).

Today, the fungal kingdom is represented by 120,000 accepted fungal species, about 8% of the

estimated total fungal diversity (Hawksworth et al. 2017). However, despite such great

diversity, the harvested timber is an object of interest for fungi of three main phyla of fungi:

Basidiomycota, Ascomycota and Zygomycota, in Figure 3.3 that are saprotrophs (saprophytes)

and grow on non-living substrates (Deacon 2006).

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Figure 3.3. Fungal Kingdom structure, retrieved from Tree of Life web project

(Blackwell et al. 2019).

The characterisation of the fungi regarding wooden material damage is based on the

terminology that came from forest pathology of the decay description. Here, the decay refers to

the tree's degradation by various biological agents where the action is caused by fungi in most

cases (Zabel et al. 2020). The division of decay fungi is based on the type of decay and

constitutes of brown, white and soft rot types and mould fungi. The mechanism of brown rot

decay is based on the non-enzymatic attack of wood cell wall using hydroxyl radicals (●OH)

which is the most vigorous oxidizing agent of biological cells via the Fenton mechanism of the

reaction. By modifying lignin or producing its derivatives, the brown rot fungus reduces

abundant Fe3+ to Fe2+ that reacts further with hydrogen peroxide generated metabolically by

fungal oxidases into strongly oxidising agent (Zabel et al. 2020). Such weakened lignocellulosic

complex allows access of carbohydrate-active enzymes (CAZy) to cleave accessible

carbohydrate chemical bonds starting from cellulose, hemicellulose and modifying lignin

derivatives by Fenton type of reaction (Eastwood et al. 2011). It is proposed that brown rot is

strongly linked with a decay of conifers, although in general, brown rot constitutes about 6%

of the total amount of fungi (Eastwood et al., 2011). Despite the low presence in total quantity,

the brown rot type fungi dominate decomposers in boreal forests and are the leading decay

agent in buildings in Europe (Irbe 2010; Gabriel et al. 2017; Haas et al. 2019). That might be

explained by adaptation to more generalist behaviour regarding nutrient sources as suggested

in a modern decay model (Krah et al., 2018). Brown and white rot type fungi usually belong to

Basidiomycetes except for a small group of white-rot found in Ascomycetes (Druzhinina et al.

2016). For their optimal growth, the presence of cellulose and a limited amount of monosugars

are required (Metreveli et al. 2021). The white-rot fungi have duplicated amount of CAZy

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enzymes and oxidoreductases due to nutritional evolutional adaptation to angiosperms. Such

sophistication in decomposition evolved the appearance of selective lignin-degrading enzymes

in that type of rot (Kubicek 2012). Brown and white rot types of fungi are responsible for

structural lignin degradation, and the recommendations for such biodeterioration prevention is

to avoid ground contact and moisture intrusion (Boverket 2021). A bad example of ongoing

decay is shown in Figure 3.4.

Figure 3.4. The example of the wooden beam decay in ground contact

Soft rot and mould fungi come from the Ascomycota and Zygomycota branches and act on

polysaccharides backbone by various enzymatic systems to obtain low molecular nutrients

(Druzhinina et al. 2016). According to Kubicek (Kubicek 2012), soft rot fungi could be divided

into two groups that form longitudinal cavities in the secondary cell wall and those that

deteriorate the whole secondary wall (Kubicek 2012). An example of the soft rot from the

Penicillium type of fungi is presented in Figure 3.5 below.

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Figure 3.5. The growth of Penicillium type of the fungi from wooden splinters washed in sodium

hypochlorite solution before placing on nutrient media for fungal growth.

3.3 FUNGI IN THE BUILT ENVIRONMENT The interactions of fungi and the built environment begins from processing forest raw materials

to the final products (NUTEK 1993). Here, the fungal colonization of wood material may occur

during the whole processing and production chain. The arrival of mycelium and adhesion of the

spores of air-borne fungi occurs by acid-base and unipolar interactions, with further production

of sticky mucilage of different origin (glycoproteins or polysaccharides) that glues the spore to

the surface (Jones 1994; Nicholson et al. 1991; Bardage et al. 1998). After adhesion, the spore

responds to favourable climate conditions such as relative humidity (RH) and temperature.

The phenomenon of fungal adhesion to the surface and the extra hyphal matrix structures

(exudates and biofilms) of the fungi are the main actors regulating its adherence, germination

and growth (Jones 1994). Most of the research in the fungal biology area is done at the cellular

level, and almost no information exists about the chemical characteristics of adhesion molecules

(Lipke 2018). Studies of the outdoor weathering tests of wood and surface changes discovered

the influence of abiotic factors and fungi on the decrease in hydrophobicity of the wooden

surface (Žlahtič et al. 2016). For example, the study of known common outdoor colonizers such

as Aureobasidium pullulans and its polysaccharide (pullulan) that promotes adhesion of

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blastospores to water-borne paints was done by Bardage in 1998 (Bardage and Bjurman 1998),

while the control of that adhesion by uronic acid-based polymer and pullulan was discovered

(Pouliot et al. 2005).

Prerequisites for fungal spore germination (see Figure 3.6) are sufficient environmental

conditions and dependent on many physical and chemical factors and their interactions. Most

essential are water, oxygen, optimal temperature range, nutrient substrate availability and

nitrogen-containing compounds, vitamins and macro and microelement, and optimum in pH

range (Carlile 2001).

Figure 3.6. Aspergillus sp. conidia formation (Zeeshan 2016).

Recent studies revealed that a limiting relative humidity level below even 60-70% exists for

some species (Stevenson et al. 2017; Johansson et al., 2012). Regarding the optimal

temperature, some fungal structures can resist extreme conditions to some extent; the threshold

for surviving in the heat is 30 min at 60-63°C, but the survival time for fungal asci (Figure 3.6)

treated at 85-90°C is 4.5 min (Tournas 1994). Additionally, the ratio between RH and

temperature is critical for fungal development in excess of water. Fluctuations of the RH and

temperature create water in voids on material surfaces which in combination with fungal

adaptation and regulation of microclimate dynamics by exudates, and accumulation of

osmolytes or secreting hygroscopic polysaccharides makes the elimination of fungal growth

quite a complex task due to resistance to toxic chemicals and weathering of them (Bjarløv et al.

2016; Kang et al. 2016; Nik et al. 2012).

The availability of nutrients for germinated spores is necessary for new colony development.

Fungi use special receptors for sensing nutrients in the external environment (Dijck et al., 2017).

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The preferred nutrient source is glucose, but the metabolic plasticity of the fungi to use other

sources to survive in harsh conditions has been developed over evolution. It could explain the

availability of a collection of secreted carbohydrate-active enzymes (CAZymes), allowing fungi

to deconstruct and utilize heterogeneous lignocellulose material (Dijck et al., 2017). Figure 3.7

describes the fungal preferences regarding utilization efficiency and complexity of nutrients.

Figure 3.7. The nutrient requirement by fungi (Deacon 2006).

Wooden material under the influence of abiotic and biotic factors is an ecological niche for

microbial communities. Fungi are using unique life-history strategies to combat the

environment they occupy (Boddy et al. 2016). From an ecosystem perspective, an

anthropogenic load caused perturbations in natural habitats and reduced microbial biodiversity,

resulting in emerging microbial pathogens appearing in humans, wildlife, and agriculture

(Fisher et al. 2020; Ogaki et al. 2020). Continuous urbanisation and increased commercial

forestry and agriculture depreciate natural soil biodiversity that is more evident now in aerial

bio-aerosols (Abrego et al. 2020). The aerial dispersion dynamics of the fungal structures are

regulated by multiple factors such as meteorology, plants, animals, insects and humans and

accumulate variability to the wooden material properties (Aylor 2017; Golan et al. 2017).

Additional variability to the wood as a building material added by long-term exposure in various

climatic and microclimatic conditions resulted in gradual deterioration (Mattsson 2017) and

fungal nutritional adaptation to substrate diversity (Morel et al. 2013). Here the understanding

of climate processes influence on wood chemistry should be linked to global and continental

scales of airborne dispersal routes of spores of the fungi colonizing wooden surface (Aylor

2017; Brown et al. 2002).

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Figures 3.8 and 3.9 below represent images from sampling from the wooden surface by

transparent tape and fungi growing from wooden splints on malt-extract agar. The pollen and

fungal spores stained by lactophenol blue are seen in dark blue colour in Figure 3.8.

Figure 3.8. Microscopic image from sampling by tape from the historical site (shingle of

church roof) after long term-exposure to environmental conditions (the scale bar is 50 µm)

Figure 3.9. Example of growth of different fungal species from wooden splinters surface

of which was sterilized by sodium hypochlorite solution

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An essential impact of fungi on the indoor environment from building material is presented.

Here the microbial communities’ dynamics are significantly influenced by microbial

communities of air, water and surface reservoirs (up to 70% due to external outdoor conditions)

(National Academies of Science US 2017). The example of degraded insulation material is

presented in Figure 3.10 below.

Figure 3.10. The degraded insulation, photo taken in the Anderstorp area, Skellefteå, Sweden.

Moreover, according to Anticimex report about defects and damage to the house's roof 2021

(Lindström 2021), every 10th roof of a small residential house built 100 years ago and until

now was with defects. In addition, several studies have already mentioned the risk of fungal

growth due to unsatisfactory hydrothermal performance in the roofs (Bjarløv et al. 2016;

Buxbaum et al. 2015; Buxbaum et al. 2013; Geving et al. 2010).

3.4 WOOD PROTECTION METHODS AGAINST BIODETERIORATION As previously discussed, untreated wood may be subject to biodeterioration. According to

Morrell (Morrell 2005), four main factors influence biological growth (oxygen, temperature,

moisture and food source). Restriction of one or more of those factors can control the biological

deterioration of wood. Oxygen, as well as temperature, maybe almost impossible to control at

outdoor exposure. Therefore, the primary attention related to moisture protection or wood

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preservation has been done by the addition of chemical substances to inhibit fungal growth.

However, maintaining moisture control to prevent fungal growth can be very difficult in

wooden buildings, especially during the construction period (Olsson 2019). Another approach

to control moisture is building by design, but it is out of the scope of current work (Brischke et

al., 2015). So, the primary method for protecting wooden materials remains wood preservation

(Sandberg 2021). Wood preservation has been already reported for several centuries, as

documented with historical wooden churches. There, tar produced by dry distillation of resin-

rich wood was applied to protect the wood from biodeterioration and has been applied since the

early modern period of human history (Skanser 2020). The coal revolution 200 years ago

resulted in the application of creosote or penta (oilborne pentachlorphenol) for wood

preservation, the primary industrial wood preservation method until the early 1960s (Schultz

2008).

The industrial use of oxides of arsenic, chromium and copper was developed in parallel in the

1930s. All these treatments can be considered 1st generation industrial wood preservation

systems (Schultz 2008). These treatments are now considered very toxic to humans and the

environment and were replaced by copper-rich treatments, recognized as 2nd generation

preservative treatments (Schultz 2008). However, their use has been shown to have limited

effectiveness, since fungi can tolerate various metals and metalloids by different biochemical

pathways (Druzhinina et al. 2016), whilst there have been warnings about copper-resistant fungi

reported in the scientific literature (Guillénet al. 2009; Hastrup et al. 2005; Green et al. 2005;

Weissman et al. 2000; Cervantes et al. 1994) and effect on mould fungi is poorly known.

Under such circumstances, some European countries shifted their requirements towards entirely

organic protective solutions, leading to 3rd generation of wood treatment products appearing on

the market. However, keeping in mind the biodegrading potential of the fungi, the development

of fungal resistance is a serious issue not only in wood material science but in agriculture, food

security and, more dangerously, where it can target human health (Berman et al. 2020; Roilides

et al. 2019; Fisher et al. 2018). Moreover, the Regulation (EU) No 528/2012 of the EU

Parliament and the Council concerning biocidal products (European Parliament and of the

Council 2012) mandates

“…the harmonisation of the rules on the making available on the market and the use of biocidal products, whilst ensuring a high level of protection of both human and animal health and the environment. The provisions of this Regulation are underpinned by the precautionary principle, the aim of which is to safeguard the health of humans, the health of animals and the environment. Particular attention shall be paid to the protection of vulnerable groups.”

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As a result of concerns regarding the environmental impact of organic preservatives, there has

been an increase in considering wood modification as a 4th generation wood protection system,

aimed to improve the material properties of the wood whilst not causing environmental danger

in use or during demolition (Hill 2006; Brischke et al. 2016, Sandberg 2021). Wood

modification is a broad concept, and recently a detailed review of the current applications

published (Sandberg 2021). Briefly, there are four main types of modification, and their mode

of action based on chemical treatment, thermally-based treatment, treatment with

electromagnetic radiation and treatments based on the intelligent use of biological processes.

When knowledge about resistance of those treatment against mould fungi is limited. The

transition towards 5th generation treatments regarding the intelligent use of biological processes

occurred recently. The scientists proposed many compositions based on natural products, but

the industrial application was achieved to our current knowledge only with fungal-based wood

finishes (Nieuwenhuijzen et al. 2018) having a brand name Xyhlo (Xyhlo Biofinish 2021).

The traditional wood science understanding for wood modifications has been based on a

production of homogeneous, steady, predictable and reproducible material. However, the

scientific community has noted the failure of standard testing procedures of biodeterioration

due to poor predictability and reproducibility resulting from short-term to long-term exposure

tests (Kutnik et al. 2014). In addition, the concept of a fresh interactive approach to studying

wood through a “dynamic surface envelop”, which considers bulk and surface chemistry, is

more relevant nowadays and leads to designing modern wood modification via single

applications (Rowell 2021). The implication of this should be a data-driven approach since

there are no similar environmental conditions for construction sites. A holistic concept

considering physical, chemical and biological changes should be applied during the design of

wooden material applications to prolong service life, meet the demand for occupants health and

meet current regulations (Mattsson 2017). One possible action could be done by monitoring the

biodiversity of the fungal population; that is DNA-based survey which will enable the

possibility to identify patterns and align them with any microenvironmental influence on the

durability of wooden material against biodeterioration (Norbäck et al. 2011; Ovaskainen et al.

2020; Mueller, et al. 2004). Some cases have been reported about the resistance of wooden

material toward biodeterioration in moisture-enriched conditions (Austigard et al. 2020) and

the influence of wood-anatomical features on durability (Ligne et al. 2021). Moreover, the

fundamental aspects of fungal growth on wood should be switched from traditional single fungi

studies towards fungal colonisation patterns and multiple scale factors influencing that

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colonisation, keeping in mind the dynamic nature of aerial bioaerosols (Šantl-Temkiv et al.

2020; Buiarelli et al. 2013; Kummer et al. 2008), which are responsible for 70% of indoor

microbial populations (Adams et al. 2013). The effect of antagonistic microbial interactions

should also be considered in parallel with inter- and intraspecies interactions to find targets

for biodeterioration prevention (Fabiola Soto et al. 2018).

However, as mentioned in a review (Lipke 2018), the shortage of fundamental funding and a

dramatic shift in research towards human pathogens has moved the attention of fungal

researchers away from studies relating to the environmental processes of degradation. Such

essentials at the interface of wood and air are the adhesion of fungi to surfaces, the composition

of the extracellular matrix, community development under the different influence of abiotic

factors and nutrient availability. Are there any antifungal extractive molecules that are

available, for example, on the wood surface? Is it possible to get more precise durability at the

wood species level for a particular fungal genus, or is it possible to retain the confidence of data

stated in European Standard EN 350:2016 given that, for example, Scots pine sapwood exhibits

durability from 3 to 5 to Basidiomycetes but not to mould fungi? The durability of wood against

outdoor and indoor mould types and their metabolic activity investigation in response to

nutrient preferences in those conditions should also be concerned for potential risks and benefits

in the assessment procedures of wood material in the future.

In summary, there is no need “to re-invent the bicycle”, but look towards other industries such

as food and agriculture that deal with biological objects and environmental exposure and adopt

their best practices for the protection of wood against biodeterioration in practical uses.

Nevertheless, for a fundamental understanding, there is a need to modelling all discussed factors

and their influence on wood material properties under environmental exposure to

biodeterioration, including all influencing factors together and such an approach requires the

application of computer methods for theoretical and experimental modelling of dynamical

processes on a wooden surface and in the bulk of the material (Epstein 2008). Furthermore, it

is necessary to apply the complexity of methods and models that describe fungal growth on the

wood, as discussed below.

3.5 METHODS AND EVALUATIONS DESCRIBING FUNGAL GROWTH ON WOOD To understand the fundamental properties of fungal growth, the research community has

applied many testing methods and evaluating procedures to describe fungal growth on wood.

The task is quite challenging since a growing fungal colony on wood has a 3D structure.

39

The ergosterol content is helpful as a fungal biomarker since it is a unique sterol that maintains

cell membrane integrity (Lau et al. 2006; Mille-Lindblom et al. 2004; Hippelein et al. 2004;

Bjurman 1994; Newell et al. 1988). However, the general procedure for its determination is

quite complex and requires a sample preparation that complicates its use in an industrial

continuous testing. Another compound produced by fungi is nitrosamine (Olofsson et al. 2016),

which forms part of the structure of chitin, and is not unique just to fungi. Phospholipid fatty

acids have been used to estimate fungal biomass (Frostegård et al. 1996) but require

chromatographic separation, as with all markers mentioned above. Fluorescein diacetate has

been evaluated as a marker for fungal activity by estimating the wood's total esterase activity

(Verma et al., 2008). However, for some wood-decaying fungi, the hydrolysis of fluorescein

diacetate was not as effective as for other microbial species (Olsen et al. 1994). Molecular

biology development had led to the appearance of methods of fungal biomass quantification

aimed to determine the ratio between DNA and ergosterol content (Song et al. 2014); DNA,

chitin and mass loss (Eikenes et al. 2005); ergosterol, phospholipid fatty acid 18:2ω6.9 and

accepted fungal barcode internal transcribed spacer (ITS) (Baldrian et al. 2013). Another

method for assessing general biological contamination, including fungi, which is widely used

in hygiene control, is based on ATP measurement, but detection limits are pretty low

(Shaughnessy et al. 2013; Amodio et al. 2014; Nante et al. 2017) and requires evaluation for

use in wood regarding fungal growth.

Nevertheless, all these methods need calibration, verification, reliability and predictability.

Therefore, the main indexes for the fungal virulence estimation are still mass loss for rot fungi,

visual inspection and surface coverage for mould fungi (Boyle et al. 2011). Furthermore, those

indexes are applied when wood material should meet criteria described in standard procedures

for durability assessment and recognized by regulatory bodies.

There are only six standards related to mould growth terminology at the international level,

particularly on the International Standard Organization (ISO) web page. However, only one of

these was indirectly related to wood material ISO 13788:2012 (ISO 2012) and provided

recommendations regarding “simplified calculation methods for the internal surface

temperature of a building component or building element below which mould growth is likely,

given the internal temperature and relative humidity during installation process”. Another

international standardisation organisation ASTM International, formerly known as the

American Society for Testing and Material, applies only one standard related to mould and

unseasoned lumber, ASTM D4445-10 (ASTM International 2019). The evaluation of the

40

biodeterioration only related to the surface intensity of mould growth and visible discolouration

practices to score by using a scale from 0 to 5 where the 5 is maximum intensity. The same

pattern based on visual surface evaluation has been employed in ASTM D7855/D7855M - 13

standard to determine the intensity of mould growth on coated building products (ASTM

International 2013). That is very similar to ISO 4628-1:2016 (ISO 2016) and EU standard

harmonized in Sweden SS-EN 16492:2014 (SIS 2014).

On the European level, the main regulating document for wooden constructions is Eurocode 5:

Design of timber structures SS-EN 1995-1-1:2004 (SIS 2004), where the resistance to

biological attack is limited by “adequate durability for the particular hazard” or selected

preservative treatment. This regulates and ensures wood material quality and consumers’

assurance. Here, the main requirements for testing are described in the SS-EN 350:2016 (SIS

2016), whilst the testing procedures CEN/TS 15083-1 (CEN 2005) for Basidiomycetes was

withdrawn and CEN/TS 15083-2 (CEN 2005) for soft rotting fungi is still valid. Currently

updated, the SS EN 113:2020 is the main testing procedure for Basidiomycete rot-like fungi, in

preservative-treated wood, and is also used to evaluate thermally modified wood (SIS 2020a;

2020b). Procedures for susceptibility of wood to heavily sporulating fungi and wood

preservative efficiency includes soft rotting fungi, other inhabiting micro-organisms with main

assessment index as a mass loss and is regulated by SS-EN 807:2009 (SIS 2009). However,

that standard does not describe microbial colonization, and community development on the

wood and the decay mechanism is still unknown (Råberg et al. 2005). Finally, SS-EN 152:2011

(SIS 2011) is a standard that describes a procedure for testing the resistance of wood

preservatives against staining fungi using species from genus Aureobasidium, but not for other

micro-fungi with different physiological requirements for growth. An additional standard, SS-

EN 15457:2014 (SIS 2014), allows for assessing five fungal strains but only applies to paint

and varnish evaluations.

At the European level, the durability of wood and wood-based products in the European Union

is under the supervision of the European Committee for Standardization, and specifically the

committee CEN/TC 38. Their technical report SIS CEN/TR 16816:2016 (SIS 2017)

summarized guidance on the utilization and improvement of existing methods to estimate the

service life of wood and wood-based products.

It has been noted that current individual standards have poor applicability for modern

requirements of end-users. That was confirmed by Meyer-Veltrup, in a study of 24 different

test methods (Meyer-Veltrup et al. 2017), where differences in moisture and decay development

41

were recorded between testing methods. Therefore, scientists are still looking for efficient

evaluation methods that allow the prediction of material behaviour.

There is a need for empirical modelling based on the experimental conditions to apply to all

factors simultaneously. Since moisture and temperature areas are considered the primary

influencing parameters of decay, several studies have focused on prediction models for wood

in different environmental conditions. For example, Viitanen developed and validated a model

based on laboratory studies of timber under varied and fluctuating humidity conditions,

temperature and time, where the mould index was used for material evaluation (Viitanen et al.

2015; Viitanen 1997; Viitanen et al. 2010). The ERA-40 weather atlas for the period from 1979

to 2001 was used to model decay risk. In another approach, the Scheffer index has been used

for decay prediction (Scheffer 1971). Assessment of the actual decay is based on mass loss or

“by knife test” could be found in the EN 252 standard (SIS 2014).

Limiting moisture conditions for mould fungi growth on the wood material surface in a climate

has been used by Johansson (Johansson et al. 2012; Johansson et al. 2017; Johansson et al.

2013; Johansson 2012) where mould index about surface coverage was applied for the

evaluation. The drawback of the approaches mentioned above are the critical values for material

assessment based on the external environmental condition, but not on the intrinsic nature of the

wooden material and the availability of nutrients for fungi in those environments.

A meta-analysis done by Johansson (Johansson et al. 2017) indicated the importance of surface

structure, wood species and sawing pattern, and the variability of the wood characteristics and

how these were significant challenges for defining standardized specifications in the

susceptibility of wood to mould growth during testing. The wetting ability and durability model

during an outdoor performance of wood was developed from data obtained from laboratory

tests performed in Norway, Slovenia and Germany, and field tests performed in Norway

(Meyer-Veltrup et al. 2017). Such a coordinated activity with different geographical locations

is a way to evaluate wooden performance in different climate conditions.

Intrinsic properties such as the influence of process parameters for drying wood surface and

attempts to link it to mould growth were first pointed out in 1990 by Boutelje in studies at the

Swedish University of Agricultural Sciences in Uppsala (Boutelje 1990; Theander et al. 1993).

Following that, Terziev attributed the importance of felling time and the influence of low

molecular sugars and nitrogen migration on the intensity of mould fungi growth (Terziev et al.

1997; Terziev et al. 2007; Terziev 2009) since Scots pine has sugar-enriched sapwood, which

attracts mould fungi.

42

Attention to the influence of wood drying temperature on durability was shown in studies of

Sehlstedt-Persson (Sehlstedt-Persson et al. 2010). The study detected that an increase in the

drying temperature reduced durability to brown rot fungus. Furthermore, the total phenolic

content was lowered with drying temperature, with degradation of phenols occurring, and the

possibility for reducing decay was discussed (Sehlstedt-Persson et al. 2010). As a result,

investigations into whether different drying temperatures and board arrangements during drying

could force and have a direct influence on the migration of nutrients towards the wood surface

was undertaken. Thus, the monosaccharide and disaccharide content influence on mould growth

during outdoor testing was studied, along with the link to low molecular compound distribution

related to drying conditions. A solution to this was the planing of nutrient-rich gradients to

prevent mould growth (Sehlstedt-Persson et al., 2011).

Furthermore, tests on Norway spruce allowed a comparison with the migration pattern of Scots

pine in order to study wood species behaviour during drying (Karlsson et al., 2012).

Furthermore, the degradation of saccharides on the surface by contact heat treatment reduced

mould growth intensity, probably due to producing some toxic chemical compounds for fungi

during such short-term high-temperature treatment (Ahmed et al., 2013). All the above studies

focused on hydrophilic components such as sugars and their degradation. However, having a

holistic overview, the hydrophobic fractions or lipophilic extractives and their influence on

mould growth should be considered, and where the knowledge gap is evident.

The modern transition toward society digitalization raises demand for fast and non-destructive

solutions in the wood quality assessment, like surface sensing and determining chemical

compositions or contaminants. To achieve this, infrared (IR) and near-infrared (NIR)

spectroscopic techniques seem to be promising techniques for developing the prediction models

that can distinguish fungal deterioration on wood without applying complex destructive

measurements.

The IR technique allows detecting fundamental molecular vibration, and here sample

preparation might be required. Lecellier (Lecellier et al., 2015) successfully applied FTIR via

developing a spectral library to identify ca. 500 mycelial biomasses of single fungal strains with

relatively high precision for some strains. The presence of characteristic fundamental vibrations

and the relatively high-resolution power of IR allows the creation of individual fingerprints of

chemical compounds, with the authors noting the need for complete chemical profiling of those

strains. However, it does not truly work for ATR detection (Jelle et al. 2012). Another

spectroscopic technique, the combination of visible and near-infra red spectroscopy, has been

43

shown to be user friendly in terms of sample preparation and simplicity of application. It has

been widely tested for the evaluation of heterogeneous samples of biological origin. However,

such applications usually collect large arrays of data that should be appropriately processed and

analysed using multivariate calibration, analysis and chemometric techniques (Workman et al.

2012).

There are two ways to do that by using hyperspectral imaging technique (HSI) and

spectrometers. Hyperspectral imaging combines image and spectra analysis, but its application

in IR range is quite difficult for microbiological applications due to signal interference by water

or low microbial concentration. It is quite challenging to detect below 100 colony-forming units

per millilitre (Gowen et al., 2015). However, for the moisture content dynamics in wood, a new

approach using HSI was proposed recently using a GUI interface (Stefansson et al., 2021). The

attempt to use HSI for fungal development and individual fungi evaluation was made for the

Fusarium genus (Williams et al. 2012b; 2012a; 2012c), whereby it was possible to efficiently

measure fungal growth in pure culture in one media, at a constant temperature and by

integration of images as a sum of pixels over time, thus allowing the construction of microbial

growth curves.

A large volume of published studies describes the application of NIR spectroscopy, particularly

for wood performance evaluation. The fundamentals of NIR band assignments for wood and

wood components were summarized by Schwanninger (Schwanninger et al. 2011) and the

summary of research in wood presented by Tsuchikawa and Schwanninger (Tsuchikawa 2007;

Tsuchikawa et al. 2013). The practical solutions based on many years of research work

regarding trees, wood and derived products assessment were summarized in a tutorial by

Sandak (Sandak et al. 2016), which has led to several research groups undertaking successful

applications of NIR spectroscopy for wood modification quality evaluation. Quality evaluation

and the link to colour changes, mass loss of heat-treatment by NIR was investigated by

Todorovic (Todorović et al. 2020). Popescu (Popescu et al. 2018) separated structural changes

in modified wood in relative humidity fluctuations by using dendrograms. To measure the effect

of weathering phenomenon, it was suggested that analysis of the absorption band on

hemicellulose was a key factor for classification (Tsuchikawa et al. 2003). However, xylograms

and spectral filters developed (Sandak et al. 2016) to visualize the degradation/modification

process provided an insight into applying more unusual technique like aquaphotomics

(Tsenkova 2009) or optical characteristics (Tsuchikawa 1998) in future research and methods

were found to model water interference in spectroscopic techniques studies using NIR.

44

However, it was out of scope in our current study. For biodeterioration and fungal load

estimation to be experimentally determined by NIR, the ergosterol content was used to build

calibration models (Börjessonet al. 2007; Porep et al. 2014; Mancinelli et al. 2014), but all

authors stated the dependence of calibration models accuracy from the heterogeneous properties

of the samples, as well as the influence of other internal and external changes. When measuring

wood decay by NIR spectroscopy, there is an overlap of the water bands with wood-related

bands, which was mentioned in the review of Fackler (Fackler et al. 2012). Sandak (Sandak et

al. 2013) defined evidence of separation of different fungal decay on wood by NIR spectroscopy

but indicated the need for further development of the spectral database for fungi-wood analysis.

However, for the early signs of decay, it seems the application of NIR spectroscopy is not

reliable (Green et al., 2012). Leinonen (Leinonen et al. 2008) highlighted problematical issues

to include mass loss as a variable in the calibration models but suggested focusing on the

extractives and relation to decay resistance as an alternative to precise decay tests. Whilst the

main interest has been related to measurements of decay rot fungi from standard decay tests,

the actual applications in historical constructions, for example, is still a challenge due to the

diversity of various environmental and biological factors. The relevance of NIR applications

for wood-staining fungi and spectral regions significant to fungal detection were identified in a

study conducted by Via (Via et al., 2007). A broader perspective has been adopted by Burund

(Burud et al. 2014) by using HSI, whereby it was possible to detect and quantify two early

primary colonizers (A. pullulans and C. cladosporoides) during weathering, with results

suggesting it was possible to identify a small number of wavelengths related to the interesting

regions instead of keeping whole big data files. That might be applied to non-destructive testing

like imaging and multispectral approaches, instead of a hyperspectral approach, might be

promising in large industrial applications, as has been shown for early disease prevention in

sectors other than the wood industry (Fahrentrapp et al. 2019). Missing information was found

regarding separation by NIR of wood from conventional drying and thermal modification. As

well as limited information exists regarding evaluation of wood contaminated by different

mould fungi.

45

4. MATERIALS AND METHODS

4.1 WOOD PLANING DEPTH, IMPREGNATION AND EXTRACTIVES INFLUENCE ON THE FUNGAL ATTACK 4.1.1 WOOD SPECIMENS PREPARATION FOR COPPER IMPREGNATION The main wood species in the study was Scots pine (Pinus sylvestris L.) sawn boards with a

high sapwood ratio selected directly after sawing at a local sawmill in North Sweden. Sample

preparation is described in detail in Paper I

The specimens were divided into four randomly selected batches, and planing depths of 0, 0.7,

2.0 and 4.0 mm were machined. The planing depth of cross-sections took into account any

degree of sample cupping as defined according to Figure 4.1.

1) the planing of pith side

2) thickness measurement immediately above the pith

3) the planing of the sapwood side

4) thickness measurement immediately above the pith

Figure 4.1. Planing scheme and definition of planing depth taking any cupping of cross-

section into account.

4.1.2 IMPREGNATION AND EVALUATION OF IMPREGNATION RESULTS After weighing all the samples, the batches were transported to an impregnation plant, where

the four batches were impregnated with a copper salt solution in one run in an industrial

autoclave. The impregnating agent was Celcure AC 800 (Koppers, Helsingborg, Sweden).

According to the Nordic Wood Preservation Council, the impregnation was performed

according to current routines in NTR class AB (Nordic Wood Preservation Council 2012) to

meet product requirements for industrially protected wood according to the Nordic Wood

Preservation Council, which requires full sapwood penetration in Scots pine.

46

4.1.3 WOOD SPECIMENS PREPARATION FOR DRYING PROCEDURES EVALUATION Green sideboards from Scots pine (Pinus sylvetris L.), consisting only of sapwood, were taken

from a sawmill in Norrbotten, Sweden. The boards were divided into two groups for air-drying

and kiln-drying. For air-drying performance, boards were dried indoors on stickers at a

temperature of 20°C (RH about 10%). Kiln-drying was performed in a small-scale laboratory

kiln with air circulation according to the design in Figure 4.2. The detailed procedure is

described in Paper II.

Figure 4.2. Drying and board preparation scheme. (A) double-stacking of boards with the

sapwood side of each board facing outwards in each pair, (B) stacking of boards in the

laboratory kiln, and (C) planing depth scheme for specimens: (a) unplaned, (b) 0-0.25 mm,

(c) 0.25-0.75 mm, and (d) 0.75-1.75 mm planing depth.

4.1.4 WOOD SPECIMENS PREPARATION AFTER THERMAL MODIFICATION Two specie of exotic African wood, namely iroko (Milicia excelsa (Welw.) C.C. Berg) and padauk

(Pterocarpus soyauxii Taub.), were harvested in Gabon (Sablík et al. 2016). The dimension of

specimens was H20 x W20 x L300 mm. Moisture content before thermal treatment was 3±1%. The

thermal modification was achieved according to the Thermowood® process (International

ThermoWood Association 2003). The thermal treatment temperatures used in the study were 20,

160, 180 and 210°C. According to the procedure in Paper IV, the extractives content and chemical

analysis were performed and described shortly below.

47

4.1.5 WOOD EXTRACTIVES CHEMICAL ANALYSIS For the extraction, shavings from collected fractions from different planing depths were used -

the detailed protocol described in Papers II and III.

The wood amount used for extraction was 1.0 g, and extractions were done using SCAN-CM

49:03 standard method (SCAN 2003). The total lipophilic extractives content was given in mg

g-1 of dry mass after evaporation of acetone and drying of the extracted solution.

Fatty acids and resin acids in the acetone extract were analysed using GC-MS after

trimethylsilylation according to Sjöström (Sjöström and Alén 1999) with 1-methylnaphthalene

dissolved in pyridine as internal standard (Lai 1992). The resin acids and fatty acids were

identified using NIST Mass Spectral Library (Johnson 2014), and a quantitative estimation was

made by comparing the peak areas with that for the internal standard. HPLC quantified the

sucrose, glucose and fructose contents by building calibration curves using corresponding

reference substances described more thoroughly in the corresponding paper. The Folin-

Chocalteu reagent determined the total phenolic content according to a procedure described by

Julkunen-Tiitto (Julkunen-Tiitto 1985). Nitrogen content was determined by the Kjeldahl

method, whilst analysis of ash content was done thermogravimetrically after full combustion.

The measurement for African wood was done by pH meter Metrohm 744 after treatment of the

sample with a sodium chloride solution to exchange protons in less accessible carboxylic groups in

the wood by sodium ions (Sithole 2005).

4.1.6 TEST DESIGN FOR WOOD MATERIAL BIODETERIORATION In the experiment with different planing depths followed by copper impregnation, the

specimens were tested by two methods of fungal exposure: indirect and direct. The indirect

method was based on the spontaneous emission of spores by using open Petri plates with pure

cultures placed on the bottom of a plastic box (Figure 4.3a). The direct method was based on

applying four isolated fungi spore suspension in a particular concentration of spore/ml directly

onto the wood surface (Figure 4.3b). Specimens were hung in two separate plastic boxes with

the open wall sealed with an air-transparent polymeric membrane and kept at 20±2°C and the

relative humidity of 90±5% (Paper I).

Four mould fungi were used from the culture collection at Wood Science and Engineering,

Luleå University of Technology. The initial MC and final equilibrium MC of wood samples

were recorded. Images of the mould growth on the wood surface were obtained by digital

photography, and the growth was assessed in two ways using ImageJ software: as the

percentage of the area of fungi from the total area of the board and as the covered area in mm2

48

of fungal growth (Schindelin et al. 2012). The individual mould species on the wood surfaces

were identified with the naked eye and an optical microscope to identify the morphological

characteristics of each fungus.

a) b) Figure 4.3. Mould growth tests: a) indirect and b) direct methods

Two white rot strains (441 and JPEI) of Trametes versicolor (L.) Lloyd was obtained from the

Aloha Medicinals Mushroom Culture Bank, Carson City, NV, USA. The virulence of each

fungal strain was assessed from the mass loss of treated and non-treated samples, determined

according to standard SS-EN 113 (SIS 2020).

The mould fungi influence on the sapwood surfaces of the kiln and air-dried Scots pine side

boards was studied in Paper III. Air drying was performed indoors in the laboratory, whereas

boards from Scots pine were kiln dried for 44 hours to a moisture content of 16%. Mould growth

on the sapwood surface was tested by applying the spore suspension on the specimens by air

brush, the suspensions containing fungal specie were from the culture collection, Division of

Wood Science and Engineering, Lulea University of Technology, Skellefteå, Sweden.

The samples were kept at 22°C and 90%RH for 30 days in a chamber with a filter to exchange

air. Visual inspection of the studied samples was classified into a percentage of the covered

area and used as a response variable (Figure 4.4).

Figure 4.4. Air-dried (left) and kiln-dried (right) Scots pine sapwood boards after mould test.

49

4.2 WOOD THERMAL MODIFICATION, PROTECTION BY GRAS AND BIOCIDES UNDER FUNGAL ATTACK The method for the methacrylic resin impregnation of dried and thermally modified wood by

applying the heating-and-cooling method is described in Paper V, VI.

The localized surface modification method was developed for Scots pine sapwood in Paper V

by Elustondo (Elustondo et al. 2016). The treatment solutions (water, rapeseed oil and

biodiesel) with different densities and kinematic viscosity were tested for their possibility to

create a permanent protective layer below the wooden surface by a heating-and-cooling method

and a compression-and-expansion method as a comparison.

The inhibition of mould fungi growth by pure methacrylate resin was studied in Paper VI. A

commercial methacrylate resin and an accelerator (Cactus Juice Stabilizing Resin) were

purchased from Turntex, LLC (San Marcos, TX, USA). A heat-sensitive accelerator (2,2’-

azobisisobutyronitrile), which on heating generates radical initiators, was mixed with the resin

(1:50) prior to the submersion coating of the wood specimens. The filter paper 00K with a

diameter of 55 mm was soaked in the resin and heated at 103°C for 30 min for curing. The

fungal test procedure was done according to the procedure described in EN 15457:2014 (SIS

2014). The filter paper, after resin curing, was placed on a Petri plate with malt extract agar.

The mixture of spore suspension of fungi was applied to the filter paper treated with

methacrylate resin and no treatment as a control. The fungal cultures used in the test were from

the culture collection, Wood Science and Engineering, Luleå University of Technology,

Skellefteå, Sweden.

As described in Paper VII, the effect of methacrylate resin treatment and wood was tested on

small wooden blocks treated with methacrylate. After kiln drying and thermally modifying the

Thermo-D process, wooden blocks treated with methacrylate resin specimens were placed on

Petri plates with malt-extract agar. One Petri plate contained paired specimens: treated with

methacrylate and non-treated. The spore suspension of four different fungi was sprayed with an

airbrush over the specimens. The fungi from Culture collection, Wood Science and

Engineering, Luleå University of Technology, Skellefteå, Sweden, were applied in the test. The

Petri plates were incubated in a climate chamber at 24°C and 90%RH. The visual evaluation of

fungal growth was assessed after seven days of exposure. All specimens in the Petri dishes were

stored for two months.

The evaluation of direct and indirect inoculation by fungal cultures for the effectiveness of

GRAS compounds against biodeterioration in the box test was done, as described in Paper VIII.

The GRAS compounds contained: bacteria Bacillus amyloliquefaciens (trade company

50

ABITEP, Germany) applied in concentration 1% in water solution; silicon, potassium, natural

parts of plants and water (trade company SIOO, Sweden) applied in 2 step treatment 1st by

treating with potassium silicate ca. 32% and second treatment with polymerizable alkoxysilane;

N-alkylbensyldimethylammonium chloride (trade company JAPE, Sweden) was applied in

concentration 20%. In addition, the moisture content of treated wood samples before and after

the test was recorded.

The fungal cultures used in the test methods were from the culture collection, Division of Wood

Science and Engineering, Luleå University of Technology, Skellefteå, Sweden.

Two (direct and indirect) inoculation methods were applied. Spraying of spore suspension on

wood samples and placing open Petri plates contained growing fungi on the bottom of the

plastic boxes with hanged tested samples above were according to design in Figure 4.3. The

detailed methodology of inoculation is described in Paper I and Paper VIII. The evaluation

was done by mould grade – a general index describing the severity of the fungal growth on the

wooden surface according to standard EN 16492:2014 (SIS 2014) and area that was covered by

single fungus in % to the whole area that evaluated microscopically and by applying ImageJ

software (Schindelin et al. 2012).

The effectiveness of the commercially available biocidal treatments on wood was studied in

Paper IX. The treatments were: treatment 1 - pigmented primer with tetramethylol

acetylenediurea and iodopropnyl butyl carbamate (IPBC), trade name HFlex from AkzoNobel,

Sweden; treatment 2 - paint with a mixture of three biocides (IPBC, benzisothiazolinone (BIT),

methylisothiazolinone (MIT) and 5-chloro-2-methyl-1,2-thiazol-3-one (CMIT)/MIT mixture),

trade name Performance from AkzoNobel, Sweden; treatment 3 - paint with iodopropynyl butyl

carbamate (IPBC), trade name Teknoshield from Teknos, Sweden; treatment 4 - laqvin Tone

contained three biocides 1,2-benzisotiazol-3(2H)-on, 2-oktyl-2H-isotiazol-3-on, 2-Methyl-4-

isothiazolin-3-one, Laqvin Ton from Sherwin-Williams Sweden AB, Sweden.

The fungal test was performed in 90 mm Petri plates. Scots pine sapwood and heartwood, and

spruce were used in the study. Two samples (treated and untreated control) were placed on the

plate with a specified distance between each other, and between, a 5 mm fungal inoculum was

placed (Figure 4.5). Five pure cultures of fungi species were used in the study from the culture

collection, Division of Wood Science and Engineering, Luleå University of Technology,

Skellefteå, Sweden. The specimens were exposed at a temperature of 24°C and relative

humidity of 90%. The mould fungi growth was evaluated by the size of the inhibition zone

51

between treatment and fungi measured from the nearest border at four positions, and the average

value was presented in mm.

Figure 4.5. Example of the assessment in Paper IX.

4.3. SPECTROSCOPIC CHARACTERISATION OF WOOD UNDER FUNGAL ATTACK Spectroscopic observation was done by using hyperspectral imaging (HSI) technique in Paper

X and portable near-infrared (NIR) spectroscopy in Paper XI and XII. Figure 4.6 shows the

HSI setup where the camera could be seen in Figure 4.6a and the conveyer belt that moves the

samples during measurement in Figure 4.6b. The samples were positioned on the belt and the

detailed procedure is described in Paper X. The point-based microNIR OnSite Spectrometer

(VIAVI Solutions Inc., San Jose, CA, USA) with NIR wavelengths from 900 to 1650 nm for

the direct evaluation of fungal contamination on the wooden surface with step 6 nm was used

(Figure 4.7).

52

a) b)

Figure 4.6. Test set-up: (a) Spectral Camera with (b) conveyer belt for samples movement.

Figure 4.7. MicroNIR portable spectrometer.

The assessment by portable NIR was done for the dried boards with different planing depths

and grown fungi on them in Paper XI, and weathered outdoor samples with protective

treatments from Paper IX described in Paper IX.

4.4 STATISTICAL ANALYSIS USED IN THE THESIS The univariate and multivariate statistical analyses were used for data analysis to estimate the

variability and spread of the data in all papers. For the ANOVA and non-parametric data

analysis in Paper I, II, IV, V, VI, VII, IX, the SPSS 20 (IBM, USA) software was used. The

multivariate analysis used in Paper III, IV VIII, XI, XII was performed by SIMCA (Umetrics,

Umeå, Sweden) and according to methods described by Eriksson (Eriksson et al. 2013). The

multivariate image analysis of obtained spectra in Paper X was performed using Evince

software version 2.7.9 software, Prediktera, Umeå, Sweden.

53

5. RESULTS AND DISCUSSION

5.1 WOOD PLANING DEPTH, IMPREGNATION AND EXTRACTIVES INFLUENCE ON FUNGAL ATTACK Paper I examined the impact of planing depth and impregnation by copper containing solution

on the fungal infestation. Strong evidence of and influence of planing depth and impregnation

solution uptake was found during impregnation, and apparently, the direct and indirect way

fungi was introduced to the biodeterioration test significantly influenced mould area growth on

the wooden surface, as shown in Table 5.1 below.

Table 5.1. Mean value of mould areas and moisture content (MC) of Scots pine sapwood

boards treated by different fungal inoculation methods.

Planing depth,

mm

Mould area

(%)

Mould area

(mm2)

MC before

test %

MC after test, %

Indirect fungal inoculation

0 32.4a 2222.8a 9.6 20.0

0.7 0a 0a 9.5 17.4

2.0 0a 0a 9.5 20.5

4.0 0a 0a 9.6 20.5

Direct fungal inoculation

0 24.5a 1659.01a 9.6 21.8

0.7 0.4a 25.9a 9.7 21.8

2.0 0.1a 3.5a 9.6 21.4

4.0 0a 2.4a 9.6 22.1 a - significant mean difference, p˂0,05

The pattern of fungal surface colonisation differs between two inoculation methods employed.

The fungal species Paecilomyces variotti and Aspergillus niger were dominating in the indirect

contamination (Paper I). However, when direct spraying was done, only the favoured growth

of Aspergillus niger was obtained. Perhaps Paesylomyces variotti, which produces conidia as

chains that quickly become airborne, could favour a more natural infection by air-flow and

adhesion to the surface (indirect). In contrast, Aspergillus niger conidia form a single dry spore

and thrive better when administered in excess of water (Adan and Samson 2011). Figure 5.1

below presents examples of the visual appearance of the contamination pattern between the two

applied methods.

54

Indirect Direct

Figure. 5.1. The surface appearance of samples inoculated by the direct and indirect methods.

The tendency to produce more aerial mycelia at the lower edges of boards was observed in the

natural (indirect) contamination method (see red arrow in Figure 5.1). Such tissue development

could be a response to extreme environment where fungi develop a biofilm able to absorb water

vapour from the surrounding atmosphere or that the fungi initiate aerial morphogenesis to

produce more reproductive tissue for further survival and new habitat colonization (van

Laarhoven et al. 2016; Pypker et al. 2017; Segers et al. 2017) or higher moisture content due to

capillary suction of axial fibres at the ends of the specimens.

55

Even after impregnation by biocidal copper, the unplaned surface gave the highest fungal

growth and revealed importance of the nutrient availability probably on the wooden surface and

surface performance. However, the effect of the planing on fungal virulence was evident not

only for the mould fungi but also for white rot such as Trametes versicolor. Despite the general

inhibition of degradation by copper impregnation, the trend to highest mass loss was found in

unplaned and non-treated by copper impregnated specimens, as summarized in Table 5.2 below.

Table 5.2. Mean mass loss of Scots pine sapwood boards treated with two strains of

Trametes versicolor according to EN 113 (1997).

Parameter Mass loss, %

Strain 441

Group 1 (planning depth 0 mm) 4.4a

Group 2 (planning depth 0.7 mm) 3.7a



Control (without impregnation) 40.6a

Strain JPEI

Group 1 (planning depth 0 mm) 4.8ab

Group 2 (planning depth 0.7 mm) 3.3ab



Control (without impregnation) 43.9a a - significant mean difference between impregnated and non-impregnated (control) groups, p˂0.05

b - significant mean difference between impregnated groups, p˂0.05

It was seen that planing depth influenced the biodeterioration pattern for the mould and the rot

fungi. To further understand factors influencing mould growth, the study of the extractives

distribution during different drying types was undertaken, as described in Paper II. Acetone

soluble extractives and total phenolic content demonstrated significant prevalence in kiln-dried

than in the air-dried samples (Figure 5.2). The most enriched part of the board was the upper

layer of the kiln-dried planks (0-0.25 mm) which could be related to the formation of an

evaporation front during the capillary drying phase, around ten tracheidы in width below the

wood surface (Sehlstedt-Persson 2008).

56

a) b)

Figure 5.2. Acetone soluble extractives and phenolic content of kiln-dried in isolated

planed fractions from Scots pine sapwood boards in mm of planning depth: (a) Total

extractives content, and (b) phenolic content of dried mass.

The kiln-drying process influences surface properties of the boards by enrichment of acetone

soluble extractives, phenols, nitrogen and ash content, as shown in Paper III and Figure 5.3

and Table 5.3 below. The nitrogen and ash content was 1.8 and 2.8 times higher in kiln-dried

than in air-dried specimens, respectively. Therefore, the total amount of studied compounds in

kiln-dried samples was estimated to be 69% higher than in air-dried ones (Table 5.3).

Table 5.3. Content of extractives (% owen dry weight) in the surface layer of dried boards

of Scots pine sapwood.

Analysed compounds Air-dried Kiln-dried

Aceton soluble extractives

Phenols

Nitrogen

Ash

3.594

0.044

0.149

0.329

5.707

0.081

0.267

0.906

Total amount 4.116 6.961

The most dominating free sugars (saccharose, glucose, and fructose) form food reserves in

Scots pine (Fischer and Höll 1992) and were also analysed. Data extracted in Figure 5.3 shows

duplicated enrichment by major migrated individual compounds of different origins in Paper

II and III that were measured in kiln dried compare to air dried samples.

57

Figure 5.3. The mean of glycerol, fatty and resin acids, saccharose, glucose and fructose

in the air- and kiln-dried samples at a different depth (0-0.25, 0.25-0.75, 0.75-1.25 mm)

from the surface.

Migration of the sugars and nitrogenous compounds was first demonstrated by Terziev (Terziev

2009), and findings herein provide evidence about translocations and higher contents of sugars

in kiln- than in air-dried samples. It is a fact that wood contains inorganic components, and

particularly pine species have different inorganic inclusions predominantly of potassium,

calcium and magnesium (Fengel et al. 1989). All those metals regulate numerous biological

functions (Banci 2013), and perhaps nutritional enrichment of inorganic and discussed above

organic compounds could create favourable conditions on the wooden surface for mould fungi

growth in our test.

The observation of fungal presence on the wooden surface after mould exposure was performed

by scanning electron microscopy; in the area, 50 µm in depth from the wooden surface, the

tissue was occupied by hyphae, as shown in Figure 5.4. That supports previous studies about

0

5

10

15

20

25

30

35

0-0.25 0.25-0.75 0.75-1.75 0-0.25 0.25-0.75 0.75-1.75

air-dried kiln-dried

(mg/

g of

dry

wei

ght)

Glycerol Palmitic acid Oleic acid Linoleic acid

Stearic acid Pimaric acid Isopimaric acid Dehydroabietic acid

Abietic acid Saccharose Glucose Fructose

58

forming an evaporation front in the drying process allocated below the surface (Sehlstedt-

Persson 2008). Somehow, mould fungi use those nutritional gradients for more intense

development.

Figure 5.4. Scanning electron microscopy image of the wooden surface with massive

fungal growth.

Since data contained various variables (X), the general influence and discovery of a pattern in

data from Figures 5.2 and 5.3 and Table 5.3 were subjected to multivariate data analysis where

the mould area and drying type was used as responses (Y).

Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) techniques was

best performing regarding separation of systematic variation of variables (X) into two parts,

one of which is correlated (predictive) to response (Y) and another part that is uncorrelated

(orthogonal) to Y as it was shown by Eriksson (Eriksson et al. 2013). The variables were centred

and unit variance scaled, and the OPLS-DA model contained one predictive and one orthogonal

component. As a result, the model could predict (R2X predictive) 17.9% of data variation, with

a general (R2X orthogonal) of 33.4%.

The spread of the data in the kiln-dried boards was higher than in the air-dried, probably due to

more intensive forces driven during the faster drying process, as seen in the score plot in Figure

5.5 below.

59

a)

b)

Figure 5.5. The OPLS model: (a) Score plot retrieved from data collected and grouped by

kiln-dried (red) and air-dried (blue) boards and (b) loading plot provided the projection of

variables (green) and response (blue).

60

The VIP variable total extractives, glucose and total phenols content contributed to the model

mostly (Figure 5.6). The remaining VIP variables had a high standard deviation at 95% of a

confidence interval, as shown in Figure 5.6.

Figure 5.6. VIP plot of the OPLS model.

During more harsh thermal treatments like the Thermo-D processing of softwoods, more severe

changes in extractives content and composition occur, such as removing volatile terpenes and

forming new compounds such as aldehydes, acids and phenols (Alén et al. 2002; Windeisen et

al. 2007; Karlsson et al. 2012; Karlsson et al. 2014), which may hamper mould growth. For the

evaluation of extractives and the remaining amount after thermal modification, two species of

exotic African wood were chosen for evaluation, namely and iroko respectively, since they are

already naturally quite rich in antioxidant compounds that are capable of hindering radical

formation during thermal modification (Hill et al. 2021). The total extractives and phenols in

untreated wood in studied samples corresponded to previously studied data (Saha et al., 2013;

Nagawa et al., 2015). The increase in temperature during thermal modification significantly

changed the total extractive content, as shown in Table 5.4 below. With the highest treatment

temperature (210°C), the wood contained the smallest amount of extractives and soluble

phenols. For padauk, a linear decrease of phenols with treatment temperature and time was

strongly evident. For hardwood species like birch, a significantly lower pH has been found in

thermally modified than in untreated wood (Dagbro et al., 2011). In the case of iroko, pH was

slightly higher as for birch, but the thermal treatment of iroko did not reduce the pH.

61

Comparably high content of ash was found in the iroko samples. The biomineralization

properties of that tree could explain higher pH and ash content for iroko, which leads to an

accumulation of mineral carbon. Calcium carbonate has been found in iroko, and such a

compound could work as a buffering agent for the formed acids during the thermal modification

process (Cailleau et al., 2004).

Table 5.4. Results of chemical tests of thermally modified iroko and padouk

Thermal

modification Total

extractives

[%]

Total soluble

phenols

[mgTA/g dry w]

pH Ash content in wood

[%]

Iroko

20 7.8 27.5 5.5 2.0

160 10.4 30.4 5.3 2.3

180 8.1 24.6 5.2 1.9

210 4.8 11.8 5.2 1.9

Padouk

20 11.1 27.4 3.7 0.2

160 9.9 27.1 3.7 0.2

180 9.5 22.5 3.8 0.3

210 7.5 22.0 3.8 0.4

5.2 WOOD THERMAL MODIFICATION, PROTECTION BY GRAS AND BIOCIDES UNDER FUNGAL ATTACK The wooden surface is essential for protection against deterioration, acting as an initial barrier

or buffer to the changes in the bulk of the material. Elustondo (Elustondo et al. 2016) in Paper

V developed the localised surface modification by using varied density and viscosity treatment

solutions and the heating-and-cooling method as an alternative to other traditional methods of

protection solution introducing into the wood. The treatment allowed the creation of a protective

layer under the wooden surface.

Karlsson (Karlsson et al. 2018) used that method for protecting thermally modified wood in

Paper VI. Here the protective properties of pure methacrylic resin were tested regarding

resistance to the growth of mould fungi. As could be seen, the sample treated with resin was

not attacked after exposure for three days to mould fungi in contrast to what was observed for

62

the untreated filter paper (Figure 5.7). Mould fungi used in the test could be identified on the

filter paper surface. The inclusion of Penicillium sp. and Aspergillus sp. used in the test s could

be seen in the control sample in Figure 5.7a. Whilst treated sample did not show any fungal

growth visually (Figure 5.7b), indicating that methacrylic resin could be efficient in reducing

the mould fungi growth of wooden panels without severely influencing the appearance of the

wood structure due to toxicity to the fungi (Ansteinsson et al. 2013; Dormer et al. 1998; Leggat

and Kedjarune 2003).

a) b) Figure 5.7. Filter paper treated without (a) and with methacrylic resin (b) after mould test.

A further test of the methacrylate resin applied on the wood regarding its protective effect was

done in Paper VII (Kim et al. 2020). In laboratory conditions, specimens treated with

methacrylate from kiln and thermally modified wood were exposed to selected mould fungi in

Petri plates. The methacrylic resin significantly delayed fungal colonisation, as can be seen in

Table 5.5 below. The Aspergillus niger was dominating, and the colonisation pattern is

presented in Figure 5.8. Table 5.5. The proportion of specimens’ surfaces of untreated and thermally modified Scots

pine treated with methacrylate where the surfaces were covered with mould after 7 days

Scots Pine Sapwood (%)* TMT (%)*

Untreated 100 (0) 78.2 (23.82)

Methacrylate resin-treated 19.7 (28.53) 6.5 (9.97)

* Standard deviation in parentheses

63

Figure 5.8. Example of Petri dishes showing specimens sprayed with a mixture of moulds

and evaluated after one week of conditioning: a) pine treated with methacrylate resin, b)

untreated pine, c) thermally modified pine treated with methacrylate resin, and d) thermally

modified pine (the red arrows indicate less mould in the surrounding nutrient medium)

The spraying technique chosen in the present study combined with the freely available water

provided a favourable environment for Aspergillus niger, but less for other fungal species, such

as Aureobasidium pullulans and Cladosporium cladosporioides, or a “dry adhesion” fungus

such as Penicillium commune, which all commonly grow on timber outdoors (Nicholson and

Epstein 1991). However, in longer-term exposure, it could be seen that fungi overgrew the

protected areas, and further outdoor exposure found vulnerability of the methacrylic resin

treatment towards the oxidative weathering exposure and possible leaching, due to increased

water solubility giving potential threat to the environment (Dormer et al. 1998; Kim et al. 2020).

The search of efficient wood surface treatments between GRAS compounds and a new

approach for fungal growth evaluation was done in Paper VIII. The two fungal inoculation

strategies, direct by spraying and indirect by placing Petri plates at the bottom of the study box,

were applied. The moisture content of the studied specimens was higher by approx. 11-13%

compared to the initial moisture content. However, the direct inoculation method treatment by

bacteria gave significantly lower moisture content increase after fungal testing. The mould

grade and mould area were used as responses. The most effective was SIOO treatment with ca.

two times lower mould grade than reference samples in both methods. The index mould area

gave a good overview of the fungal community development on the wooden surface and a

greater understanding of surface colonisation. Different micro conditions around every

specimen might explain high variability in data. However, to draw a general conclusion is quite

a

b

c

d

64

difficult by using only univariate data analysis, and here multivariate data modelling methods

had to be applied, which are presented in Table 5.6 below.

Table 5.6. Summary of multivariate analysis (MVA) of the effect of treatment of pine boards

with GRAS compounds.

№ Model PC Obs. R2X R2Y Q2(cum)

1 PCA 2 80 0.448 -0.0139

2 PLS-DA 2 80 0.404 0.36 0.319 Treatment class

3 OPLS-DA 2+1+0 80 0.534 0.394 0.336 Treatment class

4 PLS 3 80 0.49 0.843 0.75 Y mould grade

5 PLS 3 79 0.519 0.847 0.766 Y mould grade

Several PLS models were constructed, and PLS model number five in Table 5.6 showed the

best performance in predicting mould grade. In this model, the mould area and type of treatment

were among the most influencing variables. The correlation between mould areas for

Aspergillus sp. to the indirect method of fungal contamination was the highest, and the mould

areas of Penicillium sp. and Paecilomyces sp. were more influenced by the type of treatment

with GRAS. In general, the three-component PLS model demonstrated quite strong

performance and could predict about 77% of data variation (model number 5 in Table 5.6).

The evaluation of the effectiveness of commercially available wood protective products was

investigated in Paper IX. Here, wood samples of spruce, hearwood and sapwood of pine were

treated with wood protecting products (treatment 1-4) and untreated samples were tested in a

Petri plate, and the size of the inhibition zone was determined as described in the materials and

methods in section 4.2. After four days of incubation with selected individual moulds, fungal

growth was observed in all untreated samples. It was found that Trichoderma sp. and

Penicillium sp. had maximum resistance to the treatments. Specimens coated by a transparent

lacquer treatment 4 (Laqvin) were attacked by those fungi already after eight days, whilst fungal

colonies reached samples with treatment 1 (H-Flex) and treatment 2 (Performance) after 22

days of incubation for all types of wood. An effect on most common outdoor appearing fungi

Aureobasidium sp. and Cladosporium sp. for HFlex and Performance treatments was evident.

Table 5.7 presents the size of the inhibition zone between growing fungus and treated specimens

after 22 days of the experiment.

65

Table 5.7. The average value of the size of the inhibition zone between growing

fungi and treated specimens (mm).

Treatment 1 Treatment 2 Treatment 3 Treatment 4

Aureobasidium sp.

Pine Sap 7.17 4.17 7.25 0

Pine Heart 11.08 4.58 8.33 0

Spruce 8.42 5.33 8.08 0

Trichoderma sp.

Pine Sap 0 0 0 0

Pine Heart 0 0 0 0

Spruce 0 0 0 0

Aspergillus sp.

Pine Sap 6.17 3.25 6.33 0

Pine Heart 6.92 2.83 5.08 0

Spruce 6.17 3.17 8.58 0

Cladosporium sp.

Pine Sap 7.58 1.33 2.58 0

Pine Heart 7.50 1.33 1.67 0

Spruce 7.50 1.33 4.42 0

Penicillium sp.

Pine Sap 0.83 1.33 1.75 0

Pine Heart 0.83 0.5 0.08 0

Spruce 1.75 0 0.44 0

66

5.3 SPECTROSCOPIC CHARACTERISATION OF WOOD UNDER FUNGAL ATTACK

5.3.1 HYPERSPECTRAL ANALYSIS OF THE SURFACE The non-destructive evaluation by hyperspectral imaging (HSI) technique to see the difference

in group separation of dried and differently thermally modified wood was tested in Paper X.

Previous studies in Paper II, and III were able to clearly distinguish differences in extractives

between differently dried wood by wet chemistry methods and statistical tools. A set of

specimens from the air and kiln-dried pine sapwood boards and pine boards thermally modified

according to Thermo-D conditions and WTT processes was explored at the macroscale. The

merged pseudo coloured image of the specimens can be seen in Figure 5.9 below.

a) b) c) d) e)

Figure 5.9: Merged digital images of the specimens from a) air/kiln dried Scots pine

sapwood, and from Scots pine boards, b) Thermo-D planed surface, c) Thermo-D rip-cut

surface, d) WTT planed surface and e) WTT rip cut surface.

A multivariate PCA model was applied to evaluate differences in the combined hyperspectral

image of specimens in NIR region (shown in Figure 5.10) to identify the primary distinctions

among the samples and see the dataset's grouping pattern. The PCA model explained the data

variance where the index R2X_cum=0.97 explained the variance of the X matrix presented by

NIR wavelength numbers. After cleaning (background and edge effect data points removed),

the PCA model was improved, with R2X_cum=0.99461, giving four principal components

(PC). The first and second principal components (PC1 and PC2) gave 94% of the total sum of

squares (%SS) and 4.34%SS of the data variability. In Figure 5.10, five clusters obtained from

the HSI absorbance images could be distinguished (numbered arrows 2 to 6). These clusters are

related to particular areas in the wood surfaces from Figure 5.10, as described below.

67

Figure 5.10. Score plot of near-infrared hyperspectral imaging after background image

cleaning of PC 1 against PC2 of samples in Figure 5.9 above.

Six zones indicated by the arrow in the score plot in Figure 5.10 related to the specimens and

surface characteristics presented in Figure 5.9:

1) The colour difference is determined in knots due to extractives and a higher density

than the surroundings. The area indicated by a circle in the score plot is related to the

knots and latewood “bow” close to the knot on the upper right-hand side of the

surfaces of Thermo-D modified wood (Figure 5.9).

2) The rip-cut surface of specimens from Thermo-D and WTT processes is represented

by the area indicated by the yellow arrow.

3) Almost all rip-cut surfaces of the WTT process and unexpectedly, the areas in air-

dried samples represented by the area indicated by the violet arrow.

4) Air-dried and kiln-dried specimens represented by the area indicated by the red arrow.

5) The planed surface of the WTT modification specimen represented by the area

indicated by the green arrow.

6) Planed surfaces of thermally modified specimens (Thermo-D and WTT processes)

represented by the area indicated by the blue arrow.

From the PCA plot above, it is clear that HSI could distinguish different thermal modification

processes. However, the air and kiln-dried specimens form only one group in Figure 5.10, and

no grouping pattern was observed but the chemical difference was observed in Paper II and III

by wet chemistry analysis. Previous studies revealed the influence of extractives during near-

68

infrared analysis for the differentiation of wood chips containing heartwood, sapwood and knots

(Lestander et al. 2012). A negative influence of extractives on classification and mapping of

the chemical composition of similar wood products by NIR-based imaging was found by

Thumm (Thumm et al. 2016). Furthermore, one specimen in Figure 5.9a had fungal inclusions.

However, the fungal stained area was hidden and could not be distinguished by the HSI method.

The discriminant analysis of areas 2, 3 and 5, 6 from Figure 5.10 defined apparent differences

between planed and rip-cut surfaces, but slightly less for different thermal modification

processes like Thermo-D and WTT. The model parameters R2 was 99%, and Q2 was 77% and

were entirely satisfactory for an exploratory study. The primary differentiation for thermally

modified specimens was due to the wavelength band at 2100 nm. This absorption band might

correspond to the stretching of O-C=O in hemicellulose containing acetyl groups

(Schwanninger et al., 2011).

5.3.2 NIR MODELLING OF KILN/AIR-DRIED WOOD UNDER FUNGAL ATTACK The search for efficient evaluations of the air- and kiln- dried boards and subsequent mould

fungi attack on it forwarded attention to point-based in direct contact but non-destructive

measuring tools such as portable microNIR spectrometer which were evaluated in Paper XI.

The obtained results demonstrated superior performance for the analysis method of moulded

surfaces. It was possible to build a PCA model from obtained near-infrared spectra that

demonstrated the differentiation between samples with No mould and with Mould classes, and

the most outstanding results were obtained during plotting PC2 against PC3, as shown in Figure

5.11 below. After observing the clear grouping pattern with the PCA model, the Soft

Independent Modelling of Class Analogy (SIMCA) was applied to identify local models around

those classes. The multivariate models’ parameters are presented in Table 5.8.

Table 5.8. Multivariate models of NIR spectra of air and kiln-dried boards No mould and

Mould Classes. Data pre-processing - Centred + UV scaled.

Type Comp R2X_cum Q2X_cum Eig.

PCA 3 0.997 0.997 1.1

SIMCA

Class “No mould” 3 0.999 0.999 0.1

Class “Mould” 4 0.998 0.998 0.2

69

Figure 5.11. The score plot PC2 against PC3 were green - no mould class and blue - mould

class from the air- and kiln-dried Scots pine sapwood exposed to mould.

As a result of the SIMCA application, two local models have been obtained: a 3-component

around class No mould and a 4-component around class Mould. Moreover, the performance of

the local models was improved, and eigenvalues that represent self-projection of eigenvector

was reduced due to a good data fit (Eriksson et al., 2013).

Analysis of Coomans’ plot shows a graphical presentation of the developed SIMCA model in

Figure 5.12. Overlapping spectra for air-dried and kiln-dried samples is represented by the area

highlighted by green colour dots and supports the inability to entirely separate air/kiln dried

specimens by HSI spectroscopy. Spectra from class Mould, highlighted in blue, differed

significantly from class No mould supporting the application of portable NIR for measurement

of such type of the specimens since data points do not overlap in Figure 5.12.

70

Figure 5.12. Coomans’ Plot where green - class No mould, blue – class Mould and red –

class No class.

The SIMCA model demonstrated good prediction up to 95% for class Mould with a general

performance of 93.4% of correct prediction for all the datasets. Only six observations (No

classes) out of a total of 151 observations, representing about 4% of the data the program

algorithm, could not be classified, as shown in Table 5.9.

Table 5.9. Prediction table from SIMCA model of near-infrared spectra of Scots pine

specimens

Classes Total

Observations

No classes Class No mould Class Mould

No classes 6 (3.97%) 2 (33.3%) 4 (66.7%)

Class No mould 70 (46.4%) 70 (100%)

Class Mould 75 (49.7%) 4 (5.33%) 71 (94.7%)

Predicted 151 (100%) 0 (0%) 76 (50.3%) 75 (49.7%)

- correctly 141 (93.4%)

-not correctly 10 (6.62%)

The previous paper evaluated the capacity of NIR for predicting the mould fungi on the wooden

surface in a laboratory-driven test. However, in natural environmental conditions, the wooden

surface undergoes complex physical, chemical and biological changes, which may obscure the

results. Therefore, the evaluation of the treated wooden surface after weathering in outdoor

71

conditions is presented in Paper XII. The surface treatments on wood samples tested in the

single fungi laboratory tests in Petri plates is presented in Paper IX.

The treatments with pigments performed better against biodeterioration than treatments with

biocide without pigments as well as for the non-treated reference in outdoor tests. The general

appearance of the specimens from the outdoor test from Paper XII is presented in Table 5.10

below. The surface undergoes significant changes, and the differences from different angles of

exposure could be seen.

Table 5.10. The final pictures of the treated Scots pine boards after 115 days of exposure

before drying. Angle 90 is exposed vertically facing north, angle 180 is exposed

horizontally, and angle 45 is exposed in a 45o angle facing south direction.

Angle H T P C

90

180

45

72

As described in the literature review, NIR is quite sensitive to the water content, and here the

NIR measurements using MicroNIR were taken directly after exposure, dried after exposure

and non-exposed samples.

The surface coating influences weathering and detection in the NIR region, and treatments with

pigmented coatings were relatively resistant to weathering than transparent and non-treated

surfaces.

A PCA model was built with vital parameters describing and predicting 99.9% of the data from

outdoor exposure of surface treated boards. The partial least squares regression (PLS) model

with Y variables (Treatment, Wood Specie, Day, Angel of Exposure, Type of Exposure)

contained as many as 21 principal components with about 80% of prediction value (Q2 in Table

5.11). The model was used to find the best variable to contribute to the model performance. The

variable Type of Exposure mainly contributed to the model performance after the diagnostic of

the PLS model. Therefore, that variable (Type of Exposure) was used as a response in the PLS-

DA model since that model provided maximum separation, and the obtained new model had

only eight components (Eriksson et al., 2013). The parameters of all models developed are

described in Table 5.11.

Table 5.11. The multivariate models' parameters achieved from data of surface-treated

boards exposed to outdoor conditions were obtained by MicroNIR.

№ Name Components Observations R2X(cum) R2Y(cum) Q2(cum)

1 PCA 3 1166 0.999 0.999

2 PLS 21 1166 1 0.799 0.788

3 PLS-DA 8 1166 1 0.845 0.844

The PLS-DA model developed for variable Type of exposure gave almost 85% of the prediction,

and the classification table of the PLS-DA model demonstrates the proportion of correctly

classified observations in the prediction set. The NIR spectra in different treatments could be

predicted to more than 98% in total, as presented in Table 5.12.

73

Table 5.12. Classification table PLS-DA of nonexposed, exposed and exposed and dried

surface-treated boards from outdoor exposed conditions studied by MicroNIR.

(green - correct prediction, yellow -misclassification)

Variable Number Correct

(%)

After

exposure

Dried

after

exposure

No

exposure

No class

(YPred≤)

After exposure 599 100 599 0 0 0

Dried after exposure 367 95.91 0 352 15 0

Non exposure 200 100 0 0 200 0

No class 0 0 0 0 0

Total 1166 98.71 599 352 215 0

The fifteen data points from specimens dried after exposure were assigned to class “no

exposure”, probably due to minimal degradation after weathering test.

75

6. CONCLUSIONS

The study of mould fungi attack on copper impregnated Scots pine sapwood has shown that

fungal growth is regulated to a greater extent by planing depth than by the infection method.

Air-borne contaminants can heavily occupy the unplaned surfaces, but the extent of such

occupation could be reduced with planing and impregnation of boards with preservative

solutions. However, even after impregnation by toxic compounds, it is necessary to process the

wood surface to avoid or hinder micro fungi settlement.

Despite the vulnerability of the differently planed and impregnated wood towards mould fungi,

the resistance of that wood towards white-rot Trametes versicolor was found to be relatively

steady and sufficient with the mass loss for all planing depths below 5%, thereby characterizing

that wood as durability class 1 – very durable, while the same fungi aggressively degraded non-

impregnated wood with a mass loss of over 40% making it not durable (class 5).

The distribution, quantity and nature of lipophilic substances beneath the surface in the air- and

kiln-dried Scots pine (Pinus sylvestris L.) sapwood boards were studied for influence on mould

fungi growth as nutrient and inhibition source. The concentration of total extractives was

significantly higher in studied areas of kiln-dried than in air-dried samples and was higher close

to the surface than in the layers beneath. The scatter in the values for the lipophilic extractives

was high in both drying types, being highest for linoleic acid and slightly lower for palmitic,

oleic and stearic acids. The amount of fatty acids was low in kiln-dried boards, probably due to

a more substantial degradation resulting from the high temperature employed. The most

abundant resin acid was dehydroabietic acid, followed by pimaric, isopimaric, and abietic acids

in both drying types. During kiln-drying, a migration front is created at a depth of 0.25 mm with

a thickness of about 0.5 mm.

Studies were committed towards an understanding of the influence of extractives and other

migrating compounds in unplaned Scots pine sapwood surfaces on mould growth on those

surfaces of timber subjected to air and kiln drying by developing a multivariate Orthogonal

Partial Least Squares (OPLS) regression model. The model described 51% variability in

descriptive X and 69% in Y with the prediction power of 55%. Results indicated that total

acetone soluble compounds and sugars like glucose contributed to the mould growth model,

whereas fatty acids were negatively influencing mould growth.

The thermal modification at different temperatures of exotic African wood influenced the

amount of acetone soluble extractives, pH, and ash content. Thus, iroko wood has, to some

extent, buffering capacity and a different pattern of chemical changes compared to padouk,

76

which provides evidence of the need to study each wood variety for chemistry and process

conditions instead of drawing general conclusions.

The open process of wood treatment like heating-and-cooling has a potential for wood

performance improvement by developing a protective layer beneath the surface. However, the

applied methacrylic resin demonstrated effectiveness during laboratory testing but did not

perform efficiently during outdoor tests.

The test of commercial GRAS compounds and biocidal treatments in laboratory conditions

revealed a moderate inhibition effect in protection against biodeterioration. The main

conclusion from those tests is the deepening of understanding of the fungal community

interactions during different inoculation scenarios like cohabitation of Penicillium sp. and

Paecylomyces sp. and individual fungi virulence like Trichoderma sp. resisting complex

biocidal solutions.

The HSI imaging could be efficiently applied to classify thermally modified wood but failed

for air- and kiln-dried wood. A PCA model determined that regions with high absorbance were

related to extractives with phenolic groups and aliphatic hydrocarbons. The sharp wavelength

band at 2135 nm was found by multivariate analysis to be useful for multivariate calibration.

This peak represented the most considerable variation that characterises the knot area and can

be related to regions in wood rich in hydrocarbons and phenols, and can perhaps be used for

future multivariate calibration.

To check the applicability of portable sensors for mould detection, microNIR OnSite

spectrometry was used to identify the differences in wood surfaces dried in kiln and air. The

obtained principal component analysis (PCA) could describe and predict 99.7% of the

spectroscopic data obtained. No influence of the drying method nor planed depth was

discovered during classification. The significance was discovered only for separating two

classes where the first one is No mould and the second one Mould as independent data points.

The SIMCA model could predict no mould and mould class boards 93.4% correctly.

The surface treatments containing commercial biocides evaluated by the NIR technology above

and a developed PLS-DA model provided almost 99% of prediction for outdoor weathering of

commercial coatings with biocides. That might be applicable for service life prediction of

wooden surfaces in future applications.

77

7. FUTURE WORK

Increasing application of wood in constructions is expanding ecological niches and habitats for

saprotrophic fungi. Despite a common belief that mould fungi are dangerous, many clinically

significant fungi are currently limited to 700 species (Hoog et al. 2020). Natural fungal

biodiversity constitutes about a million or more species worldwide. A more consolidated

approach should be applied to the understanding of long-term fungal dynamics. Evaluation of

their particular hazards to inhabitants of wooden buildings should be done, based on the wet

chemistry study, non-targeted analysis of wood chemical constituents and growing fungi, to

understand the chemical variability of the studied systems. Due to climate change, some

emerging pathogens might show up. Broad vision and direction should be toward data-driven

investigations of wood surface colonisers via a holistic view described below.

Figure 6.1. The holistic approach to understanding the interaction of fungi and wood

Nowadays, next-generation sequencing of microbial populations from wooden material

surfaces directly in and after environmental exposure combined with metagenomics study

allows achieving a pretty high accuracy to describe microbial populations dynamic and link to

the biodeterioration processes. The same could be applied for high-resolution mass

spectrometry combined with chemometric tools, which will allow improvements in the

chemical profiling and detection of influencing fungal growth compounds and produced fungal

metabolites on the surface and in the bulk of wooden material.

The new generation of portable NIR sensors has excellent potential to be used in chemo- and

phenotyping of industrially produced timber. Proper creation of spectroscopic libraries

78

followed by the data-driven investigation of wooden material properties allows evidence-based

conclusions and predictions. The multivariate analysis of obtained data could link wooden

fingerprints to the mould growth risk and service life scenario. The fundamental understanding

of such properties formation will lead to developing targeted and innovative wood protection

solutions with minimum environmental impact.

The main challenge to overcome is the complexity, variability and diversity of environmentally

formed fungi-wood matrices that require studying multiple analytes with different chemical

properties and associated degradation processes. Nevertheless, we are step a close to such a

solution with the research expertise developed today.

79

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Paper I

PEER-REVIEWED ARTICLE bioresources.com

Myronycheva et al. (2018). “Cu-treated Scots pine,” BioResources 13(4), 8787-8801. 8787

Growth of Mold and Rot Fungi on Copper-impregnated Scots Pine Sapwood: Influence of Planing Depth and Inoculation Pattern

Olena Myronycheva, Margot Sehlstedt-Persson, Olov Karlsson,* and Dick Sandberg

The biocidal properties of an industrially used copper-based preservative were evaluated at different planing depths on exposure of pine wood to mold fungi in direct and indirect contamination methods, with simultaneous verification of white rot fungi virulence on wood. The preservative was an aqueous solution of copper carbonate, 2-aminoethanol, and quaternary ammonium compounds. Full cell preservative impregnation efficiency against visual mold fungi growth was tested on sapwood surfaces planed to different depths before impregnation. The virulence of two white rot fungal strains of Trametes versicolor (441 and JPEI) against the dried non-impregnated and impregnated wood samples was also tested. The unplaned surface of impregnated timber was occupied by air-borne contaminants, such as Paecilomyces variotti and Aspergillus niger up to 30%, and, even after impregnation it was necessary to process the surface to avoid micro-fungi settlement. The virulence of the tested rot fungi strains was confirmed by the aggressive degradation of non-impregnated wood with a mass loss of over 40%. Both Trametes sp. strains degraded the preservative-impregnated wood with a mass loss of 3.1% to 4.8%, but degradation by the JPEI strain was more intensive and more dependent on planing depth than the other strain (441).

Keywords: Scots pine sapwood; Impregnation; Planing depth; Mold; Rot; Fungi; Test

Contact information: Wood Science and Engineering Division, Luleå University of Technology,

Forskargatan 1, SE-931 87 Skellefteå, Sweden; *Corresponding author: [email protected]

INTRODUCTION

The biodegradation of wood is a critical factor in determining its performance,

especially for wood exposed to humid and wet conditions in both indoor and outdoor

environments. To protect against biological damage, a large number of preservation

methods for wood products have been developed and used. For instance, the impregnation

of wood using copper-salt solutions has been used to prevent biodeterioration since 1718

(Unger et al. 2001). Today, copper salts are the dominant preservative for wood products,

but compounds, such as chromated copper arsenate (CCA), are not allowed in Europe and

the USA due to their environmental toxicity. The leaching of copper ions from impregnated

wood can lead to various health disorders and accumulation in aquatic species (Peres and

Pihan 1991; Tchounwou et al. 2012). Therefore, researchers and the saw-mill industry are

trying to develop safer combinations and other solutions for wood impregnation with

copper-containing chemicals to reduce environmental risk (ECHA 2011). One method of

reducing the copper ion concentration in impregnating liquid is the effective combination

of organic and biodegradable biocides.

The biocidal action of copper-based treatments could be explained by separate

effects of hydro-peroxide radical formation due to the reaction of hydrogen peroxide and

copper ions, binding to the cell membranes, and decreasing membrane fluidity (Cervantes



and Gutierrez-Corona 1994; Suwalsky et al. 1998; Borkow and Gabbay 2005). However,

some fungi have developed systems to overcome the biocidal action of copper-salt

preservatives. The general mechanisms of fungal resistance to heavy metals proposed by

Nies (1999) suggest decreasing the concentration of heavy metals in wood via effluxion of

such compounds, resulting in isolation by creating complex compounds or reduction to a

less toxic (oxidation) state. After several years of research, it was found that such a

treatment is only partially effective with soft rots, whereas brown rot fungi Fibroporia

radiculosa can detoxify copper compounds by forming oxalates (Ohno et al. 2015). As a

part of global geochemical processes, fungi can leach, recover, detoxify, and remediate

metal compounds (Gadd 2017).

Primary wood-colonizing fungi are a number of ascomycetes (deuteromycetes) and

basidiomycetes, which are initially transmitted by air to wood surfaces. The ascomycetes

on wood decompose mainly cellulosic fractions when basidiomycetes or rot fungi

decompose lignino-cellulosic matrix of wood (Crous et al. 2018). The spores germinate

under favorable moisture conditions in the presence of low-molecular nutrients. Rot fungi

usually invade wood at later stages of decomposition from soil or insect contamination

(Zabel and Morrell 1992). The diversity of fungi is enormous, but mold fungi associated

with bio-based building materials are restricted to air-borne, xerotolerant, or xerophilic

types. Most of these fungi are also clinically important, as they are known to be mycotoxin

producers (Samson and Houbraken 2010) and can cause substantial deterioration in indoor

environments. The physical and chemical properties of wood surfaces and the surrounding

humidity are the main factors that determine the suitability of conditions for fungal

development. Wood surface conditions are influenced by the migration of extractives

towards the surface during the drying process and enrichment of the surface by soluble

nutrients required for the growth of microorganisms (Terziev and Nilsson 2005; Terziev

and Boutelje 2007; Terziev 2009).

Pressure impregnation, like the full-cell, Lowry, and Rüping methods, is the most

common way to treat pine sapwood boards with solutions of copper salts (Reinprecht

2016). During impregnation the preservative penetrates into the wood via various pore

systems (Ahmed et al. 2012) and is assisted by a pressure gradient of between 10 bars and

15 bars. The copper-salt complexes are also believed to be able to move into the cell wall

of wood through a diffusion process. The ability of copper ions and ethanolamine to form

strong interactions with phenols and carboxylic acids in the wood stabilizes the structure

(Ruddick et al. 2005; Zhang and Kamdem 2007). However, the impregnation process

sometimes fails when there is an uneven distribution of chemicals in the cell structure due

to the morphological nature of wood, the extractives, and the relocation of the extractives

during the drying process before impregnation as was studied by using histochemical

methods (Ahmed et al. 2012).

Various test methods have been designed to evaluate bio-based building materials

with respect to their vulnerability to fungal damage in various applications. The reliability

and variation within the results of such tests are discussed widely, especially when

evaluating mold tests (Kutnik et al. 2014). The majority of studies of vulnerability of

building material to fungal growth focused on influence of temperature and relative

humidity in different combinations but not to properties of material itself and relevant

processing technologies such as drying and planning. The hypothesis proposed in this work

is that by removal of wood surface material enriched with migrated nutrients together with

impregnated biocides it is possible to lower fungal growth and increase durability of wood

material. Therefore, this study aims to explore the impregnation efficiency by studies on



the biocidal properties of industrially used copper-based preservative treated wood at

different planing depths during the exposure to mold and white rot fungi. The influence of

different test methods were also performed.

EXPERIMENTAL Materials Wood samples

A total of 42 center-sawn Scots pine (Pinus sylvestris L.) boards (50 mm × 125

mm) with the maximum possible sapwood ratio were selected directly after sawing in a

Kåge sawmill in Västerbotten county in northern Sweden. Each board was further divided

from the top end into up to four 1-m-long samples in order to enclose the heartwood on the

upper flat side. In addition, 50-mm-long pieces were taken each sample to determine the

average raw moisture content (MC) (oven-dry method) and average density ρ0,raw. A total

of 96 samples, end-sealed with Sikaflex 221 (Sika Sverige AB, Spånga, Sweden), were

randomly distributed into four equivalent batches that were dried and conditioned in two

identical series in a small-scale laboratory kiln, using an industrial schedule with a target

MC of 18%. The drying schedule used in the experiments is shown in Fig 1.

Fig. 1. Drying schedule with a 3 h conditioning stage

After drying, the specimens were conditioned for 3 h and cooled. The MC of each

sample was measured using an electrical moisture meter. The four batches were then

randomly selected for machine setting planing depths of 0 mm, 0.7 mm, 2.0 mm, and 4.0

mm. The planing depths were measured with a caliper for each specimen before and after

planing. Planing depth, taking failure of cross-section into account, is defined in Fig. 2.

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30 35 40 45 50 55

Tem

pera

ture

(°C

)

Time (h)Temperature of dry bulp



1) planing of pith side 2) thickness measurement immediately above the pith 3) planing of the sapwood side 4) thickness measurement immediately above the pith

Fig. 2. Planing schematic and definition of planing depth with cupping of cross-section

Methods Impregnation and evaluation of impregnation results

After all the samples were weighed, the batches were transported to the

impregnation plant where the four batches were impregnated with copper salt solutions in

one run in an industrial autoclave. The impregnating agent was Celcure AC 800 (copper

carbonate 17.3%, 2-aminoethanol 35%, N-alkyl(C12-16)-N-benzyl-N,N-dimethyl-

ammonium chlorides 4.8%, and water) supplied by Koppers in Helsingborg, Sweden. The

impregnating agent was diluted with water to a concentration of 3.4% and a liquid

temperature of 15 °C.

Impregnation was performed according to the current standards of NTR class AB

(Nordic Wood Preservation Council 2012) to meet the product requirements for

industrially protected wood according to the Nordic Wood Preservation Council, which

requires full sapwood penetration in Scots pine. The process included the following stages:

1) Wood material placed in an autoclave was pre-vacuumed to 90% for 20 min; 2)

Preservative fluid was inserted and an overpressure of 1400 kPa was maintained for 40

min; and 3) The material was vacuumed for 20 min.

The subsequent fixation/drying that is usually included in industrial production was

not performed in this case. Immediately after impregnation, the samples were transported

to the laboratory, where each sample was weighed to record the impregnation agent uptake,

which was calculated as the wet weight increase per unit volume.

Each sample was then cut into four parts, with as few knots in the cross-section as

possible and then stacked on top of each other with all of the cross-sections turned so that

they were approximately 25 cm from the surface (Fig. 3). After stacking, all the cross-

sections were brushed with a heartwood-sensitive reagent to obtain a distinct boundary

between the softwood and the heartwood. The impregnated sapwood was identifed as the

green-colored areas, the heartwood was identified as the reddish areas, and impregnation

failure was identified as the wood-colored areas. The heartwood proportion was calculated,

and the impregnation agent percentage uptake in the sapwood was estimated according to



the proportion of cross-cut area of sapwood to the uptake of impregnation agent, with the

assumption that there was no uptake of impregnation fluid in the heartwood.,

a) b) Fig. 3. a) Batches immediately after impregnation; b) Examples of cross-sections of individual specimens divided into 1-m lengths, showing impregnation results and heartwood dyed with a heartwood reagent

Fungal test

The specimens for the mold fungi test included only knot-free sapwood parts taken

from the upper sapwood side and cut into two replicates with a size of approximately 110

mm × 60 mm × 10 mm. The replicates were suspended in plastic holders in two separate

plastic boxes with dimensions of 600 mm × 300 mm × 350 mm with the open wall sealed

via an air-transparent polymeric membrane, as shown in Fig. 4. The climate in each box

with indirect inoculation was T= 21.1 ± 0.1 °C and RH = 90 ± 1%, for each box with direct

innoculation climate was T = 22.1 ± 0.1 °C and RH = 89 ± 5%.

The four mold fungi of Penicillium commune Thom, C., Aspergillus niger van

Tieghem, Paecilomyces variotii Bainier, and Mucor plumbeus Bonord. (Culture collection,

Wood Science and Engineering, Luleå University of Technology) cultivated on malt-

extract agar (Merck, Darmstadt, Germany) were used for fungal inoculation. Two methods

of inoculation were used, including the indirect method using open petri plates with pure

cultures placed on the bottom of the box according to ASTM D7855/D7855M - 13 (2013),

and a direct method where a suspension of the four fungi in a concentration of 106

spore/mL, prepared according to SS-EN 15457:2014 (2014), was sprayed directly onto the

wood surface. To evaluate the influence of MC, the initial MC and final equilibrium MC

were recorded. Images of mold growth on the surface were obtained via digital

photography by WolfVision VZ-8light4, and the growth was assessed in two ways using

ImageJ software (National Institute of Mental Health, version 1.51j8, Bethesda, Maryland,

USA). In particular, growth was assessed as the percentage of the total area of the board

that was fungi and as the covered area of fungal growth (Schindelin et al. 2012). The

individual mold species on the wood surfaces were identified with the naked eye and an

optical microscope to identify the morphological characteristics of each fungi (Samson

and Houbraken 2010).

https://en.wikipedia.org/wiki/Charles_Thom

https://en.wikipedia.org/wiki/Phillippe_%C3%89douard_L%C3%A9on_van_Tieghem

https://en.wikipedia.org/wiki/Phillippe_%C3%89douard_L%C3%A9on_van_Tieghem

https://en.wikipedia.org/wiki/Bonord.



a) b) Fig. 4. Mold growth tests: a) indirect and b) direct methods

Two white rot strains (441 and JPEI) of Trametes versicolor (L.) Lloyd were

obtained from the Aloha Medicinals Mushroom Culture Bank in Carson City, NV, USA.

All the cultures were stored on solidified malt extract agar at 4 °C ± 1 °C with visual control

of their purity and activity. The virulence of each fungal strain was assessed by the mass

loss of the treated and non-treated wood samples, determined according to the SS-EN 113

(1996) standard with each sample having dimensions of 30 mm × 15 mm × 5 mm. The

experimental design and test methods are explained in the Table 1.

Table 1. Experimental Design

Experiment Test method Number of replicates at planning depth

Group 1

(0 mm)

Group 2 (0.7 mm)

Group 3 (2.3 mm)

Group 4 (4.0 mm)

Mould test

ASTM D7855/D7855M-13 5 5 5 5

SS-EN 15457:2014 5 5 5 5

Rot test

SS-EN 113 Trametes versicolor 441

20 20 20 20

SS-EN 113 Trametes versicolor JPEI

20 20 20 20

A complete data matrix for the mold test comprising of totally 40 observations (2

methods × 4 groups × 5 replicates) was obtained for each treatment method (indirect and

direct contamination) and each group (planing depth).

The analysis of the white rot test included a data matrix for the dry mass loss from

totally 200 observations (2 fungi × 5 batches × 20 replicates). A univariate analysis was

performed to study the influence of the planing depth on fungal growth after impregnation

by comparing their mean differences using IBM SPSS Statistics 20.0 (Armonk, NY, USA)

software. This approach makes it possible to summarize and compare differences across

one or more factors, or categorical variables.



RESULTS AND DISCUSSION Penetration of Preservation Solution

This experiment was designed to evaluate fungal infestation on dried and

impregnated wood. Samples planed to a depth of 4 mm (group 4) had the highest uptake

of preservative solution (Table 1). In the wood that was not planed before impregnation

(group 1), the amount of preservative solution in the wood after impregnation was 9% less

than that of group 4, which had greater planing depth. (Table 2). No statistically significant

difference in the uptake of impregnation solution was observed between the samples planed

to depths of 0.7 mm and 2.3 mm, which could be of interest for the many sawmills that are

striving to achieve lower planing depths. Even though the heartwood content was slightly

higher and the density statistically significant higher for the boards in group 3 than for the

boards in other groups, no influence on boards uptake of impregnation solution was

observed.

Table 2. Scots Pine Sapwood Properties and Uptake of Preservation Solution During Impregnation for Groups with Different Planning Depth

Parameter Group 1 Group 2 Group 3 Group 4

Planing depth (mm) 0a 0.7a 2.3a 4.0a

Heartwood content (%) 39 39 41 38

Density (kg/m3) Green Dried

813a 393a

804a 386a

768a 402a

812a 372a

Moisture content (%) Green Dried

107.7a 17.5

108.9a 17.8

91.7a 19.0

118.8a 18.0

Impregnation uptake in sapwood (kg/m3)

584 615a 626 643

a - Significant mean difference between groups (p ˂ 0.05)

The penetration of impregnation agents starts from the surface of material, and the

properties of the surface are vitally important for impregnation retention (Terziev 2005).

The design of the impregnation agent used in this experiment allows ethanolamine to

increase the solubility of copper salts in the impregnation liquid (Forest Products

Laboratory 2010) and thereby penetrate deeper into the wood. Penetration is believed to

take place through membranes in various pits into the wood, and the authors’ results

demonstrated the influence of the surface of the material on impregnation agent retention.

Fungal Test The fungal test was designed to be similar to the primary colonization of molds. To

mimic such an environment, the authors applied fungal species (Penicillium sp.,

Aspergillus sp., Paecilomyces sp., and Mucor sp.) tolerant to dry conditions that had

previously been isolated from dried wood. This made it possible to identify fungal

colonization during the infection process in both the wet and natural states of innoculation.

The determination of mean mold area at a similar initial MC of the boards revealed the

importance of both the method of inoculation and the planing depth (Table 3). The moisture



content (MC) of the samples with direct inoculation was higher in 10% in average than the

MC after natural airborne inoculation, which was probably due to the higher climate

fluctuations. No difference in the initial MC was detected between the groups for the

different mold inoculation methods (Table 2).

Table 3. Mean of Mold Areas and MC of Scots Pine Sapwood Boards Treated by Different Fungal Inoculation Methods

Sample Group No.

Mold Area (%)

Mold Area (mm2)

MC Before Test (%)

MC After Test (%)

Indirect Fungal Inoculation

1 32.4a 2222.8a 9.6 20.0

2 0a 0a 9.5 17.4

3 0a 0a 9.5 20.5

4 0a 0a 9.6 20.5

Direct Fungal Inoculation

1 24.5a 1659.01a 9.6 21.8

2 0.4a 25.9a 9.7 21.8

3 0.1a 3.5a 9.6 21.4

4 0a 2.4a 9.6 22.1 a - Significant mean difference between the groups (p ˂ 0.05)

Figure 5 shows the colonization patterns of the boards with both inoculation

methods. The fungal colonies growing on the wood surface were different. The

Paecilomyces variotti and to a smaller extent the Aspergillus niger were dominating other

species via indirect contamination. However, after spraying the molds, most of the wood

surface was occupied by A. niger. Perhaps P. variotti, which produces conidia as chains

that easily become airborne, would be better equipped for a more natural infection, whereas

A. niger conidia forms a single dry spore and functions better in an excess of water (Adan

and Samson 2011).

The tendency to produce more aerial mycelia at the lower edges of boards was

observed in the natural contamination method (see red arrow in Fig. 5). The authors suggest

that the fungi here initiated more tissue development to absorb water vapor from the

surrounding atmosphere (Pypker et al. 2017). Alternatively, the fungi might initiate aerial

morphogenesis to produce more reproduction tissue for further survival and new habitat

colonization under extreme conditions, such as low water content environment (Van

Laarhoven et al. 2016; Segers et al. 2017).

Thus, it was evident that the application method influenced the susceptibility of the

wood materials to mold attack, which was in line with previous studies on gypsum (Bekker

et al. 2012). The authors confirmed that the planing depth influenced the mold growth

development even in the preservative-treated wood (see Table 2). The addition of water to

the wood samples during spraying increased the amount of water and stimulate fungal

growth, especially when the wood moisture content stays below the fiber saturation point

and the surrounding atmosphere contains a large amount of water vapor. That was similar

to the finding of Van Laarhoven et al. (2015) that water content stimulates early

colonization and the hyphal extension rate of mold fungi. The dominance of the fungus A.

niger over other isolates could be explained by its ability to adhere more strongly to the

wood surface than other anamorphic fungal species (El Abed et al. 2010). To the authors’ knowledge, the growth of fungal species on wood surfaces of Mucor plumbeus has not

been discovered after either indirect or direct inoculation.



Fig. 5. Surface appearance of samples inoculated by the direct and indirect inoculation methods. The red arrow highlights the aerial mycelia.

This study is the first to show the differences in contamination of impregnated

wooden surfaces by common indoor fungi. To some extent, the planing depth and planing

itself influenced mold growth (Terziev 2009; Sehlstedt-Persson et al. 2016; Johansson et

al. 2017). Particular attention must be given to the indirect method of testing, where

medically essential Paecilomyces variotti showed a dominant coverage of the wood

surface. The rougher unplaned surface in group 1 contained more microvoids with water,

which were favorable for spore germination (Johansson et al. 2017). Molds were observed

only after the direct inoculation of planed wood surfaces, and mainly on the wood surface

closer to the original wood surface than deeper into the material. However, further study is

necessary to clarify the availability of nutrients for filament germination, hyphal growth,

Indirect Direct



and sporulation on the surface. The authors’ previous study revealed that mold fungi

occupied only approximately 100 µm of the wood surface in depth and that the fungal

growth was associated with the migration of nutrients during wood drying (Sehlstedt-

Persson et al. 2011; Ahmed et al. 2013). However, limited information has been presented

about fungal community development on the wood surface. For example, in the authors’ past study, all three fungi had the same spore size (Samson and Houbraken 2010).

A much lower colonization was observed in the planed than unplaned surfaces

(Table 3). Enrichment of nutrients takes place during artificial drying (Terziev et al. 2009);

however still they are present in a dried wood deeper than at 0.7 mm. It is possible that

water-soluble nutrients are dissolved and migrate along with the impregnating solution into

the wood during impregnation treatment changing the gradients of nutrients formed in the

wood during the wood drying. Surface roughness could also be of importance since indirect

inoculation colonized the planed wood surface less than direct (even though the unplaned

surface showed more colonization for indirect than direct inoculations).

The white rot fungus (Trametes versicolor) is an essential fungus for testing the

resistance of preservative-treated wood material to biodegradation according to EN113

(1996). The virulence of the two strains of white rot fungus (Trametes versicolor) on

impregnated and non impregnated samples was evaluated after 16 weeks of exposure

(Table 3). Those results may also be of importance for further use in biodegradation assays.

The general rule in the assessment of new strains is that the mass loss should be greater

than 20%, according to SS-EN 113 (1996). During the trial, the fungi demonstrated

aggressive behavior, and there was a mass loss of approximately 44% for strain JPEI and

41% for strain 441 in the wood samples without impregnation, which met the criteria stated

above (Table 3).

Table 4. Mean Mass Loss of Scots Pine Sapwood Boards Treated with Two Strains of Trametes versicolor According to SS-EN 113 (1996)

Parameter Mass loss (%)

Strain 441

Group 1 (planning depth 0 mm) 4.4a




Control (without impregnation) 40.6a

Strain JPEI

Group 1 (planning depth 0 mm) 4.8ab




Control (without impregnation) 43.9a a - Significant mean difference between impregnated groups and control (without impregnation), p ˂ 0.05; b - The significant mean difference between impregnated groups of samples, p ˂ 0.05

The mass loss of impregnated samples treated with strain 441 showed a significant

difference from that of the control samples without the impregnation agent, but there was

no significant difference between the impregnated batches with different planing depths.



For samples degraded by strain JPEI, significant mean differences were found between the

samples also treated with impregnation (Table 4).

White rot fungus (T. versicolor) is a selective degrader of lignocellulosic material,

particularly in the early stages of decay (Shirkavand et al. 2017). The primary target of the

attack is hemicellulose, but lignin and cellulose are also degraded (Chen et al. 2017). The

effect of planing depth on mass loss is shown in Table 3. The mass loss was lower at a

deeper planing depth for strain JPEI, but not for 441. The difference in the extent of

degradation of wood by the two white rot fungi may have been due to phylogenetic

variability within the species. Intraspecies variability in genetic and phenological traits are

evident in the fungal kingdom and can cause fungicide failure (Georgopoulos and

Skylakakis 1986; Nilsson et al. 2008). The agar media in the bottles developed a greenish

color at the end of the test. It was possible that the microorganisms assisted the leaching of

copper from the impregnated wood samples, which may be a result of a detoxification

mechanism via copper-transporting ATPase pumps (Weissman et al. 2000; Hall 2002;

Hastrup et al. 2005; Ohno et al. 2015).

CONCLUSIONS

1. The mold fungal damage of copper-impregnated Scots pine sapwood was influenced

to a greater extent by the planing depth compared to the infection method.

2. The unplaned surface of impregnated timber can be occupied by air-borne

contaminants, such as Paecilomyces variotti and Aspergillus niger, up to 30%, but the

extent of mold coverage was much less on the planed impregnated timber. Even after

impregnation by toxic compounds, the wood surface required processing to avoid or

hinder micro-fungi settlement.

3. The new strains of white rot fungus (Trametes versicolor) quite aggressively degraded

the non-impregnated wood with a mass loss of over 40%.

4. Both strains of white rot fungus (T. versicolor) degraded the wood impregnated with

an industrially used copper-based preservative, with an estimated mass loss of 3% to

5%. One of the strains (JPEI) was more incoherent and sensitive to planing depth.

ACKNOWLEDGMENTS

The authors thank Martinsons in Skellefteå, Sweden for providing test materials

and are grateful for the financial support of the Swedish Research Council for the

Environment Agricultural Sciences and Spatial Planning (FORMAS) projects:

“Experimental studies of capillary phenomena in bio-based materials” (942-2016-64) and

“Fungal growth on modified wood-based products under subarctic conditions” (2017-419).

In addition, the authors wish to express their gratitude for the support of COST Action

FP1407.



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received and accepted: October 3, 2018; Published: October 19, 2018.

DOI: 10.15376/biores.13.4.8787-8801

Paper II

RESEARCH ARTICLE

Distribution of low-molecular lipophilic

extractives beneath the surface of air- and

kiln-dried Scots pine sapwood boards

Olena Myronycheva, Olov KarlssonID*, Margot Sehlstedt-Persson, Micael Ohman,

Dick Sandberg

Division of Wood Science and Engineering, LuleåUniversity of Technology, Skellefteå, Sweden

* [email protected]

Abstract

During industrial wood drying, extractives migrate towards the wood surfaces and make the

material more susceptible to photo/biodegradation. The present work provides information

about the distribution, quantity and nature of lipophilic substances beneath the surface in

air- and kiln-dried Scots pine (Pinus sylvestris L.) sapwood boards. Samples were taken

from knot-free sapwood surfaces and the composition of lipophilic extractives, phenols and

low-molecular fatty/resin acids layers at different nominal depths below the surface was

studied gravimetrically, by UV-spectrometry and by gas chromatography-mass spectrome-

try (GC-MS). The concentration of total extractives was significantly higher in kiln-dried than

in air-dried samples and was higher close to the surface than in the layers beneath. The

scatter in the values for the lipophilic extractives was high in both drying types, being highest

for linoleic acid and slightly lower for palmitic, oleic and stearic acids. The amount of fatty

acids was low in kiln-dried boards, probably due to a stronger degradation due to the high

temperature employed. The most abundant resin acid was dehydroabietic acid followed by

pimaric, isopimaric, and abietic acids in both drying types. It is concluded that during kiln-

drying a migration front is created at a depth of 0.25 mm with a thickness of about 0.5 mm.

Introduction

Wood is dried industrially to remove water to achieve a more durable and dimensionally stable

light-weight material. Drying and especially thermal modification influence the presence and

structure of the constituents of the wood material, and subsequently the characteristics and

service-life of the final wood products. During drying, complex changes in the properties of

wood occur, initiated by heat and mass flows towards the wood surfaces and by degradation of

wood constituents [1]. One mechanism is a migration of non-structural wood-cell compounds

such as polar and non-polar extractives. Contamination of both hydrophilic and hydrophobic

components beneath and at the wood surface by the migration of extractives during drying

reduces the adhesion of surface coatings [2,3], affects the speed of moisture absorption [4],

PLOS ONE | https://doi.org/10.1371/journal.pone.0204212 October 10, 2018 1 / 12

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OPENACCESS

Citation: Myronycheva O, Karlsson O, Sehlstedt-

Persson M, Ohman M, Sandberg D (2018)

Distribution of low-molecular lipophilic extractives

beneath the surface of air- and kiln-dried Scots

pine sapwood boards. PLoS ONE 13(10):

e0204212. https://doi.org/10.1371/journal.

pone.0204212

Editor: Martina Lahmann, Bangor University,

UNITED KINGDOM

Received: May 5, 2017

Accepted: September 5, 2018

Published: October 10, 2018

Copyright: © 2018 Myronycheva et al. This is an

open access article distributed under the terms of

the Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information

files.

Funding: This work received support from the

Swedish Research Council for the Environment,

Agricultural Sciences and Spatial Planning

(FORMAS) project “Experimental studies of

capillary phenomena in bio-based materials” 942-

2016-64, “Fungal growth on modified wood-based

products under subarctic conditions” 2017-00419,

http://orcid.org/0000-0002-7711-9267


http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0204212&domain=pdf&date_stamp=2018-10-10








http://creativecommons.org/licenses/by/4.0/

leads to an uneven distribution of preservatives during impregnation [5], and as a consequence

may reduce the durability of the wood product.

Scots pine (Pinus sylvestris L.) is one of the major raw materials for the forest products

industries in the Nordic countries. Scots pine is naturally highly enriched in extractives, which

influence the modification of sawn timber and the properties of the final product. The migra-

tion of hydrophilic extractive fractions to the sapwood surface during drying and thermal

modification has been widely described [6–9]. The movement of liphophilic extractives during

drying decreases the permeability and can lead to failure in impregnation [5,10]. Besides, the

hydrophobic fractions that remain from nutrient reserves and natural defensive systems

against pathogens in the living tree can be used as natural protection of wood products against

damage by environmental influences.

The main wood constituents are cellulose, hemicelluloses, lignin, and to a minor part

water-soluble and insoluble extracellular and low-molecular-weight compounds called

“extractives” [11,12]. The extractives are located in various tree compartments: parenchyma

and epithelial cells, resin canals, middle lamellae, intercellular spaces and the cell walls of tra-

cheids, libriform fibers and vessels [13]. The main compounds in softwoods are resin acids,

other terpenoids, fats, fatty acids, steryl esters, sterols, phenolic substances, glycosides, sugars,

starch, and proteins [14] with seasonal and geographical variations in distribution pattern

[15–17]. The content of extractives has been reported to be higher in knots than in stem wood,

with noted variation within the tree and between trees and a certain decrease in amount along

the radial direction of the tree [18].

The resins in Scots pine are primary lipid-soluble blends of volatile and non-volatile sec-

ondary compounds of terpenoid origin, produced in subcellular regions of epithelial cells and

then transported and stored in the resin duct [19]. The volatile fractions of the mono C10 and

sesquiterpene C15 resins give fluidity to resins and plasticise the more viscous C20 diterpenes.

Scots pine possesses the high level of constitutive resins in an interconnected system of resin

ducts located in the stem wood with long-lived epithelial cells from the initial state with domi-

nance in heartwood. The non-volatile resin fraction of Scots pine is usually characterized by

diterpene resin acids of the abietane and pimarane structural types [20]. The predominant

phenolic compounds are flavonoids, stilbenes and lignans. Their main function is to prevent

oxidation of biomolecules by abiotic and biotic substances attacking the trees [21]. The pino-

sylvin stilbenes are the predominant phenolic compounds in Scots pine heartwood and also

exist in sapwood [22]. Triglycerides, such as esters of glycerol and three fatty acids C16 and C18

with varying degrees of unsaturation and sterol/triterpenoid esters in Scots pine, are stored in

sapwood with higher concentrations of free fatty acids then in the heartwood, probably related

to degradation of triacylglycerols during heartwood formation [23]. Unsaturated fatty acids

such as oleic, linoleic, linolenic and eicosatrienoic, and the saturated palmitic and stearic fatty

acids, are the most abundant components of the triacylglycerol fraction [23,24].

Green wood retains metabolic activity after harvesting and processing, and this initiates a

natural anatomical response to injuries that trees sustain while exposed to pathogenic attack or

mechanical damage. It results in the release of constitutive defense agents and activates com-

plex changes in non-structural elements of Scots pine wood [25]. The moisture flux during the

drying process re-locates low-molecular sugars [7,26] beneath the sawn timber surface with

the formation of a so-called drying shell and kiln brown stain [6] that can be explained by the

hypothesis of an evaporative front (about 10 cell rows) of capillary water beneath the surface

[27].

Despite the large amount of research in recent decades into the extractives in Scots pine,

only limited and their fragmented information can be found regarding the distributionof

hydrophobic/lipophilic extractives and migration towards the surface during drying or

Extractives in dried pine surface


PI-Dr. Olov Karlsson; COST Action FP1407

“ModWoodLife” project “Understanding wood

modification through an integrated scientific and

environmental impact approach” PI-Prof. Dick

Sandberg.

Competing interests: The authors have declared

that no competing interests exist.


thermal treatment. Moreover, the accumulation and differences in chemical composition of

extractives in relation to the contribution of compound migration remains unclear. The pres-

ent work has sought to contribute information about the distribution, quantity and nature of

some of the hydrophobic substances in air- and kiln-dried Scots pine sapwood.

Material and methods

Specimen preparation and drying procedure

A total of 10 green sideboards from 10 different Scots pine (Pinus sylvetris L.) trees with cross-

sectional dimensions of 220 x 25 mm, and a length of about 4.5 metres were taken directly

after sawing from a sawmill in Norrbotten County in Sweden during the last week of Septem-

ber 2015. The trees were felled, bucked to logs and transported to the sawmill during Septem-

ber. Each board was cut into four pieces with a length of 0.96 m and the moisture content

(green state) was determined according to EN 13183–1 [28]. The boards consisted only of

sapwood.

No permits were required for the study, which complied with all relevant regulations. The

study did not involve endangered or protected species.

The boards were divided into two groups for air-drying and kiln-drying as follows: ten ran-

domly selected boards were single stacked and dried indoors on stickers at a temperature of

20˚C (RH about 10%) and used as reference for extractives analysis as air-dried samples. The

average moisture content after drying for 30 days was 4.6%.

Another 64 boards were kiln-dried in a small-scale laboratory kiln with air circulation. All

the boards were double-stacked with the sapwood sides of each pair turned outwards in order

to get a high flow of moisture from the inner part of the boards to the sapwood surface that

could enrich the surfaces with as much extractives as possible during drying (Fig 1). The dry-

ing schedule used is shown in Fig 2 and the moisture content (MC) is shown in Table 1.

The kiln-drying was performed without a conditioning phase in order to prevent influence

on distribution of extractives after drying.

Extraction of extractives and analysis

All the air-dried boards and 10 randomly selected kiln-dried boards were cut to a length of 250

mm and a width of 100 mm, and then split into four specimens (Fig 1C). These specimens

were used for the determination of the distribution of lipophilic extractives. The sapwood

sides of three of four specimens were planed three times to depths of 0.25, 0.75 and 1.75 mm

from the original un-planed surface. 3.5 g of the shavings from each planing were milled in a

Fritch planetary mill and 0.5 g was used for moisture content determination, 1.0 g was used for

the extraction of lipophilic compounds, and the remainder was left untreated and stored at a

temperature of -20˚C.

The extraction was done using acetone (VWR Chemicals: 20165.323, 75 ml) in a mini-

Soxhlet extractor according to the SCAN-CM 49:03 standard method [29]. The total lipophilic

extractives content was given in mg g-1 of dry mass after evaporation of the acetone and drying

of the extracted solution.

Fatty acids and resin acids in the acetone extract were analysed using GC-MS after tri-

methylsilylation. The silylation with 100 μl of BSTFA and 50 μl of TMSCl was performed in an

oven at 70˚C for 20 minutes according to Sjostrom [30] with 1-methylnaphthalene dissolved

in pyridine as internal standard [31].

A SUPELCO SLB-5 MS capillary column (L × I.D. 30 m × 0.25 mm, df 0.25 μm) was used

starting at a temperature of 100˚C (1 min initial) and finishing at 270˚C (2 min final) at a rate

10˚C/min with an entire analysis cycle of 25 minutes. The injector initial temperature was




270˚C with interface temperature 290˚C and fan temperature was 50˚C, control mode was

split. The scan group started from 40 m/z and end 500 m/z with scan speed 1000.

The resin acid and fatty acids were identified by NIST Mass Spectral Library [32] and a

quantitative estimation was made by comparing the peak areas with that for the internal stan-

dard. Retention time (RT) was: hexanoic acid (RT ~ 5.6 min.), heptanal (RT ~ 6.2 min.), glyc-

erol (RT ~ 8.6 min.), internal standard (RT ~ 9.4 min.), palmitic acid (RT ~ 17.9 min.), oleic

acid (RT ~ 19.6 min.), linoleic acid (RT ~ 19.4 min.), stearic acid (RT ~ 19.7 min.), pimaric

Fig 1. Drying and board preparation scheme. (A) double-stacking of boards with the sapwood side of each board

facing outwards in each pair, (B) stacking of boards in the laboratory kiln, and (C) planing depth scheme for

specimens: (a) unplaned, (b) 0–0.25 mm, (c) 0.25–0.75 mm, and (d) 0.75–1.75 mm planing depth.

https://doi.org/10.1371/journal.pone.0204212.g001

Fig 2. Drying schedule showing dry- and wet-bulb temperatures. The total drying time was 44 hours including a 1.7

hours heating phase, and a 5 hour cooling phase.







acid (RT ~ 20.4 min.), isopimaric acid (RT ~ 20.8 min.), dehydroabietic acid (RT ~ 21.2 min.),

abietic acid (RT ~ 21.5 min.).

The total phenolics content was determined by Folin-Ciocalteu (FC) assay according to a

procedure described by Julkunen-Tiitto [33]. The absorption was measured after 40 minutes

at 735 nm against that of a mixture containing all the reagents except for the sample. Tannic

acid (VWR Chemicals; 83510.260) was used as a standard and the results are expressed in mg

(TAE) g-1 dry mass of wood. The protocol was deposited in protocols.io, where it can be

assigned via link http://dx.doi.org/10.17504/protocols.io.phqdj5w.

Data analysis

The complete data matrix with 60 observations and 9 variables as measurement parameters

was based on 10 specimens in each drying method with 3 planing depths Table 2.

The relative mean difference (Δ mean in %) was determined by subtracting the mean of the

kiln-dried variable from that of the air-dried variable and dividing by the air-dried mean, the

coefficient of variation (CV in %) was used to describe the dispersion around the mean. The

variables measured were: extractives, phenolics, glycerol, palmitic oleic-, linoleic- and stearic

fatty acids, pimaric-, isopimaric-, dehydroabietic-, abietic resin acids. A univariate analysis was

used to evaluate the differences between the groups of variables, using the IBM SPSS Statistics

20 [34] software.

Results and discussion

Distribution of extractives

The total extractives content in the three regions beneath the sapwood surface is shown in Fig

3A and 3B. The extractives content in the surface layers (i.e. 0–0.25 mm depth) of the boards

was at the same level (32.2–56.6 mg g-1) as that reported by Dorado and Normark for the ace-

tone extract from Scots pine sapwood [35,36,37]. The kiln-dried specimens showed a greater

amount of total extractives in the surface layer than the air-dried samples, and the extractives

content was lower in the two deepest layers (0.25–1.75 mm) than in the region closest to the

surface (Fig 3).

The presence of a water evaporation front in capillaries near the wood surface during the

drying process has been deduced from observations such as the enrichment of nutrients and

reaction products some cell rows below the wood surface [6–9]. This can be explained by the

movement of water-soluble nutrients together with the capillary flow of water during the early

phases of wood drying and that they or their reaction products remain after evaporation of the

Table 1. Moisture content of the kiln-dried bards.

Moisture content Mean±SD (SE), %

Before drying 116±19.6 (6.2)

After drying 14±3.8 (1.2)

https://doi.org/10.1371/journal.pone.0204212.t001

Table 2. Sample groups with 60 observations.

Planing depth

(mm)

No. of replicates

Air-dried Kiln-dried

0–0.25 10 10

0.25–0.75 10 10

0.75–1.75 10 10




http://protocols.io

http://dx.doi.org/10.17504/protocols.io.phqdj5w




capillary water. It is also possible that the observed enrichment of extractives in the uppermost

layer is due to the migration of extractives during the capillary-transport phase of the drying,

especially under kiln-drying conditions when the temperature in the wood is higher and the

solubility and volatility of lipophilic substances is also higher [27,38]. It could not, however, be

excluded that degradation of extractives takes place near the wood surface during drying or

that the migration rate is related to the properties of migrating compounds. These issues are

further discussed below.

Phenols in Scots pine are mainly found in the heartwood but also to some extent in the sap-

wood [22]. The phenolic content in the surface layers (i.e. 0–0.25 mm depth) seemed to be

higher after kiln drying than after air drying at room temperature (Fig 3B). The scatter of the

data was fairly large, but lower contents of phenols were observed in the lower surface layers.

The significance, expressed as p-value, of the difference in the content of extractive materials

between the layers was assessed by a F-test (Table 3). Low p-values (�0.05) were found for

extractives and phenols for both drying types. This suggests a clear difference in total extrac-

tives and phenolic content at different planing depths for both drying types and that also phe-

nols migrate during both air-and kiln-drying of Scots pine. Weak boundary layer could be

formed by enrichment of extractives on wood surfaces and hinder efficient adhesion with

especially hydrophilic treatments. It is well known that linseed oil could be used to increase

Fig 3. Total extractives content (A) and phenolic content (B) in air-dried and kiln-dried Scots pine sapwood boards.


Table 3. The p-value for the significance of difference at different planing depths between air- and kiln-dried

specimens.

Parameter Air-dried Kiln-dried

Total extractives 0.001� 0.001�

Total phenols 0.044� 0.022�

Glycerol 0.280 0.481Palmitic acid 0.385 0.460Oleic acid 0.554 0.319Linoleic acid 0.454 0.212Stearic acid 0.036� 0.183Pimaric acid 0.239 0.034�

Isopimaric acid 0.348 0.028�

Dehydroabietic acid 0.374 0.169Abietic acid 0.478 0.266

� Significance of difference between planing depth by F-test, the level of significance is p�0.05.







water repellency of wood but needs addition of biocides or active phenolic compounds to hin-

der mould growth when exposed under exterior conditions.

Thus, knowledge of extractives and their composition on wood surfaces as a function of

wood and process conditions is of great importance when exposed to exterior conditions.

The higher FC determined phenolic content in upper layer of air-dried boards could be

favoured by dissolution in products of lipophilic origin which will migrate to wood surface [2].

The chemical changes such as oxidation of unsaturated fatty acids and natural degradation of

resins during air-drying of wood occurs mainly due to that water is evaporated [1]. The evapo-

ration of water changes also the physiological conditions of the timber cell walls and lower

metabolic activity with time. Although, the degradation at kiln-drying could be more severe

due to high concentration of hot water inside the kiln, the higher content of lipophilic com-

pounds (i.e. total extractives including FC determined phenols) in kiln- than in air-dried

boards suggests that migration towards surface is favoured probably due to pressure gradients

inside the wood in the drying chamber [2]. The correlation coefficients between total extrac-

tives and FC-determined phenols for air-dried and kiln-dried specimens were 0.7 and 0.5

respectively that supports our hypothesis that performance of drying process influence not

only distribution of hydrophilic but hydrophobic (lipophilic) wood constituents as well.

According to Fig 3 we have higher extractive amounts and phenols in the external (outer

part) than internal (inner part) of boards. This could be controlled by the volatility, reactivity

and migration rate of the individual extractives or group of extractives. Sugars are non-volatile

and as they are readily soluble in water and will degrade only slowly under these drying condi-

tions they will follow the migration of warm water during the capillary phase of drying and

will be left and accumulated on the outermost part of the wood. The process could be seen as

emptying a bath tub and only when the capillary is broken and such transport is stopped, dur-

ing final drying, a receding evaporating front will be observed that is not supplied with

migrated sugars.

Triglycerides and other more lipophilic substances are hardly dissolved in the water and

should not be influenced by this water capillary process. As they are the major extractive com-

pounds, the total extractives will therefore be less influenced by this process and actually

smaller difference between the two drying processes is also seen than for monosaccharides

[7,8]. Migration aptitude of phenols could be higher than the more lipophilic ones especially if

they have not too high molecular weight and exist in glucoside form (Fig 3).

Composition of isolated extractives from air- and kiln-dried board was studied using

GC-MS as trimethylsilyl derivates (Fig 4).

For pimaric acid types, F-test analysis showed significant differences for kiln-dried boards

and also for stearic acid in air-dried boards (Table 3). Oleic, palmitic, linoleic and stearic acids

Fig 4. Chromatogram (GC-MS) of trimethylsilylated extractives from surface of kiln-dried board: 1. glycerol, 2.

internal standard, 3. palmitic acid, 4. linoleic acid, 5. oleic acid, 6. stearic acid, 7. pimaric acid, 8. isopimaric acid,

9 dehydroabietic acid and 10. abietic acid.






were found in the chromatograms (Fig 4), a finding in line with previous work; these are most

common fatty acids in the sapwood of Scots pine [23]. The distribution of fatty acids within

the surface regions was different from that of the other extractives and they were also found in

larger amounts in the air-dried than in the kiln-dried boards, Table 4. On the other hand,

more glycerol was found in the kiln-dried than in the air-dried specimens, indicating a more

extensive degradation of triglycerides to glycerol and fatty acids under those conditions. The

interpretation of the data is thus more complicated since fatty acids could be formed from the

degradation of fats, but they could also be consumed in further oxidation reactions. The unsat-

urated linoleic acid has been reported as a major free fatty acid and fatty acid in triglycerides in

Scots pine [23,24]. However, the variation in the amount of linoleic acid was high in the pres-

ent study, Table 3. This is partly because it is not fully separated from other compounds such

as oleic acid in the chromatogram (Fig 4). Additional uncertainty in linoleic acid data could be

due to its oxidation into products such as hexanoic acid and heptanal which were found in the

dried boards [39]. It is possible that oxidative degradation was greater in kiln-dried boards.

The larger amounts of the resin acids, dehydroabietic, pimaric, isopimaric and abietic acids

were found in the surface layers (0–0.25 mm) of both groups of boards, Table 5, which suggests

that resin acids migrate during both drying methods. A somewhat higher content of resin

acids was found in the kiln-dried than in the air-dried boards. The content of pimaric- and iso-

pimaric acids was clearly lower than that of dehydroabietic acid and corresponds to previously

reported data for stemwood of 82–84 year old Scots pine in northern Sweden [22].

The amount of abietic acid was found to be rather low compared to dehydroabietic acid,

Table 5. This corresponds to what has been reported for Scots pine sapwood in Northern Fin-

land [22].

Table 4. The mean, range, standard error (SE) and coefficient of variation (CV in %), Δmean of glycerol and fatty acids in air-dried and kiln-dried samples at differ-

ent depths from the surface, mg/g.

Depth (mm) Air-dried Kiln-dried Δ mean

%Mean

mg/g

Range (SE)

mg/g

(CV)

%

Mean

mg/g

Range (SE)

mg/g

(CV)

%

Glycerol0–0.25 0.43 0.12. . .1.05 (0.08) 60 0.82 0. . .1.68 (0.17) 64 +91

0.25–0.75 0.42a 0.08. . .0.80 (0.08) 62 1.03 0.28. . .3.05 (0.28) 87 +145

0.75–1.75 0.64a 0.08. . .1.61 (0.15) 72 1.28a 0. . .2.54 (0.33) 82 +100

Palmitic acid0–0.25 0.45 0. . .2.13 (0.25) 177 0.24 0. . .1.25 (0.14) 181 -47

0.25–0.75 0.11 0. . .0.36 (0.04) 120 0.09 0. . .0.47 (0.05) 163 -18

0.75–1.75 0.28 0. . .1.34 (0.16) 177 0.12 0. . .0.35 (0.04) 118 -57

Oleic acid0–0.25 3.63 0.49. . .25.05 (2.39) 208 0.73a 0. . .1.77 (0.17) 75 -80

0.25–0.75 1.35 0.61. . .3.54 (0.03) 63 1.73 0. . .7.26 (0.68) 125 +28

0.75–1.75 3.72 0.30. . .17.40 (1.80) 153 1.30 0. . .4.28 (0.38) 92 -65

Linoleic acid0–0.25 0.68 0. . .6.23 (0.62) 287 0.20a 0. . .0.48 (0.05) 80 -71

0.25–0.75 0.17a 0. . .0.45 (0.05) 91 0.09a 0. . .0.23 (0.03) 100 -47

0.75–1.75 0.07 0. . .0.56 (0.05) 236 0.09 0. . .0.67 (0.07) 233 -29

Stearic acid0–0.25 0.29 0.09. . .0.90 (0.08) 88 0.33 0. . .1.25 (0.11) 103 +14

0.25–0.75 0.13a 0. . .0.27 (0.02) 62 0.23 0.01. . .1.17 (0.11) 148 +77

0.75–1.75 0.25 0. . .2.04 (0.20) 252 0.08 0. . .0.52 (0.05) 200 -68






The content and distribution of dehydroabietic acid was found to be fairly similar in air-

dried and kiln-dried boards, Table 5. An indication of formation of the oxidized product

7-ketodehydroabietic acid was found in the mass peaks of 386, 371, 268 and 253, but the

amounts were generally low or non-detectable in the investigated samples. This compound

has been found in sawdust during storage in piles [40]. Under these conditions, microbiolog-

ical oxidation of extractives can occur raising the temperature inside the pile.

Conclusion

This study describes the distribution of lipophilic extractive chemicals after air-drying and

kiln-drying of green Scots pine sapwood. Enrichment of total extractives, phenols and pimaric

acids was observed in the surface layers (0–0.25 mm below the original surface) compared to

regions some 1.5 mm below the surface. A similar tendency was observed for abietane types of

resin acids during the drying operations. The extent of enrichment was more pronounced

after drying at a higher temperature than after drying at room temperature. These data con-

firm our assumption that migration of hydrophobic extractives occurs especially during kiln

drying at a moderate to high temperature. A higher mass ratio was found for phenols and

resin acids than for total extractives when the surface and the regions beneath were compared.

From the results we suggest that the evaporating front is related not only to conditions during

drying but also to the migration ability of the extractives.

No clear indication of the presence of an evaporation front was found in the fatty acids dis-

tribution. The critical point of our study is the understanding of processes that occur below

the surface. The fatty acids are also components of triglycerides and the amount of glycerol

and the presence of degradation products such as hexanal and hexanoic acids provide evidence

for the necessity of a more detailed and controlled experimental design in order to understand

the complexity of the chemical processes occurring during natural and artificial drying. The

Table 5. The mean, range, standard error (SE) and coefficient of variation (CV), Δmean of resin acids in air- and kiln-dried samples at different depth from the sur-

face, mg/g.

Depth (mm) Air-dried Kiln-dried Δ mean

%Mean

mg/g

Range (SE)

mg/g

CV

%

Mean

mg/g

Range (SE)

mg/g

CV

%

Pimaric acid0–0.25 1.58 0.38. . .6.69 (0.59) 119 1.94a 0.11. . .3.77 (0.40) 65 +23

0.25–0.75 0.73a 0.23. . .1.23 (0.11) 50 0.99a 0.23. . .2.00 (0.19) 62 +36

0.75–1.75 0.82 0.14. . .2.13 (0.22) 83 0.91 0. . .2.58 (0.25) 86 +11

Isopimaric asid0–0.25 1.05 0.28. . .3.34 (0.30) 90 1.49 0.10. . .3.03 (0.35) 75 +42

0.25–0.75 0.55a 0.14. . .1.13 (0.09) 55 0.77a 0.16. . .1.93 (0.16) 67 +40

0.75–1.75 0.51a 0.04. . .1.12 (0.12) 74 0.59a 0. . .1.43 (0.13) 71 +16

Dehydroabietic acid0–0.25 4.37 0.78. . .17.61 (1.56) 113 4.55a 0.32. . .7.58 (0.77) 54 +4

0.25–0.75 2.00a 0.50. . .3.19 (0.31) 48 2.89a 0.57. . .6.96 (0.62) 67 +45

0.75–1.75 2.49a 0.16. . .6.45(0.58) 73 2.65 0. . .9.65 (0.84) 100 +6

Abietic acid0–0.25 0.81 0.25. . .4.12 (0.37) 146 0.85 0.33. . .2.25 (0.18) 68 +5

0.25–0.75 0.33a 0. . .0.75 (0.06) 60 0.77 0.09. . .1.96 (0.23) 93 +133

0.75–1.75 0.56 0. . .1.89 (0.22) 121 0.44 0. . .1.17 (0.13) 93 +21

a–normally distributed according to the Kolmogorov-Smirnov test with Lilliefors significance correction, p�0.05.






development of robust predictive models for the assessment of wood properties requires more

detailed studies of the chemistry of wood drying. That makes it possible to calibrate an on-site

evaluation tool for the properties of dried timber and a better sorting for product

development.

Supporting information

S1 Table. Data set of lipophilic extractives pine from air-dried and kiln-dried sapwood

Scots pine.

(DOCX)

Acknowledgments

The authors wish to thank NORRA Timber, Skellefteå, Sweden for providing the test material.

Support from The European Regional Development Fund, the County Administration of Vas-

terbotten, the municipality of Skellefteå, and WoodCentre North is also greatly appreciated

and acknowledged. Financial support from the Swedish Research Council for the Environ-

ment, Agricultural Sciences and Spatial Planning (FORMAS) project “Experimental studies of

capillary phenomena in bio-based materials” 942-2016-64, “Fungal growth on modified wood-

based products under subarctic conditions” 2017–00419, is also gratefully acknowledged. Sup-

port from the COST Action FP1407 “ModWoodLife” project (Understanding wood modifica-

tion through an integrated scientific and environmental impact approach), is gratefully

acknowledged.

Author Contributions

Conceptualization: Dick Sandberg.

Data curation: Olena Myronycheva.

Funding acquisition: Dick Sandberg.

Methodology: Margot Sehlstedt-Persson.

Project administration: Margot Sehlstedt-Persson.

Supervision: Olov Karlsson.

Validation: Micael Ohman.

Visualization: Micael Ohman.

Writing – original draft: Olena Myronycheva.

Writing – review & editing: Olov Karlsson, Micael Ohman, Dick Sandberg.

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Paper III

Proceedings IRG Annual Meeting (ISSN 2000-8953) © 2017 The International Research Group on Wood Protection

IRG/WP 17-20629

THE INTERNATIONAL RESEARCH GROUP ON WOOD PROTECTION

Section 2 Test methodology and assessment

Multivariate modeling of mould growth in relation to extractives in

dried Scots pine sapwood

Olov Karlsson, Olena Myronycheva, Margot Sehlstedt-Persson, Micael Öhman,

Dick Sandberg

Luleå University of Technology

Wood Science and Engineering

Forskargatan 1

SE-931 87, Skellefteå, Sweden

Poster paper prepared for the IRG48 Scientific Conference on Wood Protection

Ghent, Belgium

4-8 June 2017

IRG SECRETARIAT

Box 5609

SE-114 86 Stockholm

Sweden

www.irg-wp.com

Disclaimer

The opinions expressed in this document are those of the author(s) and

are not necessarily the opinions or policy of the IRG Organization.

Multivariate Modeling of Mould Growth in Relation to Extractives

in Dried Scots Pine Sapwood

Olov Karlsson, Olena Myronycheva, Margot Sehlstedt-Persson, Micael Öhman, Dick Sandberg

Div. of Wood Sci. Eng., Luleå University of Technology, [email protected],

[email protected], [email protected], [email protected],

[email protected]

ABSTRACT

Influence of extractives on mould growth on Scots pine sapwood dried in air or in kiln was

studied. Boards were sprayed with water mixtures of spores of the fungal species Penicillium sp.,

Aspergillus sp., Mucor sp., Paecilomyces sp., Trichoderma sp., treated at a temperature of 22ºC

at 90% RH, classified into a percentage of covered area. Acetone and water extracts were

isolated and analyzed for sugars, nitrogen, ash, resin/fatty acids, glycerol, and phenols. A

multivariate Orthogonal Partial Least Squares (OPLS) regression model was developed to study

relations between the extent of mould coverage of boards and chemical content. The model

describes 51% variability in X and 69% in Y with prediction power of 55%. The results

indicated that total acetone soluble extractives and sugars like glucose contributed to increased

mould growth whereas fatty acids prevent mould growth.

Keywords: multivariate analysis, sapwood, fungi

1. INTRODUCTION

To be able to move towards a sustainable economy, further development in use of wood is of

great importance. However, some challenges need to be addressed which could hinder a

successful usage such as maintenance costs in multi-storey buildings, entrapment of moisture in

passive houses, climate changes, use of less harmful preservative and surface treatments, etc.

Outdoors, a fungal growth regarding moulds is mostly related to aesthetic issues, and increased

maintenance cost whereas indoor released toxins could influence people's health. Mould growth

on wooden boards can be linked to many factors such as moisture content and its variation due to

climate conditions, temperature, sawing pattern, wood surface roughness (Johansson et al. 2012,

Viitanen 1994). Often mould growth on sawn timber is observed near the edges which are close

to where the living tree grow, rather than in middle parts of the flat side. Higher content of

nutrients like sugars has been found in kiln-dried than in air-dried boards (Ahmed et al. 2013;

Sehlstedt-Persson et al. 2011, Terziev & Boutelje 2007). This phenomenon is due to the

migration of capillary water during the first parts of industrial drying, and that soluble

compounds follow and accumulates on the wood surface. Hindrance of such flow during drying

could explain the development of sticker marks sometimes found in moulded side-boards. The

presence of other nutrients on wooden boards such as nitrogen containing or fatty extractives has

been declared (Theander et al. 1993). The wood surface also contains other types of extractives,

and some of them are antioxidants and could influence or even hinder mould growth. The

purpose was to study the influence of the chemical composition of extractives on colonization of

selected mould fungi on Scots pine sapwood.

2. EXPERIMENTAL METHODS

Sapwood surface of ten kiln or air dried Scots pine sideboards (220L x 100W x 25T mm) were

studied. Air drying was performed indoor whereas boards from Scots pine were conventionally




3

kiln dried for 44 hours to a moisture content of 16%. Mould growth of the surfaces was tested

according to SP Method 4927:2013. A mixture of five mould fungi previously isolated from

dried boards (Sehlstedt-Persson et al. 2011) was sprayed on the board surfaces. The spore

suspension contained the following fungal species: Penicillium sp., Aspergillus sp., Mucor sp.,

Paecilomyces sp., Trichoderma sp. The samples were treated at a temperature of 22ºC at 90%

RH in a chamber with a filter for the exchange of air. Visual inspection of the studied samples

classified into a percentage of covered area used as evaluation (Fig. 1). The acetone solubles

were isolated by extraction of milled wood samples with acetone using Soxhlet apparatus

followed by drying in air. Ash content was determined gravimetrically. Sugars in water extracts

from milled wood were analyzed using HPLC (Karlsson et al. 2012). Resin acids, fatty acids,

and glycerol were analyzed after trimethylsilylation (BSTFA/TMSCl 2:1) at 70ºC for 20 minutes

of dried acetone solubles with GS/MS (SUPELCO SLB-5 MS) using 1-methylnaphtalene as an

internal standard. Folin-Ciocalteau (FC) was used to study the presence of phenols in acetone

solubles. The amount of nitrogen was determined by the Kjeldahl method. Statistical analyses

were performed using software SPSS 21 and SIMCA 14.

Figure 1: Surface view of air and kiln dried Scots pine sapwood boards after mold test

3. RESULTS AND DISCUSSION

Mould growth and extractives in sapwood surface layer

Moulds covering wood boards dried under industrial kiln conditions and in the air could be seen

in Fig. 1. Totally ten boards of each drying type were analyzed and on average 86% of the

surface of kiln dried samples was covered with mould compared with air dried in which 37% of

the surface was covered. The variability of mould covered area was relatively large within the air

dried as well as within the kiln dried boards (Fig. 2).

a) Air dried b) Kiln dried

Figure 2: Variability of the area coverage of mould (%) on sapwood of pine boards dried in (a) air or in

(b) an industrial kiln

It could be seen that mould growth in about 1/3 of the air dried boards was very low (5%), but

still, boards with high mould growth was observed (Fig. 2). Within kiln dried boards half of the

5%

20%

30% 45%

50%

60%

100%

100%

50%

60%

70%

90%

Air dried Kiln dried

4

analyzed samples were totally covered with mould (100%). Mould fungi are a primary colonizer

usually and interact with the outermost surface of the wood as could be seen in Fig. 3, where

hyphae are mostly situated on the wood surface.

Figure 3: Scanning electron micrograph of mould growth on a kiln-dried wood surface

However, as can be seen on the boards, the growth is uneven and very much related to the spatial

distribution on the wood surfaces with a sometimes quite irregular pattern (Fig. 2). The irregular

pattern of mould growth has been proposed to be related to the presence and distribution of

nutrient and sugars on the wood surface (Sehlstedt-Persson et al. 2011, Terziev & Boutelje

2007). This study also focuses on whether the extent of mould growth could be related to the

presence of also other extractives in the surface area. In Table 1, content of various extractives

isolated from the surface area of air- and kiln-dried boards is presented. It could be seen that

higher content of the sugars: glucose, fructose, and sucrose, was found in kiln dried boards than

in the ones dried in air. Nitrogen content includes compounds like proteins and could work as a

nutrient for mould (Theander et al. 1993). They were also higher in kiln dried boards than in air

dried ones (Table 1). Variability of data among these samples was large which makes it difficult

to draw definite conclusions regarding the role of these nutrients in promoting mould growth and

analysis by using multivariate tools was done (see below).

Table 1: Extractives content in the surface of dried boards of sapwood of Scots pine

Analysed compounds Air dried [%] Kiln dried [%]

Sucrose

Glucose

Fructose

Nitrogen

Aceton solubles

Palmitic acid

Oleic acid

Linoleic acid

Stearic acid

Pimaric Acid

Isopimaric acid

Dehydroabietic acid

Abietic acid

Phenols

Glycerol

Ash

0.086

0.160

0.132

0.149

3.594

0.045

0.360

0.068

0.029

0.158

0.105

0.437

0.081

0.044

0.043

0.329

0.416

0.805

0.603

0.267

5.707

0.024

0.073

0.020

0.033

0.194

0.149

0.455

0.085

0.081

0.082

0.906

Spread in data could be due to the formation of coloured Maillard and caramelization products

(Theander et al. 1993), but also to the spatial distribution of nutrients as mentioned above and

that compounds might migrate towards the wood surface to various extent during drying. The

content of acetone soluble extractives was found to be significantly higher in the kiln- than in

5

air-dried boards (Table 1). Fatty compounds are known to constitute the main part of isolated

acetone-soluble extractives in the sapwood of Scots pine and could act as a nutrient and

influence fungal growth. Fatty acids have been reported to have antifungal properties (Tumlinson

& Engelberth 2008) and they were found in lesser amounts in the kiln- than in the air-dried

boards (Table 1). The lower content in the kiln dried boards could be due to a faster

degradation/evaporation at higher drying temperature during the kiln than under air-drying.

Resin acids were similar or somewhat higher in the kiln dried boards (Table 1). Phenols such as

pinosylvins mostly found in the heartwood of Scots pine have antioxidant properties and may

hinder fungal colonization. Small amounts of phenols were found in the sapwood of dried boards

and data indicated that they might be higher in kiln-dried boards than in air-dried boards.

Multivariate analysis

Figure 5: Score (upper) and loading (lower) plot of OPLS model from kiln-dried (red) and air-dried (blue)

boards

In such a complex matrix of parameters, the multivariate analysis is a tool to study how each of

the factors interacts with each other. An Orthogonal Partial Least Squares (OPLS) regression

model based on the content of sugars, acetone-soluble compounds, moisture content, resin/fatty

acids, phenols, ash, nitrogen, mould coverage was developed. This type of the model allowed us

to separate systematic variation in measured chemical compounds and improve the predictive

power of the Partial Least Squared regression. In our case, the Drying type and Mould area were

selected as Y (responses), when Glycerol, Palmitic, Oleic, Linoleic, Stearic, Pimaric, Isopimaric,

Dehydroabietic, Abietic acids, Total Extractives, Total phenols, Nitrogen, Ash, Moisture content,

Saccharose, Glucose, Fructose were defined as X (variables). The developed OPLS model has

two components, defining one predictive and one orthogonal component that covers 33%

systematic variability in X variables. The model describes 51% variability in X and 69% in Y

with prediction power of 55%. Fig. 5 shows the score and loading plot from the analysis. The

analysis confirm that Drying type and Mould area strongly related to each other based on their

6

presence in the loading plot (Fig. 5). It could also be seen that Total extractives content and other

variables such as Glucose are situated quite close to Mould area in the loading plot whereas

others like Nitrogen seemed not to contribute to the developed model. A negative influence on

mould area is indicated for fatty acids such as for Oleic acid (Fig. 5).

4. CONCLUSIONS

To reach deeper in understanding on how mould grows on dried pine sapwood surfaces probably

also other characterization parameters such as spatial distribution has to be included to obtain a

more valid model. Furthermore, growth conditions of moulds depend on the species and the

microclimate on the wood surface and its physical properties.

5. REFERENCES

Ahmed, S. A., Sehlstedt-Persson, M. & Morén, T. (2013). Development of a new rapid method

for mould testing in a climate chamber: preliminary tests. Eur J Wood Wood Prod, 71(4), 451–

461.

Johansson, P., Ekstrand-Tobin, A., Svensson, T. & Bok, G. (2012). Laboratory study to

determine the critical moisture level for mould growth on building materials. Int Biodeter

Biodegr, 73, 23–32.

Karlsson, O., Yang, Q., Sehlstedt-Persson, M. & Morén, T. (2012). Heat treatments of high

temperature dried Norway spruce boards: Saccharides and furfurals in sapwood surfaces.

BioResources 7(2), 2284–2299.

Sehlstedt-Persson, M., Karlsson, O., Wamming, T. & Morèn, T. (2011). Mold Growth on

Sapwood Boards Exposed Outdoors: The Impact of Wood Drying. Forest Prod J, 61(2), 170–

179.

Terziev, N. & Boutelje, J. (2007). Effect of Felling Time and Kiln-Drying on Color and

Susceptibility of Wood to Mold and Fungal Stain During an Above-Ground Field Test. Wood

Fiber Sci, 30(4), 360–367.

Theander, O., Bjurman, J., & Boutelje, J. B. (1993). Increase in the content of low-molecular

carbohydrates at lumber surfaces during drying and correlations with nitrogen content, yellowing

and mould growth. Wood Sci Technol, 27(5), 381–389.

Tumlinson, J.H., Engelberth, J., 2008. Fatty Acid-Derived Signals that Induce or Regulate Plant

Defenses Against Herbivory, in: Schaller, A. (Ed.), Induced Plant Resistance to Herbivory.

Springer Netherlands, Dordrecht, pp. 389–407.

Viitanen, H. (1994). Factors affecting the development of biodeterioration in wooden

constructions. Mater Struc, 27(8), 483–493.

Paper IV

PROCEEDINGS

9th European Conference on Wood Modification

Burgers’ Zoo

Arnhem, The Netherlands

17-18 September 2018

In association with:

COST FP1407 ModWoodLife

Edited by:

Jos Creemers, Thomas Houben, Bôke Tjeerdsma, Holger Militz, Brigitte Junge

and Jos Gootjes

Secretariat:

SHR B.V.

Nieuwe Kanaal 9b

6709 PA Wageningen

www.ecwm9.shr.nl

http://www.ecwm9.shr.nl/

2

Legal Notice

Although all reasonable efforts were made by the editorial team to ensure the scientific

quality of the contents of these conference proceedings, the final responsibility for the

contents of individual articles therein remains with the respective authors. The editors accept

no responsibility for the information contained herein. The inclusion of information in this

publication does not constitute any form of endorsement by the editors. The editorial team is

not responsible for the contents of external websites referred to in this publication.

No permission is required to reproduce or utilise the contents of these abstracts other than in

the case of diagrams, images, or other material from other copyright holders. In such cases the

permission of the original copyright holder must be obtained. The proceedings may be sited as

Proceedings of the 9th European Conference on Wood Modification 2018, Arnhem, The

Netherlands.

Copyright © SHR B.V.

Nieuwe Kanaal 9b, 6709 PA Wageningen, The Netherlands

www.shr.nl

ISBN: 978-90-829466-1-1

http://www.shr.nl/

European Conference on Wood Modification 2018

155

Effects of thermal modification on bending properties and chemical

structure of Iroko and Padauk

Michal Kroupa1, Milan Gaff2, Olov Karlsson3, Olena Myronycheva4, Dick

Sandberg5

1Department of Wood Processing, Czech University of Life Science in Prague, Kamycka 1176,

Praha 6-Suchdol, 16521 Czech Republic [email: [email protected]] 2Department of Wood Processing, Czech University of Life Science in Prague, Kamycka 1176,

Praha 6-Suchdol, 16521 Czech Republic [email: [email protected]] 3Division of Wood Science and Engineering, Luleå University of Technology, SE-93187

Skellefteå [email: [email protected]] 4Division of Wood Science and Engineering, Luleå University of Technology, SE-93187

Skellefteå [email: [email protected]] 5Division of Wood Science and Engineering, Luleå University of Technology, SE-93187

Skellefteå [email: [email protected]]

Keywords: bending properties, Milicia excelsa, Pterocarpus soyauxii, thermal modification,

bendability, modulus of rupture, extractives, phenols, pH, Ec, ash

ABSTRACT

The thermal modification process essentially involves a controlled degradation of the

wood, primarily resulting in the degradation of hemicelluloses. In this study, thermal

modification at temperatures of 160, 180 and 210 °C was used in superheated steam.

Examined wood samples were iroko (Milicia excelsa (Welw.) C.C. Berg) and padauk

(Pterocarpus soyauxii Taub.). Testing of bending properties was done according to the

Standard ČSN 49 0116 (ASTM 04.10 WoodD143-09). The samples were loaded by three-

point bending. Chemical analysis of total extractives and phenols content, pH and Ec

changes and ash content.

The results show that with the increasing temperature of the thermal modification process,

the modulus of rupture and density decreases. The force and deflection at the limit of

proportionality increased. The thermal modification process affected the chemical

characteristics of both species. The distribution of total extractives and phenols varied

strongly for iroko, but for padauk decrease with increased treatment temperature and time

was observed. Having a more detailed assessment of wood chemical changes and

degradation products from structural and non-structural wood components and their

resistance to biological attack is necessary for the future development of sustainable

technologies for the thermal treatment of iroko and padouk.

INTRODUCTION

Commercial tropical timber species such as iroko (Milicia excelsa (Welw.) C.C. Berg)

and padauk (Pterocarpus soyauxii Taub.) possess increasing demand in various wood

products applications (International Tropical Timber Organization (ITTO) 2018a, b) that

requires to develop modern timber processing technologies. Iroko is widespread in

tropical and subtropical Africa. The colour of Iroko is usually yellow to golden or medium

brown. It is very durable and resistant to rot and insect attack. Padauk can be found in

central and tropical west Africa. Its colour can vary, ranging from pale pinkish-orange to

deep brownish red. Padauk has excellent decay resistance and is rated as a durable to very

durable species. It is also resistant to termite attacks and of another type of insect attack.



mailto:3



mailto:3


https://en.wikipedia.org/wiki/Paul_Hermann_Wilhelm_Taubert



156

The colour and texture are favoured to use these two wood species in musical instruments,

luxury furniture and other high-value products. However, new information came that

extractive rich padauk wood species can have a negative impact on human health and

cause dermatitis and allergic reactions via dust dispose of (Kiec-Swierczynska et al.

2004). For softwood species, it has been shown that thermal modification can reduce the

number of extractives and lead to the formation of less toxic substances (Poncsak et al.

2009; Esteves et al. 2011).

Thermally modified timber (TMT) is, according to CEN (2007), wood at which the

composition of the cell wall material and its physical properties are modified by exposure

to a temperature higher than 160ºC and conditions of decreased oxygen availability. The

wood is altered in such a way that at least some of the wood properties are permanently

affected through the cross-section of the timber. This product is related to heat-treated

wood, but to distinguish it from heat sterilisation at a lower temperature (≈55C) with the

purpose of killing pests in solid wood materials and preventing their transfer between

continents and regions. Main disadvantage of thermal modification is loss of strength due

to the degradation of wood constituents such as extractives, however, as advantages the

biological resistance against some micro-organisms and insects could be improved,

swelling and shrinking reduced up to 50-90 % and equilibrium moisture content could be

reduced too (Karlsson et al. 2012, 2014; Navi and Sandberg 2012).

For many tropical species, there is a lack of information about changes in properties

during thermal modification. Therefore, the purpose of this study was to understand the

influence of thermal modification on bending properties and changes of chemical

structure of iroko and padauk.

EXPERIMENTAL

Iroko (Milicia excelsa (Welw.) C.C. Berg) and padauk (Pterocarpus soyauxii Taub.) was

harvested in Gabon (Sablík et al. 2016). The dimension of specimens was 20 x 20 x 300

mm. Moisture content before thermal treatment was 3±1%. Thermal modification

achieved according to ThermoD® (International Thermowood Association 2003) process

in the thermal chamber with filled kiln capacity 0.8 m3.

The scheme of technological parameters of thermal modification process provided in

Table 1.

Table 1: Scheme for thermal modification

Test

group

No.

Heating Thermal

modification

Cooling Total

time

[h]

No. of

specimens

Time

[h]

Temp[°C] Time

[h]

Temp[°C] Time

[h]

Temp[°C]

Iroko

1 0 20 0 20 0 20 0 30

2 11.6 160 3.0 160 3.9 50 18.5 30

3 14.8 180 3.0 180 5.8 50 23.6 30

4 18.6 210 3.0 210 7.2 50 28.8 30

Padauk

5 0 20 0 20 0 20 0 30

6 10.0 160 3.0 160 2.3 50 15.3 30

7 11.7 180 3.0 180 4.1 50 18.8 30

8 15.1 210 3.0 210 4.5 50 22.6 30



157

Testing of bending properties has been done according to the standard ČSN 49 0116

(ASTM 04.10 WoodD143-09). The samples were loaded by three-point bending in a

universal testing machine UTS 50 (TIRA, Germany) according to EN 310. The loading

speed was 3 mm/min. Loading forces were measured using data logger ALMEMO 2690-

8 (Ahlborn GmbH, Germany).

The data used to create a force-deformation diagram, in which we applied the method of

accurate identification of boundary points developed by authors (Gaff et al. 2016, 2017).

The parameters used for analysis: force at the limit of proportionality FE, deflection at the

limit of proportionality YE, force at the modulus of rupture FP, deflection at the modulus

of rupture YP. The evaluating bendability did by using the minimum bend radius and

coefficient of bendability. The minimum bend radius RminB and the coefficient of

bendability KbendB are based on the bending geometry. The minimum bend radius RminC

and the coefficient of bendability KbendC are based on the basic bending equation (Gaff et

al. 2016).

For the assessment of the chemical changes during thermal modification process the

extraction of extractives for the total amount was done in mini-Soxhlet extractor by

acetone/water 95:5v/v and evaluated gravimetrically by percentage of the dry mass of

wood (Willför et al. 2006). The total phenolics content was determined by Folin-

Chiocalteu assay (Julkunen-Tiitto 1985). The absorption was measured after 20 minutes

at 735 nm against zero absorbance. Tannic acid (VWR Chemicals; 83510.260) was used

for the standard curve, and the results are expressed in mg of tannic acid per gram of dry

mass of wood (mgTA/g dry w.). The measurement of pH and Ec was done by pH meter

Metrohm 744 after treatment of the sample with a sodium chloride solution to exchange

protons in less accessible carboxylic groups in the wood by sodium ions (Sithole 2005).

The ash content of the wood was determined gravimetrically after heating at 560°C in a

muffle furnace and expressed in percentage of the dry mass of wood. The effect of

individual factors evaluated using ANOVA, specifically utilizing Fisher’s F-test in

STATISTICA 12 (Statsoft Inc., USA) and IBM SPSS 20 software (IBM 2014).

RESULTS AND DISCUSSION

The thermal modification of iroko and padauk affect the mechanical properties and

chemistry of the wood significantly. The increasing processing time reduces the modulus

of rupture and density but increases the values of force and deflection at the limit of

proportionality. However, some increasing trend of the FP is evident for the temperature

of 160°C.

The average values of the monitored mechanical characteristics listed in Table 2.


158

Table 2: Mean values of the YE, YP, FP, FE, RminB, RminC, KohB, Kohn

Thermal

Modification

YE [mm] YP [mm] FP [N] FE [N] Density

[kg/m3]

Iroko

20 2.0 5.0 2079 996 615

160 3.1 4.7 2180 1542 593

180 3.0 4.5 2015 1518 591

210 2.7 3.3 1397 1181 594

Padauk

20 2.6 6.0 3262 1749 676

160 3.7 5.4 3314 2346 691

180 3.4 4.5 2805 2174 797

210 1.7 3.7 1875 1245 657

Thermal

Modification RminB RminC KohB KohC

Iroko

20 1400 1007 0.01 0.02

160 1555 1042 0.01 0.02

180 1610 1079 0.01 0.02

210 2210 1379 0.01 0.02

Padauk

20 1346 789 0.02 0.03

160 1373 920 0.01 0.02

180 1647 1103 0.01 0.02

210 1996 1336 0.01 0.02

The data about changes of some chemical properties for thermally treated timber

presented in Table 3. The total extractives and phenols in untreated wood in our samples

corresponds to previously studied data (Saha et al. 2013; Nagawa et al. 2015). It is well

known that increasing temperature in thermal modification process affects the

significantly chemical composition of the wood. The distribution of total extractives and

phenols varied strongly for iroko but was relatively low at the most extensive treatment

(Table 3). For padauk, the decrease in the amount of extracted phenols with treatment

temperature and time was strongly evident. The unexpected phenomenon of similar pH

was observed for padauk wood with an elevation of the thermal treatment time and

temperature (Table 3). A significantly lower pH has been found in thermally modified

than in untreated birch (O. Dagbro et al. 2011). In case of iroko, pH was in similar range

as for birch, but the thermal treatment of iroko did not reduce the pH. A fairly high content

of ash was found in the iroko samples (Table 3). Higher pH and ash content for iroko

could be explained by biomineralization properties of that tree that leads to accumulation

of mineral carbon. Calcium carbonate has been found in Iroko (ref) and such compound

could work as a buffer agent for the formed acids during the thermal modification process

(Cailleau et al. 2004).


159

Table 3: Results of chemical tests

Thermal

modification Total

extractives

[%]

Total phenols

[mgTA/g dry w.]

pH Ec Ash content

[%]

Iroko

20 7.8 27.5 5.5 108.0 2.0

160 10.4 30.4 5.3 118.7 2.3

180 8.1 24.6 5.2 127.7 1.9

210 4.8 11.8 5.2 126.0 1.9

Padauk

20 11.1 27.4 3.7 213.7 0.2

160 9.9 27.1 3.7 213.3 0.2

180 9.5 22.5 3.8 208.3 0.3

210 7.5 22.0 3.8 205.0 0.4

CONCLUSIONS

In our study, we found that thermal modification process significantly affects bending

properties and acetone soluble extractives, phenols and ash content of iroko and padauk

wood as well as pH. The more detailed assessment of wood chemical changes and

degradation products from structural and non-structural wood components and resistance

to biological attack seems to be a reasonable step for future development of the sustainable

technology of iroko and padauk thermal treatment.

ACKNOWLEDGEMENTS

The authors are also much appreciated and acknowledged for financial support from the

Swedish Research Council for the Environment, Agricultural Sciences and Spatial

Planning (FORMAS) projects: “Fungal growth on modified wood-based products under

subarctic conditions” 2017-419 is gratefully acknowledged.

REFERENCES

Cailleau G, Braissant O, Verrecchia EP (2004) Biomineralization in plants as a long

term carbon sink. Naturwissenschaften 91:191–194. doi: 10.1007/s00114-004-0512-1

Esteves B, Videira R, Pereira H (2011) Chemistry and ecotoxicity of heat-treated pine

wood extractives. Wood Sci Technol 45:661–676. doi: 10.1007/s00226-010-0356-0

Gaff M, Kačík F, Domljan D, et al (2017) Bendability of Thermally Modified Oak

(Quercus robur F). In: IMPLEMENTATION OF WOOD SCIENCE IN

WOODWORKING SECTOR. University of Zagreb, Faculty of Forestry, Zagreb, pp

143–151

Gaff M, Vokatý V, Babiak M, Bal BC (2016) Coefficient of wood bendability as a

function of selected factors. Constr Build Mater 126:632–640. doi:

10.1016/j.conbuildmat.2016.09.085


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IBM (2014) SPSS Statistics. IBM, United States International ThermoWood Association

(2003) ThermoWood Handbook International Tropical Timber Organization (ITTO)

(2018a) Iroko (Milicia excelsa). In: ITTO. http://www.tropicaltimber.info/specie/iroko-

milicia-excelsa/. Accessed 13 Jun 2018

International Tropical Timber Organization (ITTO) (2018b) Padouk d`Afrique

(Pterocarpus soyauxii). In: ITTO. http://www.tropicaltimber.info/specie/padouk-

dafrique-pterocarpus-soyauxii/. Accessed 13 Jun 2018

Julkunen-Tiitto R (1985) Phenolic constituents in the leaves of northern willows: methods

for the analysis of certain phenolics. J Agric Food Chem 33:213–217. doi:

10.1021/jf00062a013

Karlsson O, Dagbro O, Granlund K (2014) Soluble degradation products in thermally

modified wood. In: Final Cost Action FP0904 Conference : “Recent Advances in the Field

of TH and THM Wood Treatment.” Luleå tekniska universitet, Skelleftea, pp 16–17

Karlsson O, Torniainen P, Dagbro O, et al (2012) Presence of water-soluble compounds

in thermally modified wood : carbohydrates and furfurals. BioResources 7:3679–3689

Kiec-Swierczynska M, Krecisz B, Swierczynska-Machura D, Palczynski C (2004)

Occupational allergic contact dermatits caused by padauk wood (Pterocarpus soyauxii

Taub.). Contact Dermatitis 50:384–385. doi: 10.1111/j.0105-1873.2004.0350k.x

Nagawa CB, Böhmdorfer S, Rosenau T (2015) Chemical composition of volatiles

extracted from indigenous tree species of Uganda: composition of bark extracts from

Psorospermum febrifugum and Milicia excelsa. Holzforschung 69:815–821. doi:

10.1515/hf-2014-0283

Navi P, Sandberg D (2012) Thermo-Hydro-Mechanical Wood Processing. CRC Press

O. Dagbro, P. Torniainen, T. Morén (2011) Thermal modification of birch using saturated

and superheated steam. In: DIVA. Norsk institutt for skog og landskap, pp 43–48

Poncsak S, Kocaefe D, Simard F, Pichette A (2009) Evolution of extractive composition

during thermal treatment of Jack pine. J Wood Chem Technol 29:251–264. doi:

10.1080/02773810902928582

Sablík P, Giagli K, Pařil P, et al (2016) Impact of extractive chemical compounds from

durable wood species on fungal decay after impregnation of nondurable wood species.

Eur J Wood Wood Prod 74:231–236. doi: 10.1007/s00107-015-0984-z

Saha J-BT, Abia D, Dumarçay S, et al (2013) Antioxidant activities, total phenolic

contents and chemical compositions of extracts from four Cameroonian woods: Padouk

(Pterocarpus soyauxii Taubb), tali (Erythrophleum suaveolens), moabi (Baillonella

toxisperma), and movingui (Distemonanthus benthamianus). Ind Crops Prod 41:71–77.

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Sithole B (2005) New Method of Measuring the pH of Wood Chips. 59th Appita Annu

Conf Exhib Inc 13th ISWFPC Int Symp Wood Fibre Pulping Chem Auckl N Z 16-19

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Willför S, Hemming J, Leppänen A-S (2006) Analysis of extractives in different pulps –

Method development, evaluation, and recommendations. Laboratory of Wood and Paper

Chemistry, Åbo Academi University

Paper V


Elustondo et al. (2017). “Wood surface modification,” BioResources 12(1), 283-295. 283

Localized Wood Surface Modification, Part I: Method Characterization Diego Elustondo,a Olena Myronycheva,a Bror Sundqvist,b and Olov Karlsson a

This study assesses the potential of an open process for treatment of European Scots pine (Pinus sylvestris) with chemicals that could potentially make the surfaces stronger, more dimensionally stable, or more durable, depending on the treatment solution. The method provides an intermediate solution between full volume impregnation by pressure treatment and superficial surface treatment by dipping. Figuratively speaking, the process creates the equivalent of a layer of coating applied below the wood surfaces rather than above. Two different techniques were compared, namely, heating-and-cooling (H&C) and compression-and-expansion (C&E). Taking into account that commercial suppliers recommend 0.15 to 0.25 L/m2 of coating in sawn wood and 0.1 to 0.15 L/m2 in planed wood surfaces, then this study demonstrates that the H&C method can impregnate an equivalent amount of solution under the surfaces in less than 15 min using treatment temperatures below 150 °C.

Keywords: Scots pine; Wood surface modification; Heating-and-cooling; Compression-and-expansion

Contact information: a: Luleå University of Technology, Division of Wood Science and Engineering,

Campus Skellefteå, Forskargatan 1, 93187 Skellefteå, Sweden; b: SP Technical Research Institute of

Sweden, Laboratorgränd 2, SE-931 77 Skellefteå, Sweden;

* Corresponding author: [email protected]

INTRODUCTION

Wood is a biodegradable material. Thus, surfaces that are exposed to the external environment (such as in cladding, decking, flooring, and furniture) experience a slow degradation that is caused by a combination of moisture, temperature, sunlight, mechanical abrasion, and biological attack (Williams 2005). This problem is exacerbated in wood that was obtained from the periphery of the logs - such as the so called "side boards" in Sweden - since sapwood has normally lower durability (Homan and Jorissen 2004). Coating is the most widely used method for protecting wood surfaces from weathering, but the time that the material can be used between maintenance cycles (service life) is limited. Typical wood cladding used in residential and low rise buildings must be re-painted and re-stained every five to seven years (Athena 2002).

This problem is leading society slowly but consistently towards a future in which a renewable material such as wood will be replaced by other much more energy-intensive industrial alternatives that may irreversibly change the planet for future generations. One possible solution is to modify the entire volume of wood by impregnation with chemicals that can make wood stronger, more stable, or more durable, depending on the application. Examples include acetylation, furfurylation, and impregnation with oils and resins (Homan and Jorissen 2004). Acetylation (impregnation with acetic anhydride) replaces the moisture-accessible hydroxyls groups in the wood with hydrophobic acetyl groups (Hill 2006). Furfurylation (impregnation with furfuryl alcohol) creates a highly branched




and cross-linked polymer bonded to the wood cell walls (Westin et al. 2003; Lande 2008). Impregnation with oils is an effective method to control the absorption of moisture in wood (Alireza 2011), while impregnation with resins refers to the application of solutions that polymerize inside the wood (Nurmi 1998; Gsöls et al. 2003). It is well known, for instance, that impregnation with phenol formaldehyde resins improve wood durability, water resistance, and dimensional stability of wood (Takahashi and Imamura 1990; Ryu et al. 1991; Deka and Saikia 2000; Hill 2006; Gabrielli and Kamke 2010).

The most widely used method for impregnating the entire volume of wood with chemicals is pressure treatment (Hunt and Garratt 1967; Ormrod and Van Dalfsen 1993; Richardson 2003). Pressure treatment is applied inside pressure chambers, typically steel cylinders (Kollmann and Côté 1984), in which the wood is first exposed to vacuum to remove the air and then to pressures in the order of 8.5 to 12 atm to force the penetration of chemicals (Forest Products Laboratory 2010). However, fully modified wood has not yet found a large number of applications, probably because of the high volume of chemicals and long processing times required to impregnate the entire volume of wood. This is reflected in the cost. It was estimated that the approximate market prices for fully acetylated wood, furfurylated wood, and wood impregnated with sodium-silicate were 10 to 4 times higher than untreated pine (Sandberg et al. 2013).

Another option for impregnating chemicals is by submerging the wood into the treatment solution and letting the chemicals penetrate by diffusion (Tamblyn 1985). This is customarily called dipping. Preservatives that can be dissolved in water or oils can be potentially absorbed by dipping. Reported treatment times have ranged from seconds or minutes (Kollmann and Côté 1984) to hours or even days (Blew 1948). In this study the objective is to test a third option that provides an intermediate solution between pressure treatment and dipping. Two different techniques were compared. These are referred to in this study as heating-and-cooling (H&C) and compression-and-expansion (C&E) methods. The H&C method consists of increasing the temperature of the wood and subsequently submerging it into treatment solution at room temperature (Crawford 1907). The C&E method consists in compressing the wood with a press and then allowing it to expand while submerged in treatment solution (Fry 1973; Inoue et al. 2008). In principle, both methods have the capacity to induce internal vacuum by reducing the density of the gases. The H&C method reduces the gas temperature at constant volume and the C&E method increases the gas volume at constant temperature. In this study the C&E method was only implemented to assess its potential in comparison with the H&C method, but the effect of the process parameters was not investigated further. The methods were implemented with European Scots pine (Pinus sylvestris) side boards which predominantly contain sapwood. EXPERIMENTAL Materials

The wood for this study was European Scots pine (Pinus sylvestris) supplied by Norra Skogsägarna sawmill in Kåge, Sweden. The boards were 19 mm thick in radial direction by 75 mm wide in the tangential direction by 5 m long in the longitudinal direction, and they contained mainly sapwood (referred as side boards in the industry). The boards thickness was reduced to approximately 18 mm with a planer, cut into 200 mm long sections free of knots and bark, and then end-sealed with commercial glue that



can stand up to 300 °C (Silikon 300 °C, Sika Norge AS, Skjetten, Norway). Twelve samples were cut from each board, thus producing 12 sets of 6 matched samples.

Before the H&C tests, samples were oven-dried at 103 °C until constant weight, in order to eliminate moisture content as a variable of the study. Three liquids were tested as examples of treatment solutions, namely: water, vegetable oil, and biodiesel. The vegetable oil was rapeseed oil bought in a local store (Coop Trading A/S, Izegem, Beligum), and the biodiesel was rapeseed oil methyl ester (RME) supplied by a local company (Ecobränsle i Karlshamn AB, Karlshamn, Sweden). The properties of these particular liquids were not measured in this study, but there are European standards for rapeseed oil and biodiesel that were used for comparison (Bernat et al. 2011). Water properties are also available in the literature (Venard and Street 1975). Table 1 reports the density and kinematic viscosity of water, rapeseed oil, and biodiesel according to the cited literature. The purpose of selecting those liquids was to assess the potential of the H&C method in terms of solution uptake and processing times. In Part II of this study, the H&C method will be implemented with resins to investigate the possibility of creating a permanent layer of protection below the wood surfaces.

Table 1. Density and Kinematic Viscosity of Water, Rapeseed Oil, and Biodiesel

Temperature Density (kg/m3) Kinematic viscosity (mm2/s)

°C Water1 Oil2 Biodiesel2 Water1 Oil2 Biodiesel2

20 998.2 914.5 879.8 1.003 74.19 6.78

80 971.8 877.7 837.2 0.364 11.58 2.16

100 958.4 865.8 822.9 0.294 7.89 1.69

120 853.7 807.5 5.86 1.36

140 839.5 791.2 4.47 1.13 1 Venard & Street (1975); 2 Bernat et al. (2011)

Methods

Four temperatures were tested for the heating-and-cooling (H&C) method, namely, 20, 90, 120, and 150 °C, where the 20 ºC test was representative of the dipping method with both wood and liquid at room temperature (Fig. 1).

Fig. 1. Set of three hot samples submerged in cold treatment solution

CT-scanner plane

Wood sample

Frame

Load



The H&C process was monitored in real time with a fourth generation Siemens SOMATOM Emotion computed tomography scanner (CT-scanner) available at Luleå University of Technology. The procedure consisted in warming up the samples in an oven for approximately 4 h to guarantee uniform temperature, and then submerging the samples in the treatment solution that was approximately at 20 ºC. The H&C process was performed inside 30 cm x 23 cm plastic boxes with enough capacity for accommodating three samples. Sets of three samples were fastened to a wood frame and metal loads were used to hold the samples submerged in the treatment solution. The metal loads were placed over the ends of the samples so that the center of the samples was unobstructed for CT-scanning.

The plastic box was inside the CT-scanner field of view when the samples were submerged (Fig. 2), but it took approximately 30 s to start the first scan. Despite this delay, the first CT-scan image was used as reference to calculate weight gain as function of the treatment time. The weight gain measured with the CT-scanner was representative of a 10 mm slice in the center of the samples (Based on the CT-scanner parameters). The weight gain was not confirmed with a balance for the entire sample because the wood frame was covering part of the surfaces close to the sample ends. Each experiment was performed two times, thus resulting in 6 replicates per treatment.

Fig. 2. Set of three samples positioned inside the CT-scanner field of view

The weight gain based on the CT-scanner measurements was calculated from the

CT-images by using ImageJ image analysis software. The procedure consisted of selecting a polygon around the cross section image of each sample and then calculating the total mass contained inside that polygon by multiplying area and average density. The average density of the first CT-image was also used to determine the initial wood density. The measured initial density was approximately 480 kg/m3 with a standard deviation of 43 kg/m3. Because the samples were first oven-dried and subsequently warmed up in an oven for approximately 4 h, it was assumed that the initial moisture content was close to 0%.

For the compression-and-expansion (C&E) tests, a laboratory press was used with a plate size of approximately 140 mm x 140 mm. The original 18 mm thick by 75 mm

Wood samples

CT-scanner



wide by 200 mm long samples were trimmed to a length of approximately 180 mm to fit into the press compartment, and no end-sealing was applied. It was speculated that end-sealing could have blocked the air from flowing out of the wood during compression. The force applied by the press was set to the maximum of 100 kN, that for 75 mm wide samples represented approximately 9.5 MPa. The liquid solution at room temperature was placed in approximately 16 cm x 23 cm aluminum pans, and the samples were maintained under the liquid with metal loads placed over the samples ends (that protruded 20 mm from the back and front of the press plate). Two aluminum bars were also placed submerged at the right and left sides of the wood samples to stop the press at exactly 12 mm thickness. Once the 12 mm thickness was reached, the press was programed to remain in that position for 10 s and then release the pressure. After releasing the pressure, the sample thickness recovered quickly (spring back), but it was allowed to remain submerged a longer time so that the total immersion time was 3 min for all experiments. Reference samples were also submerged 3 min in the solution but without any compression. The total weight gain by the C&E method was measured with a balance after removing the excess liquid from the surfaces by hand with a paper towel (Tork “Basic Papper”, brown, cat. no. 150109).

The comparison between H&C and C&E was included in this study to show that under the present experimental conditions neither of the methods resulted in considerably higher solution uptake than the other. Based on this comparison, the H&C method was selected for further characterization. In order to be conservative, the comparison probably overestimated the C&E uptake per unit of area. It was assumed that all solution absorption due to spring back occurred through the top wood area that was compressed by the press plate, thus meaning that absorption due to spring back through the bottom wood surface in contact with the container floor was assumed negligible. Based on this criterion, the absorption area was calculated as the wood width multiplied by the plate length = 75·140 mm2. Theory

The basic principle behind the proposed method is the theory of ideal gases. The theory of ideal gases postulates that the pressure of a gas is equal to R·n·T/V, where R is the ideal gas constant, n is the number of gas molecules, T is the absolute temperature, and V is the gas volume. Consequently, if the temperature reduces because the wood is cooled down or the volume increases because the wood springs back, then there will be a proportional reduction of pressure inside the wood that can produce suction of an external solution. Based on this theory, solution uptake in the H&C method is expected to be directly proportionally to the treatment temperature and reduce gradually with time as the wood cools down. RESULTS AND DISCUSSION

Figure 3 shows an example of the H&C process implemented with Scots pine side boards initially at 120 °C and submerged in rapeseed oil methyl ester (RME) at approximately 20 °C. The figure shows the initial CT-scanned density distribution of a wood sample (top image) and subsequently the differences with other CT-scanned density distributions taken at 5, 10, 15, and 29 min during the H&C process. The lighter areas represent the penetration front, where it can be observed that for scots pine side



boards the penetration front developed relatively uniformly below the wood surfaces, with some areas of course absorbing more RME than others. The core of the samples also showed a higher tendency to absorb solution, arguably because of internal tensions developed during the process. It is speculated that internal stresses may have been caused by swelling of the external wood surfaces in contact with the treatment solution, but this could not be detected with the CT-Scanner. The CT-scanner showed however that internal checks developed in some pieces when water was used as model of treatment solution, but internal checks were never observed during rapeseed oil or RME absorption.

Fig. 3. Initial CT-image and density differences after 5, 10, 15, and 29 min of RME absorption

The fact that some wood areas absorb more liquid than others may be a

disadvantage or an advantage if more protection is desired in areas with higher liquid permeability. This also makes the process more difficult to predict. Figure 4 is presented as an example of the variability observed among individual samples for the particular case of RME absorption. Similar variability among individual samples was also observed for rapeseed oil and water absorption. In the figure the solution uptake was converted to L/m2, which is a customary unit for commercial coatings. The conversion from grams to



liter was based on the average densities at 20 °C reported in Table 1, while the sample external area was calculated as 2·10·(75+18) mm2, based on the sample width (75 mm), thickness (18 mm), and 10 mm length in the longitudinal direction set by the CT-scanner parameters.

RME uptake at 20 °C RME uptake at 90 °C

RME uptake at 120 °C RME uptake at 150 °C Fig. 4. Experimental average and standard deviation of six matched samples H&C treated in RME at 20, 90, 120, and 150 °C

The average solution uptake measured in g/m2 and the standard deviation for

selected impregnation times are reported in Table 2. Each average and standard deviation represents six matched samples, with the

exception of the rapeseed oil at 120 ºC where only three matched samples were included. The first set of three samples was discarded because the oil level reduced below the wood surface during the H&C treatment.

It was evident from the data that variability between samples was high, but there were general trends that could be described mathematically. It was observed that for a fixed treatment temperature, the solution uptake rate reduced gradually with the time, while for a fixed treatment time, the solution uptake rate increases gradually with the temperature. For this study the observed solution uptake trends were described as the product of two power functions of temperature and time,

Uptake = (a ∙ ( T - T0 )m + b ) ∙ tn (1)

where T is the treatment temperature (°C), T0 is the room temperature (20 °C), t is the treatment time (min), a is a parameter quantifying the solution uptake due to temperature difference, b is a parameter quantifying the solution uptake due to diffusion at room

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 5 10 15 20 25 30

Mean

Mean +/- SD

Individual pieces

Time (min)

lite

r/m

2

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0 5 10 15 20 25 30

Mean

Mean +/- SD

Individual pieces

Time (min)li

ter/

m2

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0 5 10 15 20 25 30

Mean

Mean +/- SD

Individual pieces

Time (min)

lite

r/m

2

0.000.050.100.150.200.250.300.350.400.450.500.550.600.650.700.750.800.85

0 5 10 15 20 25 30

Mean

Mean +/- SD

Individual pieces

Time (min)

lite

r/m

2



temperature, m is a power factor describing the solution uptake rate as function of temperature, and n is a power factor describing the solution uptake rate as function of the time. Table 2. Average Solution Uptake and Standard Deviation (in parentheses) for Selected Treatment Times

Time Treatment temperature

min 20 °C 90 °C 120 °C 150 °C

RME (g/m2)

5 49.6 (25.2) 91.1 (35.1) 110.5 (15.4) 152.2 (49.6)

10 67.3 (35.5) 147.9 (48.0) 179.7 (19.9) 245.6 (77.1)

15 80.4 (42.9) 193.1 (56.4) 233.5 (25.0) 319.1 (97.4)

20 89.9 (48.8) 231.8 (62.6) 279.7 (29.5) 379.9 (110.7)

25 98.8 (54.3) 266.1 (69.2) 322.6 (34.1) 440.6 (125.6)

29 104.8 (57.6) 292.9 (73.6) 354.4 (37.1) 479.2 (134.9)

Water (g/m2)

5 39.5 (20.7) 82.6 (27.1) 139.8 (89.9) 152.8 (60.6)

10 54.2 (28.7) 109 (37.0) 188 (117.1) 202.6 (78.1)

15 64.9 (35.9) 123.4 (40.8) 217.4 (128.8) 232.6 (87.3)

20 73.8 (40.9) 135.3 (45.6) 241.2 (135.9) 257.2 (91.7)

25 79.5 (43.8) 144 (47.7) 260 (138.7) 277.8 (96.2)

29 84.3 (46.2) 149.6 (49.9) 276.3 (140.3) 296.5 (99.1)

Rapeseed oil (g/m2)

5 30 (6.6) 73.7 (13.7) 78.1 (16.9) 108.5 (43.4)

10 41.9 (8.1) 112.2 (23.1) 110.3 (26.6) 175.7 (60.1)

15 48.9 (9) 143.7 (33.5) 133.1 (30.6) 226 (71)

20 54 (10.5) 168.2 (43.3) 150.4 (36.1) 267.7 (79.3)

25 59.3 (11.2) 186.9 (50.5) 164.8 (39.7) 302.1 (87.7)

29 63.3 (11.1) 197.9 (54.1) 176.7 (41.4) 327.7 (92.9)

The parameters for Eq. 1 were determined by numerical fitting of the

experimental data based on the minimum mean square error method. The rapeseed oil absorption data at 120 °C were not used because it was contradictorily lower than the rapeseed oil data at 90 °C, likely because only three samples were used. One sample for RME at 20 °C and one sample for water at 120 °C were also excluded because they were conspicuously higher than the rest.

The numerical results of the data fitting are reported in Table 3, and the comparison between the mathematical description and the experimental data is shown in figures 5, 6, and 7 for respectively RME, water and rapeseed oil.



Fig. 5. Experimental data and mathematical description of RME absorption (after removing the data for one sample at 20 °C)

Fig. 6. Experimental data and mathematical description of water absorption (after removing the data for one sample at 120 °C)

Fig. 7. Experimental data and mathematical description of rapeseed oil absorption (after removing the data for all samples at 120 °C)

Table 3. Results of Fitting the H&C Absorption Data with Eq. 1 from 0 to 29 min and 20 to 150 ºC

Solution a · 104 b · 102 m n

RME 1.91 1.17 1.14 0.65

Water 0.11 2.44 1.78 0.41

Rapeseed oil 2.62 1.05 1.03 0.60

Table 3 indicates that the power factor m is always higher than 1, meaning that the

effect of the temperature is increasingly important as the treatment temperature increases,

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20 25 30

150C 120C

90C 20C

150C (model) 120C (model)


Time (min)

lite

r /

m2

0.0

0.1

0.2

0.3

0.4

0.5

0.6

20 30 40 50 60 70 80 90 100 110 120 130 140 150

29 min 20 min

10 min 5 min

5 min (model) 10 min (model)


Temperature (C)

lite

r /

m2

0.0

0.1

0.2

0.3

0.4

0 5 10 15 20 25 30

150C 120C

90C 20C



Time (min)

lite

r /

m2

0.0

0.1

0.1

0.2

0.2

0.3

0.3

0.4

0.4

20 30 40 50 60 70 80 90 100 110 120 130 140 150

29 min 20 min

10 min 5 min



Temperature(C)

lite

r /

m2

0.0

0.1

0.2

0.3

0.4

0 5 10 15 20 25 30

150C 90C 20C

150C (model) 90C (model) 20C (model)

Time (min)

lite

r /

m2

0.0

0.1

0.1

0.2

0.2

0.3

0.3

0.4

0.4

20 30 40 50 60 70 80 90 100 110 120 130 140 150

29 min 20 min

10 min 5 min



Temperature(C)

lite

r /

m2



while the power factor n is always lower than 1, meaning that H&C process becomes slower as time proceeds. Table 3 also shows that the fitting parameters are similar between RME and rapeseed oil, which is reasonable as the biodiesel used in this study was derived from rapeseed oil. Parameters are different for water. The power factor m is close to 1 in RME and rapeseed oil, meaning that the solution uptake rate increases proportionally to the increase of the treatment temperature, but it is much higher in water, approximately 1.8. This is explained by the formation of internal checks.

Figure 8 shows the CT-images of three samples that were exposed to H&C water absorption. This example shows that wood treated with water tended to develop internal checks, which in turn were filled with water. This was equivalent to the pattern observed in Fig. 3, where the center of the pieces tended to absorb higher amounts of solution, but in the case of water it resulted in the development of internal checks at some point of the treatment.

Fig. 8. CT-images of water absorption at 120 °C showing internal checks filled with water after 29 min of treatment

The C&E method was tested with six samples and their matched references for

only one treatment solution (RME). The results are reported in Table 4. The solution uptake based on the total area accounts for the diffusion effect through all the surfaces, included the unsealed ends, while the solution uptake through the compressed 140 mm x 75 mm area accounts for the C&E effect. Assuming that the C&E contribution is the difference between compressed and reference samples, then the average solution uptake due to C&E method was 166.1 g/m2. This was equivalent to approximately 12 min at 90 ºC, 9 min at 120 ºC, and 6 min at 150 ºC with the H&C method. The uptake results in the C&E method were in the same order of magnitude as the uptake results in the H&C method with reasonable treatment times; thus the C&E method was not further investigated. The main disadvantage of the C&E method, in the opinion of the authors, was that the samples only recovered half of the initial thickness by immediate spring back, leaving the other half to likely recover in service when the wood is exposed to moisture.



Table 4. Average Results and Standard Deviation (in parentheses) of the C&E Method Implemented with RME

Parameter Reference C&E method Difference

Press plate length (mm) -- 140

Press pressure (MPa) -- 9.5

Holding time after compression (s) -- 10

Total treatment time (min) 10 10

Board length (mm) 180 180

Board width (mm) 74.68 (0.46) 74.8 (0.39)

Board thickness (mm) 17.92 (0.12) 18.0 (0.12)

Thickness after spring back (mm) -- 15.2 (0.90)

Initial samples weight (g) 130.2 (17.4) 132.1 (15.7)

Final sample weight (g) 134.2 (17.1) 137.8 (14.0)

Uptake based on the total area (g/m2) 111.8 (36.0) 159.8 (67.0)

Uptake based on the compressed area (g/m2) 384.9 (123.7) 551.0 (232.1) 166.1 (162.2)

CONCLUSIONS 1. Based on Scots pine side boards as model for treated material, and water, rapeseed

oil, and rapeseed oil methyl ester (RME) as models for treatment solutions, this study demonstrates that the heating-and-cooling (H&C) method can be used as an intermediate technology between full volume impregnation and superficial surface coating for depositing a layer of protective chemicals below the external surfaces of wood materials.

2. It was found that absorption rate increases proportionally to the increase of treatment

temperature, but the relationship is not linear as predicted by the theory of ideal gases. The absorption rate is increasingly higher when the temperature increases, thus suggesting the influence of other possible factors such as a reduction of the solution viscosity or an increase in the wood diffusion coefficient.

ACKNOWLEDGMENTS

This project was funded by BioInnovation in partnership with VINNOVA, Formas, Energimyndigheten, and the industry members of TräCentrum Norr. A special thanks to Sveaskog for directly supporting the project and Frans and Carl Kempe Foundation 1984 for supporting a Ph.D. student involved in the project activities. Thanks very much as well to Norra Skogsägarna for providing wood for the project. REFERENCES CITED Alireza, S. M. (2011). Effect of Wood Constituents Oxidation of Unsaturated Fatty Acids,

Licentiate thesis, KTH Royal Institute of Technology, Stockholm, Sweden. Athena (2002). Maintenance, Repair and Replacement for Building Envelope Materials,

AthenaTM Sustainable Materials Institute (www.athenasmi.org/wp-content/uploads/2011/10/2_Maintenance_Repair_And_Replacement.pdf).



Bernat, E., Jordi-Roger, R., Grau, B., Antoni, R., and Rita, P. (2011). "Temperature dependence of density and viscosity of vegetable oils," Biomass & Bioenergy 42(2012),164-171. DOI: 10.1016/j.biombioe.2012.03.007

Blew, J. O. (1948). Treating Wood in Pentachlorophenol Solutions by the Cold-Soaking

Method, Report No. R1445, Forest Products Laboratory, Madison, WI. Crawford, C. G. (1907). The Open-Tank Method for the Treatment of Timber (Circular

101), US Forest Service, Washington, DC. Deka, M., and Saikia, C. N. (2000). “Chemical modification of wood with thermosetting

resin: effect on dimensional stability and strength property,” Bioresour. Technol. 73(2), 179-181. DOI: 10.1016/S0960-8524(99)00167-4

Fry, H. J. (1973). “Compression impregnation of wood veneers,” US Patent # 3950577A. Forest Products Laboratory (2010). Wood Handbook—Wood as an Engineering Material,

General Technical Report FPL-GTR-190, U.S. Department of Agriculture, Forest Products Laboratory, Madison, WI.

Gabrielli, C. P., and Kamke, F. A. (2010). “Phenol–formaldehyde impregnation of densified wood for improved dimensional stability,” Wood Sci. Technol. 44(1), 95-104. DOI: 10.1007/s00226-009-0253-6

Gsöls, I., Rätzsch, M., and Ladner, C. (2003). "Interactions between wood and melamine resins effect on dimensional stability properties and fungal attack," in: Proceedings of

the First European Conference on Wood Modification, Ghent, Belgium, pp. 221-225. Hill, C. A. S. (2006). Wood Modification: Chemical, Thermal and Other Processes, C. V.

Stevens (ed.), John Wiley & Sons, UK. Homan, W. J., and Jorissen, A. J. M. (2004). "Wood modification developments,"

HERON 49(4), 361-386. Hunt, G. M., and Garratt, G. A. (1967). Wood Preservation, 3rd Ed., McGraw-Hill, New

York, NY. Inoue, M., Adachi, K., Tsunoda, K., Rowell, R. M., and Kawai, S. (2008). "A new

procedure for treating wood," Wood Material Science & Engineering 3(1-2), 46-54. DOI: 10.1080/17480270802605495

Kollmann, F. F. P., and Côté, W. A. (1984). Principles of Wood Science and Technology.

Volume I: Solid Wood, Springer-Verlag, Berlin, Germany. Lande, S. (2008). Furfurylation of Wood – Wood Modification by the Use of Furfuryl

Alcohol, Ph.D dissertation, Norwegian University of Life Science, Ås, Norway. Nurmi, A. (1998). Natural Resins as a Potential Wood Protecting Agent (Final Report),

EC’s fourth framework programme, FAIR CT95-0089, 1995-1998. Ormrod, D. J., and Van Dalfsen, B. (1993). Wood Preservation on the Farm (Final

Report), Ministry of Agriculture, Fisheries and Food, British Columbia, Canada. Richardson, B. A. (2003). Wood Preservation (2nd Ed.), Chapman & Hall, London, UK. Ryu, J. Y., Takahashi, M., Imamura, Y., and Sato, T. (1991). “Biological resistance of

phenol-resin treated wood,” Mokuzai Gakkaishi 37(9), 852-858. Sandberg, K., Pousette, A., Karlsson, O., Sundqvist, B. (2013). Fasader i Trä för

Flervåningsbyggnader Jämförelse Mellan Material och Behandlingsmetoder (SP Rapport 2013:21), SP Technical Research Institute of Sweden, Skellefteå, Sweden.

. Takahashi, M., and Imamura, Y. (1990). Biological Resistance of Phenol-Resin Treated

Wood, Doc. No. IRG/WP 3602, International Research Group on Wood Preservation, Stockholm, Sweden.



Tamblyn, N.E. (1985). "Treatment of wood by diffusion," in: Preservation of Timber in

the Tropics, W. P. K. Findlay (ed.), Springer, Amsterdam, The Netherlands, pp. 121-140. DOI: 10.1007/978-94-017-2752-5_6

Tikkurila (2016). Commercial Information from Paint Supplier, Vantaa, Finland, (www.tikkurila.sk/pdfexport/index.phtml%3Fs%3D80.pdf).

Venard, J. K., and Street, R. L. (1975). Elementary Fluid Mechanics (5th ed.), Wiley, New York, NY.

Westin, M., Lande, S., and Schneider, M. (2003). "Furfurylation of wood - Process, properties and commercial production," in: Proceedings of the First European

Conference on Wood Modification, Ghent, Belgium, pp. 289-306. Williams, R. S. (2005). "Weathering of wood," in: Handbook of Wood Chemistry and

Wood Composites, R.M. Rowell (ed.), CRC Press, Boca Raton, FL, pp. 139-185. Article submitted: June 25, 2016; Peer review completed: October 6, 2016; Revised version received and accepted: October 30, 2016; Published: November 14, 2016. DOI: 10.15376/biores.12.1.283-295.

Paper VI

Proceedings IRG Annual Meeting (ISSN 2000-8953) © 2018 The International Research Group on Wood Protection

IRG/WP 18-40849

THE INTERNATIONAL RESEARCH GROUP ON WOOD PROTECTION

Section 4 Processes and properties

Thermally modified wood treated with methacrylate

Olov Karlsson(s)*

Olena Myronycheva

Dennis Jones

Dick Sandberg

Wood Science and Engineering, Luleå University of Technology,

Forskargatan 1

S-93197 Skellefteå, Sweden,

Diego Elustondo

Jägarvägen 11, S-93140 Skellefteå, Sweden

Paper prepared for the IRG49 Scientific Conference on Wood Protection

Johannesburg, South Africa

29 April – 3 May 2018

IRG SECRETARIAT

Box 5604

SE-114 86 Stockholm

Sweden

www.irg-wp.com

Disclaimer The opinions expressed in this document are those of the author(s) and

are not necessarily the opinions or policy of the IRG Organization.

Thermally modified timber treated with methacrylate

Olov Karlsson1, Olena Myronycheva

2, Dennis Jones

2, Diego Elustondo

3, Dick Sandberg

2

1Wood Science and Engineering, Luleå University of Technology, Forskargatan 1, SE-93197 Skellefteå, Sweden,

[email protected]

2 Wood Science and Engineering, Luleå University of Technology, Forskargatan 1, SE-93197 Skellefteå, Sweden

3 Jägarvägen 11, SE-93140 Skellefteå, Sweden

ABSTRACT

Thermally modified timber (TMT) from Scots pine sapwood similar to Thermo-D quality was

impregnated with methacrylate resin by the hot-and-cold method and subsequently cured at

elevated temperatures. The results showed that methacrylate resin could be used to reduce

colouring of painted TMT wood during accelerated weathering probably by hindering the

migration of extractives. The resin itself did not reduce greying of the unpainted wood. Hardness

was only slightly improved by treatment with the resin probably due to a higher density of the

material. Formation of blisters occurred but was reduced by treatment with the resin. Resistance

to mould growth by a mixture of Aureobasidium pullulans, Cladosporium cladosporioides,

Aspergillus versicolor, Penicillium purpurogenum was performed by applying EN-15457:2014.

Treatment with methacrylic resin hindered the colonisation of the three last mould fungi.

Keywords: TMT, Scots pine, methacrylic resin, mould, bulking, extractives.

1. INTRODUCTION

Thermal modified timber (TMT) is in many ways ideal for use in out-door conditions and its

durability could be compared with other common preservative or modification methods as long

as it is not exposed to extended periods of water or in ground contact. Mould growth is to a great

extent related to the moisture in wood and will lead to undesired colour changes and a lower

quality of wood. Leaching or migration of extractives (native or formed in the

modification/drying process) during production and end-use may lead to undesired colour

changes of wooden facades. Such behaviour could influence the ability to attract or repel

colonisation of microorganisms on wood (Karlsson et al. 2014, 2017). In this paper we study the

influence of treatment of thermally modified wood from Scots pine (Pinus sylvestris) sapwood

with methacrylic resin against a series of properties including leaching of extractives through a

painted surface as well as colonisation by moulds. Previous results have shown that a hot and

cold process in which a heated wood is immersed in a cold liquor could be used for impregnation

of wood with vegetable oil derivates (Elustondo et al. 2017) and a similar approach was applied

for impregnation with methacrylic resin (MA) followed by curing at elevated temperatures.

2. MATERIAL AND METHODS

Scots pine was kiln dried to a moisture content (MC) of 10-15% and thermally modified under

saturated steam conditions in an autoclave at 170°C only adjusting to keep constant climate

(Dagbro et al. 2010). Samples containing sapwood were carefully sawn (130*74*18 mm) and

conditioned at 20°C and RH 65%. Samples were dried in oven at 103°C and directly transferred

into a metal net basket in a 5°C solution of methacrylic resin and accelerator (Cactus Juice,

Turntex) and kept under liquid surface by application of some weights at the top of samples (Fig.

1). The vessel was equipped with a magnetic stirrer to facilitate heat and mass transfer of liquid

into the sample. Samples were taken up after 30 min at a temperature of ca. 12°C and excess of

liquid was wiped off with paper. Samples were heated standing on their cross-cut ends at 70°C

for one hour and then 90°C for one hour in a ventilated oven to reduce smoke formation during

the hardening process. During this period, any resin that migrated out of the board during the

heating was collected on the plate on which the sample is standing on. It could be added that by

touching the hardened samples, the feeling of a wooden (and not plastic) surface was apparent

and a droplet of water applied on MA treated wood was absorbed rather quickly into the

material. Separate experiments were performed by mixing either acetic acid, furfural or dried

methanol extract from the thermally modified wood with the resin, but these substances did not

seem to reduce the hardening rate of the resin. Samples were dried until constant weight at

103°C and stored at 20°C and RH 65%.

Figure 1: Hot and cold batch of methacrylate and samples of wood.

Samples were brush painted with white acrylate paint: by primer (Tinova primer exterior,

AkzoNobel) and two top coats (One super tech, Akzo Nobel) according to manufacturer’s

guidance. Samples were also brush painted twice with white polyurethane paint (two-component,

perfection AkzoNobel). The samples were allowed to dry in air as described by the manufacturer

and then conditioned at 20°C and RH 65%. Dimensions of samples were measured with a caliper

and used for density calculations. Brinell hardness was measured essentially following EN 1534

by using Zwick Roell ZwickiLine 2.5 TS universal testing machine (Neyses et al. 2016).

Accelerated weathering test was performed according to Q-Lab corporation with UV lamps

operating at 340 nm according to ISO 927-6; condensation 24 h at 45°C followed by UV-

irradiance of 0.89W/m2 at 60°C for 150 min and spraying for 30 minutes (7L/min distributed

over 8 nozzles reaching 48 samples) the irradiation and spraying was repeated 48 times and

samples were then conditioned for 24 h at 45°C. The whole cycle was repeated until the end of

the experiment, after which the samples were taken out, conditioned at 20°C and RH 65% and

studied using colorimeter (Chroma meter CR-410, Konica Minolta). L*, a* and b* coordinates

were recorded and ΔE was calculated as ΔE=((ΔL*)2+(Δa*)

2+(Δb*)

2)-1

. Gloss was studied at 65°

using glossmeter (ZGM 1110, Zehntner testing instruments). Pictures of wood samples were

taken using Iphone 4s. The ability of the MA-resin to hinder colonisation by fungi was studied

by soaking the filter paper in the resin heat for 30 min. at 103°C followed by inoculation of 0.3

ml of a mixture of fungi (Aureobasidium pullulans, Cladosporium cladosporioides, Aspergillus

versicolor, Penicillium purpurogenum) on filter paper (00K) applying EN-15457:2014.

3. RESULTS AND DISCUSSIONS

Mass and dimensions of boards samples were measured before and after treatment with the

methacrylate resin (MA) (Table 1). Mass increase was found to be slightly larger for untreated

than thermally modified wood. The density was fairly similar for the untreated material after

treatment with MA, probably due to losses during the curing. Swelling of the TMT-material

(2.4%) was observed by the treatment with the resin (Rowell and Ellis 2007). This could be due

to its penetration into cell wall of TMT favoured by a less hygroscopic wood which more readily

interacted with the organic resin than the untreated pine.

Table 1: Properties of pine sapwood (PS) and thermally modified wood (TMT) treated with methacrylate

(MA) resin.

Specimen Mass [m] Volume [10

-4m

2] Dry density [kg/m

3] Hardness [HB]

PS

PS+MA

TMT

76.5

86.8

76.4

1.66

1.66

1.703

462

521

453

1.03

1.17

0.88

TMT+MA 84.1 1.744 483 0.79

Hardness of the boards was studied and found to be slightly lower for the thermally modified

samples than the untreated samples (Table 1). Any increase in hardness of TMT when treated

with methacrylic resin may be related to the density of the material, which was higher in the

resin treated material (Table 1). Inspection of the impact on the wood created during the

hardness test showed that finer edges were produced for TMT treated with MA, indicating a less

brittle material was formed by the treatment with the resin.

Board samples were painted with white acrylic as well as with white two-component

polyurethane paint followed by exposure to accelerated weathering conditions according to ISO

927-6. It could be seen that after 4 weeks of exposure, the gloss was practically unchanged for

the TMT and untreated pine samples painted with PUR paint, whereas the gloss of acrylic paint

decreased at an observing angle of 65 degrees (Fig. 2b).

Figure 2: Change in a) colour (ΔE) and b) gloss (65o) during accelerated weathering.

Changes of colour was estimated as ΔE and can be seen Fig. 2a. Treatment with methacrylate

resin seems not to have an effect of colour of TMT and untreated painted samples painted with

PUR when exposed to the accelerated weather conditions. Boards that had not been treated with

MA-resin and painted with acrylic paint obtained a loss in colour, but not those that had been

treated with the resin as well as for untreated wood (Fig. 2). This could be due to presence of

coloured water-soluble material in TMT (Karlsson et al. 2014) that migrates through the more

porous acrylic coating during the wet exposure conditions. Both the unpainted boards that had

been treated with MA-resin and the ones without this treatment started to grey, thereby changing

the ΔE. Greying was more apparent in earlywood than latewood (Fig. 2). Blisters were formed

-40

-30

-20

-10

0

10

20

a. ΔE

MA Without

-15

-10

-5

0

5

10

b. Gloss (%)

MA Without

both in samples treated with the MA-resin as well as the one that was not treated, but at a lower

extent for samples treated resin (Fig. 3). Formation of blisters was not observed for the painted

samples.

a. b.

c. d.

Figure 3: Accelerated weathering of sapwood of pine (PS) and thermally modified timber (TMT) treated

with methacrylic resin (MA): a) PS, b) PS+MA, c) TMT, d) TMT+MA.

Finally, we studied whether the methacrylate resin could have an influence on stability of

cellulosic material against mould attack using filter paper that had been soaked in the MA-resin

and heated in an oven (Fig 4). The filter paper absorbed 2.5 times its weight of resin but most of

it evaporated during heating in oven and only 10% remained after heating for 40 min at 103°C.

As could be seen, the sample treated with resin was not attacked after exposure for three days to

mould fungi in contrast to what is observed for the untreated filter paper. Mould fungi

Cladosporium cladosporioides, Aspergillus versicolor, Penicillium purpurogenum could be

identified after the test. This indicates that methacrylic resin could be efficient in reducing the

mould growth of wooden panels without severely influencing the appearance of the wood

structure.

4. CONCLUSIONS

The results from the test showed that methacrylate resin could be used to reduce colouring of

painted TMT wood during accelerated weathering probably by hindering migration of

extractives. The resin itself did not improve greying of the unpainted wood. Hardness was only

somewhat improved by treatment with the resin probably due to a higher density of the material.

Formation of blisters occurred but was reduced by treatment with the resin. Resistance to mould

could be largely improved, however, for outdoor conditions the resin may be protected from

degradation reactions.

Fig. 4. Filter paper treated without (1) and with methacrylic resin (7) after mould test.

Acknowledgements

Financial support from the Swedish Research Council for Environment, Agricultural Sciences

and Spatial Planning (FORMAS), project “Fungal growth on modified wood-based products

under sub-arctic conditions 2017-00419”, is gratefully acknowledged. Carl-Johan Stenwall,

Uteträ AB, is acknowledge for fruitful discussions.

5. REFERENCES

Dagbro, O, Torniainen, P, Karlsson, O, Morén, T (2010): Colour responses from wood,

thermally modified in superheated steam and pressurized steam atmospheres. Wood Material

Science & Engineering, 5(3), 211-219.

Elustondo, D, Myronycheva, O, Sundqvist, B, Karlsson, O (2017): Localized Wood Surface

Modification: Part I: Method Characterization. BioResources, 12(1), 283-295.

Rowell, R M, Ellis, W D (1978): Determination of dimensional stabilization of wood using the

water-soak method. Wood Fiber Science, 2, 104-111.

Karlsson O, Myronycheva O, Sehlstedt-Persson M, Öhman M, Sandberg D (2017): Multivariate

modeling of mould growth in relation to extractives in dried Scots pine sapwood. Proceedings

IRG Annual Meeting, IRG/WP 17-20629, 6 pp.

Karlsson, O, Dagbro, O, Granlund, K (2014): Soluble degradation products in thermally

modified wood. Final COST Action FP0904 conference, March 4 19-21 Skellefteå, Sweden, pp.

16-17.

Neyses, B, Rautkari, L, Yamamoto, A, Sandberg, D (2016): Pretreatment with sodium silicate,

sodium hydroxide, ionic liquids or methylacrylate resin to reduce set-recovery and increase the

hardness of surface-densified Scots pine. iForest: Biogeosciences and Forestry, 10(5), 857-864.

Paper VII


Kim et al. (2020). “Methacrylic resin wood protection,” BioResources 15(3), 7018-7033. 7018

Methacrylic Resin for Protection of Wood from Discoloration by Mould Growth and Weathering

Injeong Kim,* Olov Karlsson, Olena Myronycheva, Dennis Jones, and Dick Sandberg

The discoloration of uncoated wood surfaces in both outdoor and indoor use in non-heated spaces has become an increasing problem in European timber constructions due to the use of less toxic substances for protection and also changes in outdoor climate conditions, necessitating the use of protective coatings. To investigate the effect of methyl methacrylic (MMA) resin for the protection of wood from discoloration and mould growth, resin-treated wood surfaces were studied in a laboratory-scale mould test, as well as in an outdoor weathering test. Non-modified Scots pine and Norway spruce were used, and some of the test materials were also thermally modified. The resin suppressed mould growth for the laboratory-scale experiments. The protective effect was considerably reduced for outdoor tests. The MA resin did not effectively prevent the wood from greying from ultraviolet (UV) radiation exposure; there was some protective effect detected on the pine. The Fourier transform infrared (FTIR) spectra of weathered specimens showed a reduction of lignin-associated absorption bands for all treatments, which corresponded to the UV degradation and greying of the wood surface. It is suggested that MMA resin may provide adequate protection against mould growth on wood without direct exposure to rain and sunshine (e.g., attics, basements, etc).

Keywords: Methacrylic resin; Scots pine; Norway spruce; Thermally modified timber; FTIR; Mould

Contact information: Wood Science and Engineering, Luleå University of Technology, Forskargatan 1, 931

87 Skellefteå, Sweden; *Corresponding author: [email protected]

INTRODUCTION

Wood can be a very durable material against different types of degradation, but its

durability depends upon the circumstances, such as the environment, where the wood is in

use, the type of wood, and how it has been protected. In constructions where wood-based

building materials are not exposed to extreme chemical or thermal environments, wood

durability is mainly related to biological, physical, and photochemical degradation

mechanisms on its exposed surfaces or its interior, which can reduce its function and/or

appearance. High durability is also important as it is often related to lowered maintenance

costs.

The biological degradation of wood in northern Europe is mainly due to wood-

destroying fungi (e.g., white-rot, brown-rot and soft-rot fungi), which can not only degrade

the structure of wood and have a significant effect on functional properties, but also

influence the appearance of the wood material by discoloration. Degradation may also

occur with discoloring or mould fungi, which as their name suggest, result in deterioration

in appearance without impacting mechanical performance. However, the presence of

discoloring and mould fungi may be a prelude to subsequent attack by wood-destroying

fungi (Brischke and Unger 2017). Mould fungi can also induce allergic reactions in humans

and, as a consequence, it is important to prevent mould growth in buildings. Mould growth



is usually related to the accessibility of nutrients and moisture to these microorganisms, as

well as ventilation and condensation (Brischke and Unger 2017). Mould can grow indoors,

especially in areas that have limited heating and airflow, such as inside the attic and

basement, as well as outside where the wood may be exposed to higher levels of humidity

or rain.

The conventional method to prevent fungal growth is to apply a toxic preservative,

such as a copper-based agent. However, due to their toxicity to humans and to the

environment, many of these preservatives are banned or need to be substituted. Alternative

wood modifications and biocides based on an organic system have been proposed to

substitute conventional preservatives. To prevent mould, compounds, such as 3-iodo-2-

propynyl butylcarbamate (IPBC), diiodomethyl-p-tolylsulfone (e.g., Amical 48), and

examples of isothiazolinones are used. However, these compounds are only capable of

protecting the wood for a short period (Schultz et al. 2007); hence, a better solution for the

protection of wood against mould is needed.

Besides mould growth, the discoloration of wood can occur due to different abiotic

factors. During outdoor exposure, wood is degraded and discolored by a combination of

ultraviolet (UV) radiation, water or moisture, and elevated temperatures. This results in

internal stresses and degradation mainly at the surfaces of the wood, resulting in reductions

of the material’s service life (Forest Products Laboratory 1975). Because of the limited

ability of radiation to penetrate into the wood, the effect of the UV degradation is limited

to a region approx. 2.5 mm beneath the wood’s surface; also, wood erosion is slow, i.e., 5

to 12 mm per 100 years (Browne and Simonson 1957; Feist and Mraz 1978).

When wood absorbs light, its color change depends on the components that interact

with the light. All major components of wood are sensitive to UV radiation; cellulose

strongly absorbs radiation between 200 to 300 nm, while lignin and polyphenols (i.e.,

extractives) strongly absorb light below 200 nm and at 280 nm. Lignin is responsible for

80 to 95% of the total light absorption coefficient for wood (Norrström 1969; Gierer and

Lin 1972; Hon 1981; Hon and Minemura 2001), and its degradation is believed to proceed

rapidly via the formation of radicals that oxidize phenolic units to chromophoric structures,

such as unsaturated and conjugated carbonyl structures (Feist and Hon 1984; Pandey and

Vuorinen 2008). These conjugated chromophoric structures are not stable to further

exposure to UV-radiation. They will be degraded and finally removed from the middle

lamella of the cell wall, resulting in wood surfaces with loosely attached fibres with low

chroma. Therefore, the color starts to change more or less directly when the surface is

exposed to UV radiation, with a grey hue developing as the lignin degradation products are

washed away (Feist and Hon 1984).

This discoloration and/or degradation is typical for uncoated wood surfaces;

however, the same behaviour occurs with thermally modified timber (TMT) surfaces. The

thermal modification process of wood is most frequently used today to give a durable and

dimensionally stable material with an initial brownish color (Jones et al. 2019). The

brownish color is related to the degradation of the hemicelluloses and lignin during the

modification process (Esteves and Pereira 2008), and the color can be varied by the choice

of wood species and process conditions (International ThermoWood Association 2003;

Bekhta and Niemz 2005). The chromophores present in TMT before and after UV radiation

exposure are not known in detail, but a study (Tolvaj et al. 2014) showed that thermal

modification processes reduced the red color discoloration during photodegradation, whilst

limiting changes in yellow color associated with lignin degradation. It was concluded that

thermal modification provided a slight improvement in photodegradative stabilization.



TMT has been found to be more durable against biological degradation than

unmodified timber, possibly due to the reduced levels of easily accessible nutrients. The

presence of quickly metabolised sugars and methanol-soluble extractives has been shown

to be lower in TMT processed under open superheated conditions versus TMT processed

under closed and saturated steam conditions (Karlsson et al. 2012a, 2012b; Sidorova et al.

2018).

Because lignin and polyphenols are the wood constituents most susceptible to

photodegradation, attempts to increase the UV-stability of lignin in wood have been

accomplished with benzophenones (Kiguchi and Evans 1998; Olsson et al. 2012) and with

UV-absorbers, such as hindered amine light stabilizers (HALS), in conjunction with

Tinuvin® lignin radical stabilisers (Schaller and Rogez 2007; Forsthuber et al. 2013).

Reactions using benzoyl chloride (Pandey and Chandrashekar 2006; Pandey and Srinivas

2013), vinyl benzoate (Jebrane et al. 2009), acetylation of wood, and treatment with certain

chromium compounds (Evans 2009) can also reduce the discoloration of weathered wood.

Iron-based salts with or without oxidants can influence the color of TMT during weathering

(Karlsson and Morén 2010). The addition of pigments into an oil coating of TMT can

improve its color stability (Jämsä et al. 2000). Silicon-based sol-gel (Mahltig et al. 2010)

and TiO2 and ZnO nanoparticles can improve color stability, but the latter can also lead to

increased cracking of the waterborne polyacrylate film coating during both outdoor and

artificial exposure (Miklečić et al. 2015). Methyl methacrylate (MMA) is known to

increase the hardness of wood; it has also been suggested that it has a stabilizing effect on

wood degradation by UV radiation (Feist et al. 1991).

MMA is a monomer that is produced on a large scale for the production of

poly(methyl methacrylate). It has been used to improve the dimensional stability of wood;

however, the effect of MMA resin is limited because the monomer does not easily penetrate

into the cell walls (Ellis and O’Dell 1999). Some studies on the application of MMA to

wood have shown an improvement of the dimensional stability, surface characteristics, and

density (Koubaa et al. 2012; Li et al. 2012).

MMA has a distinctive acrid odour that makes it difficult to handle. Karlsson et al.

(2018) have suggested applying a commercial MMA-based resin to improve the resistance

of wood to mould growth. Colonisation of such microorganisms on surfaces in wood

constructions may not only influence their aesthetic appearance but may also cause health

problem for residents.

The purpose of the present study is to investigate the possibility of applying

commercial MMA-based resin to untreated and thermally-modified wood to improve its

resistance to mould growth and to surface discoloration when exposed outdoors for a

limited period of weather exposure (for example during the construction of a wooden

building). Based on the results of the experiments, examples of the use of the treatment

system in wooden constructions are proposed.

EXPERIMENTAL

Two different tests were performed: an outdoor weathering test with wood

specimens of dimensions of 20 mm × 100 mm × 150 mm (length (L) × width (W) × height

(H)), and an in vitro mould test with wood specimens of dimensions of 45 mm × 22 mm ×

5 mm (L × W × H).



Materials For the in vitro mould test, unmodified and thermally modified Scots pine sapwood

timbers were used. The TMT used was modified in saturated steam at 170 °C; it was

obtained from Thermoplus (Arvidsjaur, Sweden). All specimens were cut to the

dimensions of 45 mm × 22 mm × 5 mm (L × W × H) and were oven-dried before testing.

For the outdoor weathering test, boards of Scots pine (Pinus sylvestris L.) and

Norway spruce (Picea abies (L.) Karst.) with cross-section dimensions of 25 mm × 105

mm and moisture content of 15% were obtained from a local sawmill (Martinsons Trä AB,

Bygdsiljum, Sweden). The boards were cut and planed to the dimensions of 150 mm × 100

mm × 20 mm (L × W × H) and were subsequently conditioned (20 °C and 65% relative

humidity (RH)) until the specimens reached a constant mass. In addition, the thermally

modified timber of Scots pine and Norway spruce, modified by the ThermoWood® process

in superheated steam at 212 °C (Dagbro et al. 2010), were obtained from Heatwood AB

(Hudiksvall, Sweden); these specimens were prepared in the same way as the unmodified

specimens.

A commercial methacrylate resin together with an accelerator (Cactus Juice

Stabilizing Resin) was purchased from Turntex, LLC (San Marcos, TX, USA). Heat-

sensitive accelerator was mixed with the resin (1:50) prior to submersion coating of the

wood specimens. Benzophenone (CAS No. 199-61-9) and malt-extract agar media (CAS

No. 8002-48-0) were purchased from Merck KGaA (Darmstadt, Germany).

Methods In vitro mould test

To observe the effect of the MMA resin treatment on wood, specimens were tested

with respect to mould growth under laboratory conditions with pure fungal cultures.

An oven-dried specimen was submerged in the resin for 10 min, which was

followed by heating at 103 °C for 1 h. The weight percentage gain (WPG) of the specimens

due to the resin treatment is shown in Table 1. Prior to placement on petri dish, all

specimens were sterilized by steam (120 °C, 152 kPa) for 20 min.

Spore suspensions (ca. 106 spores/mL) with four different fungi were produced:

Aureobasidium pullulans (De Bary) G. Arnaud, Cladosporium cladosporioides (Fresen.)

G.A. de Vries, Aspergillus niger Van Tieghem, and Penicillium commune Thom; all were

obtained from the culture collection at the division of Wood Science and Engineering,

Luleå University of Technology, Luleå, Sweden.

The specimens treated with MMA resin were paired with the untreated specimens

in the same Petri dish. The suspension produced was then sprayed onto the surfaces of each

Petri dish in the amount of 0.3 mL using an airbrush with a pressure of 2 bar. The Petri

dishes were then covered with a lid and stored in a climate chamber (24 °C and 90% RH).

The visual evaluation of fungal growth was assessed after 7 days of exposure. All

specimens in the Petri dishes were stored for two months.

Outdoor weathering test

The cross-sections of all the specimens were end-sealed with a commercial

polyurethane-based sealant, Sikaflex®, obtained from AT Connection (Sika Sverige,

Sweden). Half of the specimens from the untreated and the thermally modified timber

groups were submerged in the resin for 10 min. Unmodified spruce specimens treated with

a resin that contained 3% benzophenone were included. After immersion, the excess resin

was wiped gently from the surfaces; then the specimens were heated to cure the resin



standing on one of its cross-section surfaces in a ventilated oven at 70 °C for 1 h, followed

by 90 °C heating for 1 h (Karlsson et al. 2018). In this way, the exudation of resin from the

board and the evaporation of volatile compounds in specimens could be suppressed during

the curing treatment. The WPG due to the MMA resin after curing is shown in Table 1.

Each treatment group contained five replicates. The different test groups are described in

Table 2.

Table 1. Oven-dried Density of Specimens Before the Resin Treatment and WPG After the Curing of the MMA resin

Test Group No. of Specimens Oven-dried Density (kg/m3)*

WPG of MMA resin (%)*

Mould Test

Scots pine sapwood 5 468 (15.10) 10.5 (1.40)

Scots pine TMT 5 415 (31.11) 5.0 (0.52)

Weathering Test

Scots pine sapwood 5 507 (22.1) 11.4 (1.16)

Scots pine TMT 5 443 (49.1) 4.6 (1.40)

Spruce 5 470 (63.0) 9.4 (0.28)

Spruce TMT 5 403 (25.4) 5.2 (0.36)

* Standard deviation in parenthesis

Table 2. Test Groups for the Outdoor Weathering Test

Test Group Species Modification Treatment

P Pine sapwood Unmodified Untreated

PM Pine sapwood Unmodified Resin

S Spruce Unmodified Untreated

SM Spruce Unmodified Resin

SMU Spruce Unmodified Resin + benzophenone

TP Pine ThermoWood Untreated

TM Pine ThermoWood Resin

TS Spruce ThermoWood Untreated

TSM Spruce ThermoWood Resin

TSU Spruce ThermoWood Resin + benzophenone

The specimens were weighed and placed outdoors on racks facing south at a 45°

inclination at Luleå University of Technology (64.744453° N, 20.955569° E). The outdoor

test was performed between 11 July 2018 and 1 October 2018. On 4 September (55 days),

the specimens were placed vertically in the northern direction in the rack. The growth of

the mould was observed and visually assessed.

Color measurement

The exposed surface color of all the specimens from the outdoor weathering test

was measured with a colorimeter (Chroma meter CR-410; Konica Minolta, Tokyo, Japan).

The L*, a*, and b* color coordinates were measured from the centre of the board with a

measuring head diameter of 5 cm. The hue angle (h (°)) and chroma (C*) were computed

from the coordinates. The h value represents a visual sensation according to which a surface

appears to be similar to a perceived color; it is computed as:



h = arctan(b*/a*). (1)

The C* value is defined as the strength of the hue and it is computed as:

C* =√(𝑎∗)2 + (𝑏∗)2 . (2)

The overall color difference (ΔE) was calculated as:

ΔE = √(𝐿∗)2 + (𝑎∗)2 + (𝑏∗)2 . (3)

Weather data during the test

The weather data collected during the experiment is shown in Fig. 1., and is from a

weather station at Balderskolan, Skellefteå (64.7519° N, 20.9433° E) (Balderväder 2020).

The intensity of sun light was determined each day as the sum of 48 measurements during

24 h.

Fig. 1. The amount of sunshine and rain during the outdoor weathering test

Fourier transform infrared spectra (FTIR) spectra

To study the stability of the MMA resin and the changes in wood components as a

result of weathering, the attenuated total reflection (ATR), which used a diamond crystal,

was determined with a PerkinElmer Frontier™ FTIR spectrometer (Waltham, MA, USA).

The IR spectra over the wavenumber range of 4000 to 450 cm-1 were collected using 4

scans with a resolution of 4 cm-1. All spectra were normalised with respect to the strongest

absorption band (1023 to 1030 cm-1). Small scrapes were randomly taken from sites on the

surface of the exposed and the unexposed specimens, and the scrapes were tested applying

a similar pressure.

Statistical analysis

To determine the significance of difference between treatment groups and

measurements of different dates within groups, all results were determined with analysis

of variance (ANOVA). The significance of difference between mean values was

determined at 5% difference (p = 0.95).

0

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RESULTS AND DISCUSSION In vitro Mould Test

The investigation of fungal growth of pure cultures in a conditioning chamber

revealed that the resin treatment appreciably delayed fungal colonisation of the surfaces

(Table 3). In general, the growth of Aspergillus niger was greater than that of the other

fungal species. This observation could have been due to the method of inoculation.

According to previous studies (Myronycheva et al. 2018, 2019), the spraying technique

chosen in the present study combined with the freely available water provided a favourable

environment for Aspergillus niger, but less for other fungal species, such as Aureobasidium

pullulans and Cladosporium cladosporioides, or “dry adhesion” fungus-like Penicillium

commune, which commonly grow on timber outdoors (Fig. 2).

Table 3. Proportion of Specimens Where the Surfaces were Covered with Mould One Week After Inoculation

Scots Pine Sapwood (%)* TMT (%)*

Untreated 100 (0) 78.2 (23.82)

MMA resin-treated 19.7 (28.53) 6.5 (9.97)

* Standard deviation in parentheses

Fig. 2. Example of Petri dishes showing specimens sprayed with a mixture of moulds and evaluated after one week of conditioning: a) pine treated with MMA resin, b) untreated pine, c) thermally modified pine treated with MMA resin, and d) thermally modified pine (the red arrows indicate less mould in the surrounding nutrient medium)

Mould Growth on Specimens During Outdoor Weathering The specimens after the weathering test are shown in Figs. 3 and 4. Mould growth

was observed on unmodified pine and spruce after 41 days of exposure; however,

specimens treated with MMA resin showed no visible mould until 75 days of exposure,

which was two weeks after turning the specimens towards the north. The mould growth

can be seen in Figs. 3 and 4 as a darker area on the exposed untreated pine, and to some

extent, on the spruce, whereas the MMA-treated surfaces were less affected.

a

b

c

d



Fig. 3. Scots pine specimens before weathering (unexposed) and after 82 days of outdoor weathering. In each picture, weathered specimens are shown in the upper row (a, c, e, and g) and the same unexposed specimens are shown in the bottom row (b, d, f, and h) of untreated pine (a and b), pine treated with MMA resin (c and d), thermally modified pine (e and f), and thermally modified pine treated with MMA resin (g and h)

Fig. 4. MMA resin-treated spruce specimens (top (a and c)) and untreated spruce specimens (bottom (b and d)) of unmodified (left (a and b) and thermally modified (right (c and d)) after 82 days of outdoor weathering

Color Changes Due to Outdoor Weathering Test All the specimens showed greying after outdoor weathering (Figs. 3 and 4). This

observation was most evident for the TMT. The greatest change in color was observed after

the first seven days of exposure, even though the weather conditions for first few weeks

were fairly similar (Fig. 1). The rapid initial change followed by gradual change in color

was also observed in previous studies (Nzokou and Kamdem 2006; Tomak et al. 2018).

The MMA resin treatment provided no protection against greying (Figs. 3 and 4).

The color coordinate data agreed with the visual observation. The change in color

coordinates of the unmodified wood surfaces as a result of outdoor weathering is shown in



Fig. 5. The color change indicators (ΔE, ΔL*, and ΔC*) of specimens were more intensive

during the first than the succeeding weeks of exposure. The lightness (ΔL*) of exposed

specimens decreased during the exposure until the second week when it started to stabilize.

The darkening of the surface could be related to the formation of new oxidized lignin

degradation products (Hon and Minemura 2001). The intensity or depth of color can be

expressed as chroma (C*), which increased the first week of exposure but mostly decreased

thereafter until the final weeks of exposure. Hue (h) decreased with exposure period until

about 20 days of exposure which indicates that the color of the specimens becomes less

yellow but more red. From the 20th day to the 41st day of exposure, hue stabilized and then

started to decrease again on further exposure. The reason for this behaviour was not easily

understood but was attributed to a sudden change in the weather conditions and was not

further evaluated. Addition of a few percent benzophenone did not improve the color

stability of the MMA-treated specimens. In general, the resin-treated pine showed fewer

changes in the color coordinates than the other specimens. The lightness and total

discoloration are known to be affected by the number of water-soluble extractives that can

act as antioxidants and can protect the wood from photodegradation (Nzokou and Kamdem

2006); additionally, the resin seemed to create a physical barrier that prevented water-

soluble extractives from being washed out of the wood during outdoor exposure. However,

the effect of the MMA resin was not as evident in the spruce samples as in the pine samples

(Fig. 5).

Fig. 5. Color differences over time for pine (P), spruce (S), pine treated with MMA resin (PM), spruce treated with MMA resin (SM), and spruce treated with MMA resin and benzophenone (SMU) during outdoor weathering; a) overall color difference (ΔE), b) lightness (L*), c) chroma (C*), and d) hue angle (h)

0

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ΔE

Time of exposure (days)

P S PM SM SMU

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L*


P S PM SM SMU

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Time for exposure (days)P S PM SM SMU

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h (°)

Time for exposure (days)P S PM SM SMU

(a)

(d) (c)

(b)



The color coordinates of the thermally modified specimens changed in general

more than those of the unmodified specimens during the outdoor exposure (Fig. 6). The

thermally modified specimens showed initial rapid change of color similar to that of

unmodified specimens, followed by a continuous change in both chroma and lightness

during the test period. This tendency was also shown in previous studies (Srinivas and

Pandey 2012; Shen et al. 2016). After 55 days, the values of the coordinates were even

more stable, which may have been due to a decrease in sunlight exposure after the

specimens were turned towards the north. The fairly rapid increase in hue in contrast to the

more even decrease in hue for unmodified pine indicated a shift towards a more yellow

color, which could mean that the chromophore systems in the thermally modified

specimens were fairly quickly converted to other types, perhaps less extended ones, but

still somewhat color intensive (Figs. 3 and 4). The intensity of the color decreased,

however, with increasing exposure, which can be seen as a decrease in the chroma of the

thermally modified specimens.

Fig. 6. Color coordinates of thermally modified samples treated with or without MMA resin and exposed outdoors: pine (TP), spruce (TS), pine treated with MMA resin (TPM), spruce treated with MMA resin (TSM), and spruce treated with MMA resin and benzophenone (TSU); a) overall color difference (ΔE), b) lightness (L*), c) chroma (C*), and d) hue angle (h)

In general, thermally modified pine (TP) and spruce (TS) showed less change in

color coordinates, while the wood treated with MMA resin showed a considerably larger

color change, which could be at least partly due to the somewhat darker initial color of the

MMA-treated specimens. The specimens treated with the resin were darker than the

untreated specimens before the test (Figs. 3f and h). For example, the average L* value of

TP was 48 whereas TPM (TS treated with resin and benzophenone) was 41. However, after

0

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Time of exposure (days)TP TS TPM TSM TSU

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h (°)

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(a)

(d)

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(c)



outdoor exposure the color of the surfaces did not show any visible differences, and the

measured color coordinate values did not show any appreciable difference. However, it

seemed clear that the resin did not improve the color stability of thermally treated

specimens (Fig. 6).

FTIR Spectroscopy Study

The FTIR spectra of pine specimens before and after outdoor weathering tests are

shown in Fig. 7. The specimens treated with the resin showed an absorption around 1718

to 1719 cm-1, but the intensity of this peak decreased after weathering, which might indicate

the removal of resin (Figs. 7b and d). The intensity of lignin-associated bands, such as at

1596 cm-1, 1511 cm-1, 1268 cm-1, and 1220 cm-1 (Faix 1991, 1992; Pandey and Pitman

2003; Müller et al. 2009), either decreased or disappeared after outdoor exposure, which

may explain the greyed surfaces of the exposed specimens (Figs. 3 and 4).

Fig. 7. ATR-FTIR spectra of the surfaces of specimens before (black) and after (red) outdoor weathering for 82 days: a) pine untreated (P), b) pine treated with MMA resin (PM), c) thermally modified pine (TP), and d) thermally modified pine treated with MMA resin (TPM)

1722

2000 1800 1600 1400 1200 1000

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1719

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1723

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1718

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Wavenumber (cm-1)

(b)

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(a)



The extent of the absorption at 1722 to 1723 cm-1 was lower for the thermally

modified than for the unmodified specimens after outdoor exposure (Figs. 7a and c).

Hydrolysis of the acetyl group bond is believed to be caused by thermal modification

(Nuopponen et al. 2005), but the FTIR absorption due to ester groups in wood components

still seemed to be present, although to a lesser extent (Fig. 7c). Oxidised and water-soluble

lignin photodegradation products have been found to absorb in this region (Bejo et al.

2019). However, they contributed to a minor extent in the exposed specimens.

CONCLUSIONS 1. The methacrylate resin improved the mould resistance but not the discoloration of

wood during outdoor exposure, which was presumably because the methacrylate resin

was removed by the exposure to outdoor weathering.

2. To prevent mould growth and greying the resin treatment could be applied to wood

surfaces that are not directly exposed to sunshine and rain, but where the RH is high,

like under a roof or in an attic.

ACKNOWLEDGMENTS

Financial support from the Swedish Research Council for Environment,

Agricultural Sciences and Spatial Planning (FORMAS), project “Fungal growth on modified wood-based products under sub-arctic conditions 2017-00419,” is gratefully acknowledged.

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Article submitted: December 29, 2019; Peer review completed: March 28, 2020; Revised

version received and accepted: July 21, 2020; Published: July 24, 2020.

DOI: 10.15376/biores.15.3.7018-7033

Paper VIII

Article

Application of GRAS Compounds for the Control ofMould Growth on Scots Pine Sapwood Surfaces:Multivariate Modelling of Mould Grade

Olena Myronycheva 1 , Faksawat Poohphajai 1,2, Margot Sehlstedt-Persson 1, Tommy Vikberg 3,Olov Karlsson 1,* , Helmut Junge 4 and Dick Sandberg 1

1 Wood Science and Engineering, Luleå University of Technology, 50115 Skellefteå, Sweden2 InnoRenew CoE, 6310 Izola, Slovenia3 RISE Research Institutes of Sweden, 50115 Skellefteå, Sweden4 ABiTEP GmbH, D-12489 Berlin, Germany* Correspondence: [email protected]; Tel.: +46-910-585-705

Received: 30 May 2019; Accepted: 19 August 2019; Published: 21 August 2019��

Abstract: Research Highlights: In this study, the Generally Recognised as Safe (GRAS) compounds wereapplied in order to study mould-fungi growth on dried Scots pine sapwood. Background and Objectives:The transition to the use of more sustainable wood-material may be possible by applying GRAScompounds that can control and prevent contamination by primary colonising mould fungi. Materialsand Methods: Kiln-dried sawn timber was treated with three different GRAS compounds, and differentfungal inoculation methods applied in order to investigate differences in the development of fungalcommunities. Results: Substances based on potassium silicate significantly reduced fungal growthand mould contamination on the studied wood surfaces. By combining wood-surface treatmentswith GRAS compounds, fungal-area size as predictors and mould grade as response, a partial leastsquares (PLS) model that makes it possible to predict mould grade on wood surfaces was developed.The PLS model is a key component in the development of a smart grading-systems equipped bye.g. high-speed digital cameras for the early detection of fungal attack on wood surfaces in differentapplications. However, the measurements based on chemical characterisation should be the nextstep to take in order significantly to enhance the model and increase the range of robust applications.In the current study, a multivariate model describing the influence of each fungal-covering area onmould grade was presented for the first time.

Keywords: bacteria; potassium silicate; N-Alkylbensyldimethylammonium chloride; wood; fungi;mould area; PLS modeling

1. Introduction

The negative load of fossil- and petrol-based biocidal substances on the environment and thereduction of volatile organic compounds (VOCs) emitted while using solvent-based coatings, requirethe introduction of new concepts for wood-surface treatment for outdoor and indoor applications [1,2].A significant problem when wood is used and the moisture level is high, e.g. in outdoor conditions,is biodegradation caused primarily by fungi which have a unique nutrient-sensing mechanism [3]which makes the task of wood protection difficult. Environmental processes that affect the wood inoutdoor use result in aesthetic concerns and increased maintenance costs [4]. Fungal bio-aerosolsaccumulating on outdoor building-material surfaces may also have a significant impact on indoorbiological contamination, especially under sub-arctic conditions [5].

Toxigenic moulds, particularly ascomycetes, are ordinary food and indoor inhabitants associatedwith everyday human activity and they may present a health risk to humans. For example, information

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from public health monitoring programs related to secondary metabolites of toxigenic moulds knownas mycotoxins has raised concern about health aspects [6]. Urinary multi-biomarker analysis amongadults in Sweden revealed that “common and concurrent exposure to more than one toxin was foundin 69% of the studied population” [7]. Nowadays, mycotoxin studies face many masked mycotoxins,and the analytical methods are quite complex due to the polydiversity of compounds’ properties.When modern regulations require “minimal fungi” levels and at the same time environmentallyfriendly solutions for bio-based materials such as wood and other related products.

Wood in the living tree is a durable material with an active defence mechanism includingextractives enrichment [8–11]. The processing of a log to sawn timber was initially developed tomeet the need for a cheap and easily available building material, and the sawmill drying procedurewas aimed to lower the sawn-timber moisture content to reduce the board weight and the risk offungal attack. Already during felling, the initial immunity of the tree starts to decrease, and the woodresistance is reduced during the further processing of the tree for use in wood-based products. Previousstudies have shown, for example, that industrial wood drying leads to the migration of hydrophilicand hydrophobic substances towards the wood surface, resulting in the creation of a nutrient gradientin the sawn timber [8,9]. Despite the enrichment of extractives, the accessibility of the wood surfaceto mould attack may be greater after industrial kiln drying than in e.g. sawn timber seasoned in theopen air [10]. The enormous biodiversity and short life cycles of fungi also emphasises other issuesinvolved in wood protection. The application of traditional copper-based wood preservatives has ledto the adaptation of fungi to these compounds and the appearance of copper-tolerant rot fungi [9].Colonising mould fungi such as Aspergillus sp. and Penicillium sp. can early grow and sporulate on acopper-impregnated wood surface, but the fungi gave different growth-patterns [12]. These differencesare addressed in the present study.

Those fungi that are most economically significant and deleterious, related to food spoilage,mycotoxin production and bio-deterioration head the list of fungi under observation by the authorities.Attention has been paid to Paecilomyces spp. for several years due to its feared negative impact onthe human body. In the present study, these fungal species (all the fungi were from the same orderEurotiales and all cosmopolitans [13]) were selected for the study because of their dominance in thesimultaneous tests and because it was planned to model the growth of the selected fungi and todevelop a method to predict fungal growth after surface treatments with Generally Recognised as Safe(GRAS) compounds.

The standard fungal test uses the mould grade index in the assessment of the treatment and mostof the models for the prediction of fungal and mould growth on wooden surfaces are based on moistconditions. There is, however, no reliable information about the interactions between mould fungi onwooden surfaces under different test conditions. In a previous study [12], the mould-area index wasintroduced for the assessment of mould growth on wooden surfaces, but more information about theinfluence of Ascomycete genera on wood degradation is required in order to better understand thebiological phenomenon.

The concept of GRAS compounds used by the Food and Drug Administration (FDA) in the USAhas been said by qualified experts to guarantee the use of adequately safe substances. This could be a“green alternative” to be applied as a wood-surface treatment to prevent biological attack by harmfulmicroorganisms [14,15]. The GRAS database includes more than 370 active GRAS substances that canbe applied in a “smart way” for wood protection without any threat to humans or the environment.For example, a study of post-harvest treatment of fruits to prevent microbiological disease successfullyreported an effective control of fungal growth [16]. GRAS compounds can be grouped by their action:biocontrol agents, natural antimicrobials, decontaminating agents and physical barriers [16].

This study has investigated the efficiency of treatments using selected GRAS compounds oforganic and inorganic origin to restrict mould-fungi growth on wood surfaces of industrial kiln-driedScots pine sapwood. The primary objective was to establish criteria for modelling and predicting

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mould growth for those fungi and surface treatments using two inoculation methods and moisturechanges during the test by applying selected ascomycete fungal cultures.

2. Materials and Methods

2.1. Preparation of Wood Specimens

Five Scots pine (Pinus sylvestris L.) sideboards with cross-sectional dimensions of 30 × 200 mm anda length of 4 m were randomly selected from a sawmill production in northern Sweden (Martinsonssawmill, Kroksjön, N 64◦ 42.3113′, E 20◦ 54.4597′). The trees from which the boards originated werewinter felled, i.e. the temperature was considerably below 0 ◦C, and the boards were deeply frozenuntil they reached the kiln-drying stage. The boards were single-stacked and dried in a progressivekiln at a wet-bulb temperature of 52 ◦C and a maximum dry-bulb temperature of 67 ◦C. Ten clear woodspecimens, i.e. free from defects and knots, with dimensions of 30 × 50 × 100 mm, were taken from eachboard so that they contained only sapwood, which was verified by a heartwood reagent containingsodium nitrite and sulfanilic acid. Two of these specimens were used for density and moisture content(MC) determinations, according to EN 13183-1 [17], the other eight were used for mould tests and werecoded according to the GRAS treatment applied before the mould test (Table 1). The mean MC of theboards after kiln-drying was 15.5% ± 0.4%, and the mean density at this MC was 548 ± 12 kg/m3.

Table 1. Coding of specimens and description of the Generally Recognised as Safe (GRAS) treatmentsused in the study.

Code Active Substance for Surface Treatment Original Concentration Concentration Used

R Reference

B Bacteria Bacillus amyloliquefaciens(trade company ABITEP, Germany) 25 bln spores/mL 1%

S Silicon, potassium, natural parts of plantsand water (trade company SIOO, Sweden)

1st potassium silicate ca. 32%2nd polymerizable

alkoxylane100%

G N-Alkylbensyldimethylammonium chloride(trade company JAPE, Sweden that) 80 g/L 20%

It was five board in total A, B, C, D and E. The coding was used accordingly (see Figure 1).The eight specimens from each board for the mould test were divided into two groups: set#1 for direct,and set#2 for indirect inoculation method (Figure 1). The surfaces used for the fungal and mould testswere either the tangential surfaces in the direction of bark-side of the log (bark-side specimens) orthe opposite tangential surface in the direction of the pith (pith-side specimens). All specimens weresterilised in steam before the GRAS treatment.

In the case of treatments B and G, the stock solution of the GRAS compound at the originalconcentration was diluted with water whereas, for treatment S, it was used as delivered. Treatments Band G were deposited by spraying evenly on the bark-side and pith-side surfaces of each specimenby using an airbrush with the consumption of 0.20–0.25 L/m2. Treatment S was used as follows:a first treatment was sprayed evenly on the bark-side and pith-side surfaces of each specimen usingan airbrush with a consumption rate of 6–8 L/m2, and the boards were allowed to dry for 2 hours.A second treatment with GRAS was applied in the same way but with a consumption of 10–12 L/m2,and allowed to dry for 4 hours, than a third coating with first GRAS was applied to all surfaces of thespecimens with a consumption of 8–2.12 L/m2, and dried.

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Figure 1. The preparation of specimens from each of the five sideboards, and an example of labelling according to one of the boards: the first letter indicates the board from which the specimens were cut (A), the second letter indicates the GRAS treatment (see Table 1), and the third letter indicates the inoculation method: D indicates direct inoculation, and I indicates indirect inoculation.

In the case of treatments B and G, the stock solution of the GRAS compound at the original concentration was diluted with water whereas, for treatment S, it was used as delivered. Treatments B and G were deposited by spraying evenly on the bark-side and pith-side surfaces of each specimen by using an airbrush with the consumption of 0.20–0.25 L/m². Treatment S was used as follows: a first treatment was sprayed evenly on the bark-side and pith-side surfaces of each specimen using an airbrush with a consumption rate of 6–8 L/m2, and the boards were allowed to dry for 2 hours. A second treatment with GRAS was applied in the same way but with a consumption of 10–12 L/m2, and allowed to dry for 4 hours, than a third coating with first GRAS was applied to all surfaces of the specimens with a consumption of 8–2.12 L/m2, and dried.

2.2. Mould Test

For the direct inoculation method, a mixture of spore suspension (ca. 106–107 spores/ml) from Aspergillus niger van Tieghem, Penicillium commune Thom, C., and Paecilomyces variotii Bainier (Culture collection, Division of Wood Science and Engineering, Luleå University of Technology) was sprayed onto the bark- and pith-side surfaces of each specimen in an amount of 0.4 mL using an airbrush attached to a mini-compressor with a pressure regulator giving a working pressure of 2 bar, and the samples were hung-up in plastic twines in a closed plastic container with a base area of 40 × 42 cm2, and a height of 60 cm with a bath containing one litre of water at the bottom to maintain a high humidity.

For the indirect inoculation method, the samples were hung-up in the same way as in the direct incubation method. Five Petri dishes of each mould species Aspergillus niger van Tieghem, Penicillium commune Thom, C., and Paecilomyces variotii Bainier were randomly placed the hanging specimens at the bottom of the box (Figure 2). All the sub-cultures of fungi were incubated at 24 ± 2°C until good sporulation was achieved after seven days of growth. The choice of fungi was related to the dominant fungal species detected in simultaneous internal laboratory tests of Scots pine sapwood. A detailed description of the inoculation methods can be found in [12]. The choice of the two inoculation methods was related to the differences in processes of wood colonisation of fungi from the same order of Eurotiales.

Figure 1. The preparation of specimens from each of the five sideboards, and an example of labellingaccording to one of the boards: the first letter indicates the board from which the specimens werecut (A), the second letter indicates the GRAS treatment (see Table 1), and the third letter indicates theinoculation method: D indicates direct inoculation, and I indicates indirect inoculation.

2.2. Mould Test

For the direct inoculation method, a mixture of spore suspension (ca. 106–107 spores/ml) fromAspergillus niger van Tieghem, Penicillium commune Thom, C., and Paecilomyces variotii Bainier (Culturecollection, Division of Wood Science and Engineering, Luleå University of Technology) was sprayedonto the bark- and pith-side surfaces of each specimen in an amount of 0.4 mL using an airbrushattached to a mini-compressor with a pressure regulator giving a working pressure of 2 bar, and thesamples were hung-up in plastic twines in a closed plastic container with a base area of 40 × 42 cm2,and a height of 60 cm with a bath containing one litre of water at the bottom to maintain a high humidity.

For the indirect inoculation method, the samples were hung-up in the same way as in the directincubation method. Five Petri dishes of each mould species Aspergillus niger van Tieghem, Penicilliumcommune Thom, C., and Paecilomyces variotii Bainier were randomly placed the hanging specimensat the bottom of the box (Figure 2). All the sub-cultures of fungi were incubated at 24 ± 2 ◦C untilgood sporulation was achieved after seven days of growth. The choice of fungi was related to thedominant fungal species detected in simultaneous internal laboratory tests of Scots pine sapwood.A detailed description of the inoculation methods can be found in [12]. The choice of the two inoculationmethods was related to the differences in processes of wood colonisation of fungi from the same orderof Eurotiales.

The containers were placed in a climate chamber, and were incubated in darkness at a temperatureof 25 ± 2 ◦C and a relative humidity (RH) of 95%–100% for the first three days, and at 25 ± 2 ◦C and90% ± 3% RH for a further 25 days. The temperature and RH in the climate chamber were recordedby a logger, type AAC-2 (Intab, Sweden). The higher RH for the first three days was to give a betterwetting of the samples, and to promote a faster spore germination. Before the test, the containers andthe climate chamber were washed with 99% ethanol solution and then with 5% hydrogen peroxidesolution in order to achieve an aseptic environment.

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The containers were placed in a climate chamber, and were incubated in darkness at a temperature of 25 ± 2 °C and a relative humidity (RH) of 95%–100% for the first three days, and at 25 ± 2 °C and 90% ± 3% RH for a further 25 days. The temperature and RH in the climate chamber were recorded by a logger, type AAC-2 (Intab, Sweden). The higher RH for the first three days was to give a better wetting of the samples, and to promote a faster spore germination. Before the test, the containers and the climate chamber were washed with 99% ethanol solution and then with 5% hydrogen peroxide solution in order to achieve an aseptic environment.

Figure 2. Containers for the mould tests with hanging specimens, and Petri dishes of each mould species (right).

2.3. Mould Assessment

The grade scale used in the mould-growth assessment is presented in Table 2. The scale was modified compared to that published in standard EN 16492:2014 [18] by adding grade 4 for 51%–80%, and grade 5 for more than 80% mould growth. Examples of mould growth on wood surfaces representing the six mould grades 0–5 are shown in Figure 3.

Table 2. Grades for assessment of mould growth on wood surfaces.

Grade Description

0 No visable mould growth

1 Initial fungal growth consisting of scattered hyphae on the surface: less than 5% of the studied surface is covered by mould

2 Still scattered growth, but more apparent than in 1. Conidiophores may have started to

develop: 6% to 20% mould growth

3 Patchily distributed, massive growth, and hyphae with developed conidiophores: 21% to 50% growth

4 Heavy growth over the entire surface: 51% to 80% mould growth

5 Very heavy growth: more than 80% mould growth

After 28 days, the incubated specimens were removed from the climate chamber and weighed. The mould growth on the wood surfaces was evaluated by visual inspection and graded by two persons. The mould growth was graded by person A by the naked eyes in a laboratory environment,

Figure 2. Containers for the mould tests with hanging specimens, and Petri dishes of each mouldspecies (right).

2.3. Mould Assessment

The grade scale used in the mould-growth assessment is presented in Table 2. The scale wasmodified compared to that published in standard EN 16492:2014 [18] by adding grade 4 for 51%–80%,and grade 5 for more than 80% mould growth. Examples of mould growth on wood surfacesrepresenting the six mould grades 0–5 are shown in Figure 3.

Table 2. Grades for assessment of mould growth on wood surfaces.

Grade Description

0 No visable mould growth

1 Initial fungal growth consisting of scattered hyphae on the surface: less than 5% of thestudied surface is covered by mould

2 Still scattered growth, but more apparent than in 1. Conidiophores may have started todevelop: 6% to 20% mould growth

3 Patchily distributed, massive growth, and hyphae with developed conidiophores:21% to 50% growth

4 Heavy growth over the entire surface: 51% to 80% mould growth5 Very heavy growth: more than 80% mould growth

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whereas person B graded the growth from images at 1× magnification where the ImageJ software [19] was used for the detection of mould growth. The individual mould species detected on the wood surfaces were identified with the optical microscope to classify the morphological characteristics of each fungus.

Figure 3. The six grades of mould growth on wood surfaces.

After assessment of mould growth, the specimens were dried in an oven at a temperature of 103 °C for 24 hours to determine the MC. The coefficient of variation (CV in %) was used to describe the scatter of the data around the mean value. The variables used for the univariate and the multivariate analysis (MVA) are described in Table 3.

The data set for statistical analysis represented in the Table S1.Univariate statistical analysis (descriptive statistics, analysis of variance (ANOVA) and post hoc tests for analysis of variance) was performed in IBM SPSS Statistics 20.0 (Armonk, NY, USA). Principal component analysis (PCA), soft independent modelling by class analogy (SIMCA) and partial least squares discriminant analysis

Figure 3. The six grades of mould growth on wood surfaces.

After 28 days, the incubated specimens were removed from the climate chamber and weighed.The mould growth on the wood surfaces was evaluated by visual inspection and graded by twopersons. The mould growth was graded by person A by the naked eyes in a laboratory environment,

Forests 2019, 10, 714 6 of 16

whereas person B graded the growth from images at 1×magnification where the ImageJ software [19]was used for the detection of mould growth. The individual mould species detected on the woodsurfaces were identified with the optical microscope to classify the morphological characteristics ofeach fungus.

After assessment of mould growth, the specimens were dried in an oven at a temperature of103 ◦C for 24 hours to determine the MC. The coefficient of variation (CV in %) was used to describe thescatter of the data around the mean value. The variables used for the univariate and the multivariateanalysis (MVA) are described in Table 3.

The data set for statistical analysis represented in the Table S1. Univariate statistical analysis(descriptive statistics, analysis of variance (ANOVA) and post hoc tests for analysis of variance) wasperformed in IBM SPSS Statistics 20.0 (Armonk, NY, USA). Principal component analysis (PCA),soft independent modelling by class analogy (SIMCA) and partial least squares discriminant analysis(PLS-DA) were applied for a global overview, pattern recognition and display of relationships betweenvariables. Partial least squares regression (PLS) was used to model how the GRAS treatments influencedthe mould growth on the Scots pine sapwood surfaces. The multivariate analysis (MVA) was performedin SIMCA 14.0 (Umetrics, Umeå, Sweden).

Table 3. Description of the variables used in the univariate and multivariate analysis.

Variable Range

Boards A, B, C, D, E 1–5

Method of inoculation 1-indirect0-direct

GRAS treatment

1-R2-B3-S4-G

Surface 0-bark side1-pith side

Mould grade 0–5

Density 548 ± 12 kg/m3

The initial moisture content of the board before the fungal test 15.5% ± 0.4%

Moisture content increase 12.1% ± 1.5%

Area covered by fungus Aspergillus niger 0–87.3%

Area covered by fungus Penicillium commune 0–91.9%

Area covered by fungus Paecilomyces variotii 0–80.9%

3. Results

The local moisture content (MC) of a wood surface is one of the most important factors affectingmould-fungi spore germination, colony development and sporulation, and further spreading overthe environment [13]. Table 4 shows variation in the MC between treatments of wood surfaces andinoculation methods. The one-way ANOVA test for the indirect method of inoculation showed nodifference between treatments (B, S, G and reference R, see Table 1) regarding MC changes during thetest, but direct spraying of spore suspension resulted in a significantly lower variable MC increase fortreatment with bacteria B compared to reference material R.

Multiple comparisons using Tukey´s range test [20] was performed to analyse the influence of thedifferent treatments on mould growth, i.e. within the inoculation-method groups, between methodsof inoculation, and on bark-side and pith-side surfaces (see Tables S2 and S3). The correspondingp-values are presented in Table S2 in the supplemented material. It was not possible to show any

Forests 2019, 10, 714 7 of 16

influence of the type of treatments within the indirect inoculation group, but there were differencesbetween the treatments within the direct inoculation group.

There were only slight differences in MC between the different treatments after the mould test.There was no significant difference in the MC increase between the methods of inoculation for

groups R, B, and S, but the MC increase differed between inoculation methods for group G (Table S3 inthe supplementary material).

Table 4. The moisture content (MC) of specimens after 30 days fungal test, and the percentage increasefrom the initial MC (Mean + standard deviation (SD)).

GRASTreatment MC after the Test (%) MC Increase during the Test (%)

Indirect Fungal-Inoculation Method

R 27.3 ± 0.8 11.8 ± 1.2B 28.0 ± 2.8 12.5 ± 2.6S 27.7 ± 0.8 11.8 ± 1.0G 28.9 ± 2.2 13.4 ± 2.0

Average 27.4 ± 1.2 11.8 ± 1.0

Direct Fungal-Inoculation Method

R 27.8 ± 1.1 12.3 ± 0.8B 26.4 ± 1.0 * 10.9 ± 0.8 *S 28.1 ± 1.0 12.6 ± 0.8G 27.0 ± 1.0 11.5 ± 0.7

Average 27.9 ± 1.9 12.4 ± 1.9

* significant difference between GRAS treatments according to one way ANOVA, p < 0.05.

The variance of the fungal area and mould grade on different GRAS-treated wooden surfacesis shown in Table 5. The most effective GRAS treatment preventing mould growth was the SIOOtreatment (Group S) in both the indirect and direct inoculation methods with average mould grades of1.9 and 1.8, respectively.

The mould area was smallest for Aspergillus niger in the indirect inoculation method, but in generalit was smallest for Paecilomyces variotii (Table 5). The G treatment gave a mould area for indirectinoculation with Aspergillus sp. greater than that with the other treatments and for the reference sample,but the variation (CV) was quite high. Univariate analysis performed to statistically access differencesbetween the treatments showed little influence of the GRAS treatment on mould area.

Tukey test was used to obtain differences in mould area between the treatments within theinoculation methods. Analysis of the indirect method of inoculation revealed no influence of B treatmenton the Aspergillus niger fungal area and the R group differed only from the G group (p = 0.008).There were clear similarities in the GRAS treatments, where groups S, R and B were similar with regardto the area of Aspergillus sp. but differed from G (p = 0.001). For the Penicillium commune fungal area,it can be concluded that the samples can be divided into two groups by their similarities (Table 5).The first group contains the R and B groups and the second the S and G groups. With the Paecilomycesvariotii species the R group differed from the S group but was similar to the B and G groups and the Sgroup was found to be distinct from R and B groups but similar to the G group.

Mould growth was characterised by the mould grade index, which does not relate directly tothe mould type but includes the intensity of growth (see Figure 3). Wood surfaces treated with SIOO(group S) had the lowest mould grade index. The R group was similar to the B group, but differedfrom the S and G groups, but the S and G groups differed from each other and other treatments (R, B)also (see Table 5).

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sts

2019

,10,

714

8of

16

Tabl

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The

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Are

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105

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10.6

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204

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25 11 35

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140

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97 82 125

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199

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88 95 93

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Forests 2019, 10, 714 9 of 16

The direct method of inoculation showed significant differences between the treatments (Table 6).According to the Tukey criteria, the fungal areas of Aspergillus niger in the R, B, G groups were similar,but they differed from the S group. With Penicillium commune fungi, the R group differed only from Ggroup. The B and S groups were similar to other treatments. The Paecilomyces variotii fungal area forgroup R was similar to that of group B but different for groups S and G. Groups S and G were similarto group B, but differed from group R. Group B was similar to all the groups. With regard to mouldgrade, the R, B, and G groups were similar, but differences were observed only for the S group. Thus,treatment with SIOO was most effective in preventing the growth of mould under the conditions usedin this study.

It was expected that while drying, the nutrients, especially in the direction of the bark-side ofthe board, migrate towards the surfaces making the surface more attractive for mould growth [6].This was not, however, clearly seen in the data, but direct inoculation of Aspergillus sp. let to adistinct colonisation on the bark side. This means that this fungus had a considerable influence onits metabolism of sugars. With indirect inoculation, other factors could be more influential. The Streatment has a significant influence on hindering the spreading of Aspergillus sp. fungus on bothsides of the specimen. The S-treatment consisted of application of alkaline silicate followed by asilicon-containing aqueous-emulsion, which is thought to seal the wood surface, making it moredifficult for fungi to penetrate the wood and access nutrients.

Regarding the influence of the bark/pith side surface index on mould growth, the highest growth wasexpected where the highest nutrient contents are found, usually on the bark-side surface. However,with Paecilomyces sp. directly inoculated, less mould growth was found on the bark side of the sapwoodafter R, B and G treatment, but not after S treatment although this seemed to be the most efficienttreatment to hinder fungal growth (Table 6). Such a pattern was indicated for the S treatment whichgave the lowest mould development for both methods of inoculation.

The pattern of fungal development on the bark side for R groups of both methods showedno significant difference in mould grade for areas colonised by Aspergillus sp. The fungal areas ofPenicillium sp., Paecilomyces sp. for the bark- and pith side of sapwood surface differed significantly(p = 0.002, 0.005, and 0.031, respectively) with different methods of inoculation. Interestingly, thecomparison of fungal areas in the R group between the inoculation methods revealed a significantdifference for Aspergillus sp. and Penicillium sp. (p = 0.022 and 0.035), but not for Paecilomyces sp.

The B group mould-grade indices for bark/pith-side surfaces were not significantly different fromeach other, and showed the highest mould grade value, and the areas of Aspergillus sp., Penicilliumsp., Paecilomyces sp. in group B significantly differed between the bark-side and pith-side surfaces(p = 0.001, 0.000, and 0.001, respectively). For B group, neither indirect nor direct treatments inoculationgave any significant difference in mould grade, but there was a significant difference for fungal areas ofAspergillus sp., Penicillium sp., Paecilomyces sp. with p = 0.000; 0.000 and 0.004 respectively. A significantdifference in fungal area of pith-side surfaces after indirect and direct inoculation for the B group wasfound only for Paecilomyces sp. with p = 0.037.

With the S group there were no significant differences between bark/pith-side surfaces forAspergillus sp. but there was a distinct difference with Penicillium sp. and Paecilomyces sp. withp = 0.008 and 0.005. With the samples from G group in terms of bark/pith-side surfaces the inoculationmethod had a significant differences only for Aspergillus sp. and Penicillium sp. with p = 0.004 and0.040 respectively. The difference between inoculation methods for bark-side surfaces for group G wassignificant for the area of Penicillium sp. fungus with p = 0.044. For the pith-side surface, a significantdifference between inoculation methods was obtained only for Aspergillus sp. with p = 0.014.

The average in variable mould area showed, in general, that the Aspergillus sp. area when inoculatedindirectly was 3.6 times smaller than with the direct method (see Tables 5 and 6). The area forPenicillium sp. was the opposite of the area in the indirect method being 2.5 times larger than with thedirect method.

Fore

sts

2019

,10,

714

10of

16

Tabl

e6.

The

fung

alar

eaan

dm

ould

grad

eon

diff

eren

tGR

AS

trea

ted

surf

aces

,%.

GR

AS

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tA

sper

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usni

ger

Pen

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lium

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Gra

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Ran

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E)C

V,%

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48.7

466

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11.9

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3(8

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63 48 62

30.3

22.0

38.7

0–68

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.5)

0–68

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2.5)

6.9–

66.7

(11.

6)

89 128

67

12.0

4.2

19.9

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.5)

0–15

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10.5

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7(3

.8)

90 154

43

4.9

5.0

4.8

BBa

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dePi

thsi

de

51.9

62.6

41.3

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27.0

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10.7

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7(1

2.4)

49 33 67

27.3

19.4

35.2

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0–43

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6.5–

60.0

(10.

6)

76 83 67

6.9

3.6

10.2

0–18

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.3)

0–10

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0–18

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106

139

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5.0

5.0

5.0

SBa

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thsi

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4(1

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195

176

216

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15.0

7.9

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.0(5

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0–53

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3.5–

20.5

(3.2

)

139

147

90

0.2

0.4 0

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0(0

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0–2.

0(0

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0

306

223 0

1.8

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1.6

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61.3

69.2

53.3

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4(7

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26 17 32

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42.9

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)

316 0 224

2.3 0 4.6

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22.8

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316 0 224

4.3

4.6

4.00

Ave

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4.0

Forests 2019, 10, 714 11 of 16

The behaviour of Paecilomyces sp. was similar to that of Penicillium sp. showing a 2.6 times highervalue of fungal area after indirect rather than direct inoculation, but a value lower 3 times than thePenicillium sp. area. The mould grade averages was similar with both inoculation methods. The highvariability of the data of all the samples from 0% to 316% (see Tables 5 and 6) makes it difficult to drawrelevant conclusions based on the univariate or comparison of average data. In order to find a possibledeeper relationship between the variables, a multivariate statistical analysis was performed.

By plotting two principal components, it was possible to observe the spreading and groupingpattern in the dataset, but in PC analysis the model performance (R2) was low (see Model 1 in Table 6).Despite the weakness of model 1, it showed the correlation between Aspergillus area and the method ofinoculation (Table 3), supporting the previous observation from Table 5 that for all surface treatments,the coverage area for Aspergillus was higher when the indirect inoculation method was applied.However, with Penicillium sp., the area depended on the type of treatment with a maximum in the Rgroup for both inoculation methods and a minimum in the G group for direct inoculation (Table 5).

A stronger performance of the model was achieved by PLS-DA modelling, but the OPLS-DAclassification was even stronger (Table 7). The critical variables for explaining the model were mouldgrade and area of fungi.

Table 7. Summary of multivariate analysis (MVA).

№ Model PC Obs. R2X R2Y Q2 (cum)

1 PCA 2 80 0.448 −0.01392 PLS-DA 2 80 0.404 0.36 0.319 Treatment class3 OPLS-DA 2 + 1 + 0 80 0.534 0.394 0.336 Treatment class4 PLS 3 80 0.49 0.843 0.75 Y mould grade5 PLS 3 79 0.519 0.847 0.766 Y mould grade

A stronger performance of the model was achieved when Mould grade was used as Y (response)and other variables as X (explanatory variables)— the results of PLS regression (model 4 in Table 6)described in Table 6. However, the R2X was quite low in comparison to the Q value due to high noise.The observation of the DModX plot (the distance of observation in training set to the X model plane orhyperplane) revealed a definite outlier, and its removal marginally improved the PLS model (No 5.)listed in Table 6.

The scope and loading plot of the PLS model No. 5 is presented in Figure 4. It is evident that theAspergillus sp. is highly associated with the indirect method of inoculation (Figure 4a) whereas theareas of Penicillium sp. and Paecilomyces sp. were found to be more influenced by the type of treatment.It is also evident that there are moderate relations between these two fungi (correlation coefficient 0.7),and the Paecilomyces sp. moderately contributed to mould grade development (Figure 4b).

The variables that contributed most to the model or were essential for the projection of VIPwere the areas of Aspergillus sp., Penicillium sp., Paecilomyces sp and the mould treatment method.The regression line for PC3 is presented in Figure 5a. In Figure 5b, residuals from normal and randomdistribution and absence of outliers are demonstrated.

Forests 2019, 10, 714 12 of 16

The scope and loading plot of the PLS model No. 5 is presented in Figure 4. It is evident that the Aspergillus sp. is highly associated with the indirect method of inoculation (Figure 4a) whereas the areas of Penicillium sp. and Paecilomyces sp. were found to be more influenced by the type of treatment. It is also evident that there are moderate relations between these two fungi (correlation coefficient 0.7), and the Paecilomyces sp. moderately contributed to mould grade development (Figure 4b).

(a)

(b)

Figure 4. Partial least squares (PLS) model performance: (a) Score plot and (b) Loading plot withPC1 plotted against PC2. Colours indicate the method of inoculation: blue—indirect inoculation andred—direct inoculation.

Forests 2019, 10, 714 13 of 16

direct inoculation.

The variables that contributed most to the model or were essential for the projection of VIP were

the areas of Aspergillus sp., Penicillium sp., Paecilomyces sp and the mould treatment method. The

regression line for PC3 is presented in Figure 5a. In Figure 5b, residuals from normal and random

distribution and absence of outliers are demonstrated.

(a)

(b)

Figure 5. PLS model diagnostics: (a) observed versus predicted values of Y response (mould grade)

plot and (b) normal probability plot of residuals. Figure 5. PLS model diagnostics: (a) observed versus predicted values of Y response (mould grade)plot and (b) normal probability plot of residuals.

4. Discussion

The present study provides an understanding of the influence of GRAS compounds of organic andinorganic origin in restricting mould fungi growth on dry Scots pine sapwood surfaces. The selectedfungal species were isolated main primary colonisers [13]. Efforts were made to define criteria for

Forests 2019, 10, 714 14 of 16

multivariate modelling and the prediction of mould growth for various GRAS treatments under theinfluence of different inoculation methods.

Between selected GRAS treatments, the most effective treatment was treatment S (potassiumsilicate) which gave the greatest reduction in fungal growth in the direct method of inoculationwith the lowest mould grade. The known fact that potassium silicate has a fungicidal effect viareinforcement of the wood surface and acts as a physical barrier for the fungi improves the propertiesof the material [21–23]. The efficiency of bacterial treatment was lowest and rather stimulated fungalgrowth. This effect is surprising because the biocidal role of Bacillus sp. has been reported in woodprotection [24]. The biocidal action of quaternary ammonium compounds (group G) was found to besmall and seemed not to be useful for mould control. The non-treated reference boards showed highmould grade, but it was lower in the direct method of inoculation than with indirect inoculation due tolow fungal growth on the inner pith surface with a variation of 43%–67%. In general, the growth offungi on inner pith surfaces was lower than on the bark side surface of the samples. This was expectedbecause of the presence of extensive nutrients close to the cambium/bark. In this study, boards weresingle stacked during drying and the movement of nutrients occurred in all directions. Such a nutrientenrichment of the sapwood side influenced fungal growth even after subsequent preservative surfacetreatment [8,9,12]. Noticeable was the great variation in the data within the test results particularlywhen direct inoculation was applied (from 0% to over 300%). The equilibrium moisture content(EMC) for the reference wood sample was calculated to be ca 24.4% in our test conditions according totables [25]. At the end of the test, the MC of all samples was higher than that EMC value but the finalMC was found to have a negligible influence on fungal growth probably because the high moistureconditions are favourable for mould growth.

The method of inoculation influenced the development of Aspergillus sp. growth, the developmentof the fungal area was quite significant in the direct method of inoculation. It agrees with our previousstudy [12], and the necessity of a water reservoir for single dry spores’ germination of Aspergillus sp.was proposed [13]. The Penicillium sp. growth was more related to the type of treatment and was moreintense with the indirect method of inoculation. It is reasonable since the growth of Penicillium sp.and Paecylomyces sp. were related in our study, and previous studies have shown that, for example,Penicillium sp. spores and probably Paecylomyces sp. do not require large amounts of water forgermination and no influence on the growth of the method of inoculation was found [26]. The areasof all fungi in our study influenced the mould grade in the multivariate model the Paecylomyces sp.having most significant influence on the model performance. The three-component PLS model showedquite a strong performance and can predict about 77% of data variation (Q2 = 0.766).

5. Conclusions

This study contributes to the transition to sustainable wood materials by applying GRAScompounds that can control and prevent contamination by primary colonising mould fungi. Substancesbased on potassium silicate could be excellent sustainable substances for wood treatment. By combiningwood surface treatments with the response to a fungal attack, a PLS model makes it possible to predictthe contamination rate of wood. The model may be beneficial in the use of smart sorting systemsequipped with high-speed digital cameras in industrial use. It shows the ability to model such acomplicated phenomenon that standard tests do not provide. However, the measurements basedon chemical characterisation should be the next step to take in order to significantly enhance modelproperties and increase the range of robust applications.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4907/10/9/714/s1:Table S1: Data set used for data analysis in the article; Table S2: The p-value for the significance of moisture contentchanges after test difference between treatments in the indirect and direct method of inoculation, significantdifference p < 0.05; Table S3: The p-value for the significance of difference between treatments in both methods,significant difference p < 0.05.

http://www.mdpi.com/1999-4907/10/9/714/s1

Forests 2019, 10, 714 15 of 16

Author Contributions: Conceptualization, O.M., F.P., M.S.-P., and O.K.; methodology, O.M., M.S.-P., and O.K.;software, D.S.; validation, O.M., F.P., M.S.-P., and O.K., T.V., D.S., H.J.; formal analysis, O.M., F.P.; investigation,O.M., F.P.; resources, T.V., D.S., H.J.; data curation, O.M., F.P., M.S.-P., and O.K.; writing—original draft preparation,O.M., F.P.; writing—review and editing, O.M., F.P., M.S.-P., and O.K., T.V., D.S., H.J.; visualization, O.M..;supervision, M.S.-P., O.K.; project administration, M.S.-P., O.K.; funding acquisition, D.S., O.K.

Funding: This research was funded by the FORMAS (Swedish Research Council for the Environment AgriculturalSciences and Spatial Planning) (FORMAS), project number 419, 2017 and 942-2016-64, 2016.

Acknowledgments: Support from Martinsons Såg AB sawmill, The European Regional Development Fund, theCounty Administration of Västerbotten, the municipality of Skellefteå, and WoodCentreNorth is much appreciatedand acknowledged. Also, the authors express appreciation for the support of COST Action FP1407. The EuropeanCommission is gratefully acknowledged for funding the InnoRenew CoE project (#Grant Agreement 739574)under the Horizon2020 Widespread-Teaming program.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. European Union. European Parliament and of the Council Directive 2004/42/CE on the Limitation of Emissionsof Volatile Organic Compounds Due to the Use of Organic Solvents in Certain Paints and Varnishes and VehicleRefinishing Products and Amending Directive 1999/13/EC; European Union: Brussels, Belgium, 2004.

2. European Union. ECHA Directive 98/8/EC Concerning the Placing of Biocidal Products on the Market; EuropeanUnion: Brussels, Belgium, 2011.

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concurrent mycotoxin exposure among adults in Sweden through urinary multi-biomarker analysis. FoodChem. Toxicol. 2015, 83, 133–139. [CrossRef] [PubMed]

8. Terziev, N. Migration of Low-Molecular Sugars and Nitrogenous Compounds in Pinus sylvestris L. DuringKiln and Air Drying. Holzforsch. Int. J. Biol. Chem. Phys. Technol. Wood 2009, 49, 565–574.

9. Myronycheva, O.; Karlsson, O.; Sehlstedt-Persson, M.; Öhman, M.; Sandberg, D. Distribution of low-molecularlipophilic extractives beneath the surface of air- and kiln-dried Scots pine sapwood boards. PLoS ONE 2018,13, e0204212. [CrossRef] [PubMed]

10. Karlsson, O.; Myronycheva, O.; Sehlstedt-Persson, M.; Öhman, M.; Sandberg, D. Multivariate modeling ofmould growth in relation to extractives in dried Scots pine sapwood. In Proceedings of the 48th Conferenceof the International Research Group on Wood Protection, IRG48, Ghent, Belgium, 4–8 June 2017.

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12. Myronycheva, O.; Sehlstedt-Persson, M.; Karlsson, O.; Sandberg, D. Growth of Mold and Rot Fungi onCopper-impregnated Scots Pine Sapwood: Influence of Planing Depth and Inoculation Pattern. BioResources2018, 13, 8787–8801. [CrossRef]

13. Adnan, O.C.G.; Samson, R.A. Fundamentals of Mold Growth in Indoor Environments and Strategies for HealthyLiving; Wageningen Academic Publishers: Wageningen, The Netherlands, 2011; ISBN 978-90-8686-135-4.

14. U.S. Food and Drug Administration Generally Recognized as Safe (GRAS). Available online: https://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/ (accessed on 16 January 2019).

15. U.S. Food and Drug Administration GRAS Substances (SCOGS) Database. Available online: https://www.fda.gov/food/ingredientspackaginglabeling/gras/scogs/default.htm (accessed on 17 January 2019).

16. Romanazzi, G.; Lichter, A.; Gabler, F.M.; Smilanick, J.L. Recent advances on the use of natural and safealternatives to conventional methods to control postharvest gray mold of table grapes. Postharvest Biol.Technol. 2012, 63, 141–147. [CrossRef]

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18. CEN-European Committee for Standardization. EN 16492:2014 Paints and Varnishes-Evaluation of the SurfaceDisfigurement Caused by Fungi and Algae on Coatings; CEN-European Committee for Standardization: Brussels,Belgium, 2014.

19. Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.;Saalfeld, S.; Schmid, B.; et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012,9, 676–682. [CrossRef] [PubMed]

20. Tukey, J.W. Comparing Individual Means in the Analysis of Variance. Biometrics 1949, 5, 99–114. [CrossRef][PubMed]

21. Rodrigues, F.A.; Datnoff, L.E. Silicon and Plant Diseases; Springer International Publishing: Basel, Switzerland,2015; ISBN 978-3-319-22929-4.

22. Okon, K.E.; Lin, F.; Chen, Y.; Huang, B. Effect of silicone oil heat treatment on the chemical composition,cellulose crystalline structure and contact angle of Chinese parasol wood. Carbohydr. Polym. 2017, 164,179–185. [CrossRef] [PubMed]

23. Lahtela, V.; Kärki, T. Effects of impregnation and heat treatment on the physical and mechanical propertiesof Scots pine (Pinus sylvestris) wood. Wood Mater. Sci. Eng. 2016, 11, 217–227. [CrossRef]

24. Kreber, B.; Morrell, J.J. Ability of Selected Bacterial and Fungal Bioprotectants to Limit Fungal Stain inPonderosa Pine Sapwood. Wood Fiber Sci. 2007, 25, 23–34.

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Paper IX

2020

InnoRenew CoE International Conference2020

Integrating sustainability and health in buildings through renewable materials

InnoRenew CoE Livade 6, 6310 Izola, Slovenia

IRIC2020 Scientific CommitteeAndreja KutnarAnna SandakDavid DeVallanceIztok ŠušteršičJakub SandakMichael BurnardMiklós Krész

innorenew.eu

InnoRenew CoE is funded by the European Commission under Horizon 2020, the EU Framework Programme for Research and

Innovation (H2020 WIDESPREAD-2-Teaming #739574), and by investment funding from the Republic of Slovenia and the

European Regional Development Fund.

INTEGRATING SUSTAINABILITY AND HEALTH IN BUILDINGS THROUGH RENEWABLE MATERIALS

19

The increased concern regarding mould on wood-based building materials has raised demand for sustainable biocidal treatments to protect early contamination during the construction stage of timber buildings. Providing surface protection for all types of wood-based construction elements already at the construction site will reduce the mould-associated risk for not only the construction elements but also for the indoor climate and dwellers at the use-stage of the building. The purpose was to test the protective effect of commercial water-based treatments containing different biocides on single mould fungi growth in pure culture.

Small specimens of Scots pine, sapwood and heartwood, and Norway spruce were treated with four treatments, and a fungal test was performed in 90 mm Petri plates. Two samples (treated and untreated control) placed on the plate with a distance between each other, and between, a fungal inoculum placed. Five pure cultures of fungi species used in the study: Aureobasidium sp., Trichoderma sp., Aspergillus sp., Cladosporium sp., Penicillium sp. The specimens were exposed at the temperature of 24°C and relative humidity of 90%.

The treatments contained biocides, i.e.: treatment 1: tetramethylol acetylenediurea and io-dopropynyl butyl carbamate (IPBC); treatment 2: mixture of several biocides (IPBC, benzisothiazolinone [BIT], methylisothiazolinone [MIT] and 5-chloro-2-methyl-1,2-thiazol-3-one [CMIT/MIT mixture]); treatment 3: IPBC; and treatment 4: mixture of propiconazole and IPBC.

The fungal growth was observed in the untreated samples after four days of incubation; the specimens with treatment 4 attacked after eight days while treatment 1 and 3 had free from fungal mycelia inhibition zone after 22 days of incubation. The moisture content after the test was similar for treated samples in plates with Aureobasidium sp., Aspergillus sp., Cladosporium sp., Penicillium sp. but was significantly higher for Trichoderma sp. The type of wood did not influence fungal growth in comparison to the type of treatment.

Keywords: biocide, wood, surface treatment, fungi, mould

Acknowledgements: First Author gratefully acknowledges receiving funding from programme Bioinnovation (IPOS-DP2) and from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS), the project “Fungal growth on modified wood-based products under subarctic conditions” (2017-00419).

The Influence of Four Commercial Wood-surface Treatments on Mould-fungi Growth in a Pure Culture

Olena S. Myronycheva1, Injeong Kim1, Peter Jacobsson2, Olov Karlsson1, Margot Sehlstedt-Persson1, Dick Sandberg1

1 Luleå University of Technology, Wood Science and Engineering, [email protected], [email protected], [email protected], [email protected], [email protected] Martinsons Byggsystem AB, [email protected]

The Influence of Four Commercial Wood-surface Treatments on

Mould-fungi Growth in a Pure Culture

Olena S. Myronycheva1*, Injeong Kim1, Peter Jacobsson2, Olov Karlsson1, Margot Sehlstedt-Persson1, Dick Sandberg1

1 Luleå University of Technology, Wood Science and Engineering, Forskargatan 1, 931 87 Skelleftea, Sweden,

[email protected], [email protected], [email protected],

[email protected], [email protected] Martinsons Byggsystem AB, Skelleftea, Sweden, [email protected]

*Corresponding author

INTRODUCTION

The aim of our study was testing of the protective effect of commercial

surface coatings containing different biocides on single mould fungi

growth in pure culture.

MATERIALS AND METHODS

Scots pine sapwood, heartwood, and Norway spruce were cut into

25(T)x15(L)x5(R) [mm]. Specimens were treated with four surface

coatings containing biocides, i.e.:

Treatment 1: Pigmented primer with tetramethylol acetylenediurea and

iodopropnyl butyl carbamate (IPBC). Applied average amount of the

coating 0.2 g/g wood.

Treatment 2: Paint with a mixture of three biocides (IPBC,

benzisothiazolinone (BIT), methylisothiazolinone (MIT) and 5-chloro-

2-methyl-1,2-thiazol-3-one (CMIT)/MIT mixture). Applied average

amount of the coating 0.15 g/g wood.

Treatment 3: Paint with IPBC. Applied average amount of the coating

0.01 g/g wood.

Treatment 4: Laqvin Tone contained three biocides 1,2-benzisotiazol-

3(2H)-on, 2-oktyl-2H-isotiazol-3-on, 2-Methyl-4-isothiazolin-3-one.

Applied average amount of the coating 0.14 g/g wood.

Wood samples (treated and untreated control) were placed on the Petri

plate and 5 mm fungal inoculum placed in the centre of plate (Figure

1). Five pure cultures of fungi species were used in the study:

Aureobasidium sp., Trichoderma sp., Aspergillus sp., Cladosporium

sp., Penicillium sp.

The mould growth was estimated as the size of the inhibition zone of

treatments and measured from the nearest border four times and

average value presented in mm (Figure 2).

Inhibition

zone

Figure 1. The example of

specimens position and

inoculation pattern (start of

experiment).

Figure 2. Measurement of

the inhibition zone (red

arrows).

RESULTS

The fungal growth was observed in all three types of untreated

samples already after four days of incubation. In Table 1 size of

inhibition of growth zone of fungus to treated specimens after 22 days of

incubation is shown. Trichoderma sp. followed by Penicillium sp. was

found to be the most resistant to biocides within the tested group of

mould fungi. The specimens coated by treatment 4 were attacked already

after eight days. Treatment 1 and 3 became free from a fungal mycelia

inhibition zone after 22 days of incubation for all types of wood.

Table 1. The average value of the size of the inhibition zone of

treatments (mm).

The moisture content of treated and non treated specimens was similar

for treated samples in plates with Aureobasidium sp., Aspergillus sp.,

Cladosporium sp., Penicillium sp., but was significantly higher for

Trichoderma sp after the test.

Treatment 1 Treatment 2 Treatment 3 Treatment 4

Aureobasidium sp.

Pine Sap 7.17 4.17 7.25 0

Pine Heart 11.08 4.58 8.33 0

Spruce 8.42 5.33 8.08 0

Trichoderma sp.

Pine Sap 0 0 0 0

Pine Heart 0 0 0 0

Spruce 0 0 0 0

Aspergillus sp.

Pine Sap 6.17 3.25 6.33 0

Pine Heart 6.92 2.83 5.08 0

Spruce 6.17 3.17 8.58 0

Cladosporium sp.

Pine Sap 7.58 1.33 2.58 0

Pine Heart 7.50 1.33 1.67 0

Spruce 7.50 1.33 4.42 0

Penicillium sp.

Pine Sap 0.83 1.33 1.75 0

Pine Heart 0.83 0.5 0.08 0

Spruce 1.75 0 0.44 0

CONCLUSIONS

1. The best effect to hinder fungal growth was found for the treatment

1, 2 and 3 whereas treatment 4 performed worst. The ranking in

performance the best is a treatment 1, then treatment 3 followed by

treatment 2.

2. The type of Nordic softwood does not influence general

performance against mould attack under the used controlled

conditions.

3. The moisture content of exposed specimens after the test was

similar in the plates with fungi Aureobasidium sp., Aspergillus sp.,

Cladosporium sp., Penicillium sp. However, significantly higher for

Trichoderma sp.

Paper X

Research ArticleHyperspectral Imaging Surface Analysis for Dried andThermally Modified Wood: An Exploratory Study

Olena Myronycheva , Ekaterina Sidorova, Olle Hagman, Margot Sehlstedt-Persson,Olov Karlsson , and Dick Sandberg

Lulea University of Technology, Wood Science and Engineering, Forskargatan 1, 931 87 Skelleftea, Sweden

Correspondence should be addressed to Olena Myronycheva; [email protected]

Received 25 May 2018; Revised 13 August 2018; Accepted 16 October 2018; Published 14 November 2018

Guest Editor: Ingunn Burud

Copyright © 2018 Olena Myronycheva et al. )is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Naturally seasoned, kiln-dried, and thermally modified wood has been studied by hyperspectral near-infrared imaging between980 and 2500 nm in order to obtain spatial chemical information. Evince software was used to explore, preprocess, and analysespectral data from image pixels and link these data to chemical information via spectral wavelength assignment. A PCA modelshowed that regions with high absorbance were related to extractives with phenolic groups and aliphatic hydrocarbons. )e sharpwavelength band at 2135 nm was found by multivariate analysis to be useful for multivariate calibration. )is peak represents thelargest variation that characterizes the knot area and can be related to areas in wood rich in hydrocarbons and phenol, and it canperhaps be used for future calibration of other wood surfaces. )e discriminant analysis of thermally treated wood showed thestrongest differentiation between the planed and rip-cut wood surfaces and a fairly clear discrimination between the two thermalprocesses. )e wavelength band at 2100 nm showed the greatest difference and may correspond to stretching of C�O-O ofpolymeric acetyl groups, but this requires confirmation by chemical analysis.

1. Introduction

Increased automation in wood-manufacturing processeswould allow a more custom-oriented approach that en-courages companies in the wood industry to move fromtraditional quality grading with high-speed visual inspectioncamera systems (VIS) to more sophisticated systems for thegrading of sawn and further processed timber. )e VISmethods are in some cases not sufficiently precise and ef-ficient because of wood variability and wood-treatmentheterogeneity [1].

Near-infrared spectroscopy (NIR) is a well-knowntechnology for the nondestructive analysis of wood. )eadvantages of the NIR technology are speed and the ability tomeasure a broad range of the chemical and physicalproperties, which make the technology promising for in-dustrial use [2]. )e main challenge for developing an ef-fective predictive model based on NIR data and for itsfurther use in wood-manufacturing processes is, however, to

find the reliable absorption range of interest in a highlyheterogeneous material such as wood and to calibrate themodel for quantitative process parameters assessment [3].Portable NIR sensors use point-based measurements thathave to be developed experimentally, and they will not givea 2-dimensional spatial visualisation of the variation in NIRabsorptions from a surface.)is weakness can, at least partly,be reduced by using a more stationary technique likehyperspectral imaging for accurate data collection andmodel development and relating this information to othersources such as a portable NIR sensor.

Hyperspectral techniques can be used for the develop-ment of models that quantify the heterogenic chemicalcomposition of the wood surface. )e objective of thecurrent study was to use a hyperspectral imaging techniqueand follow the recommendations of Sandak et al. [4] to builda classification model for wood surfaces in the NIR spectralregion. Hyperspectral imaging provides spatially resolvedinformation of the wood surface in the NIR range, and

HindawiJournal of SpectroscopyVolume 2018, Article ID 7423501, 10 pageshttps://doi.org/10.1155/2018/7423501


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https://doi.org/10.1155/2018/7423501

selected wavelengths can be developing calibration modelsfor specific compounds.

Industrial drying, and especially thermal modification,of wood influences the presence and structure of the woodconstituents (cellulose, hemicelluloses, lignin, and extrac-tives), and this may in turn influence the characteristics andconsequently the service life of the woodmaterial in differentproducts. )ere are a number of difficulties in describing thechanges that take place when wood is heated. A range ofchemical reactions take place simultaneously, and sincethese include both endothermic and exothermic reactions,the determination of the onset temperatures for the differentreactions is nearly impossible. )e analysis is furthercomplicated by interactions between reactions in differentconstituents.)is means that the information obtained froman analysis of an isolated derivative of one of the compo-nents can be very different from what actually takes placeinside wood. )ere are not only interactions between thecomponents in the wood but also interactions between thewood and the treatment atmosphere [5]. Figure 1 showsa schematic representation of the changes in wood com-ponents with increasing temperature under humidconditions.

Natural seasoning of sawn timber is the oldest way ofprocessing sawn timber to remove water and increase itsdurability. Compared to drying of industrial timber in a kilnat a temperature far above 20°C, natural seasoning takesa long time and the chemical composition remains close tothe natural composition of the wood [6, 7]. In order toimprove the industrial sawmill process, kiln drying wasdeveloped in the late 1800s to provide a fast and controlleddrying process to reach the target moisture content 6–12%[8, 9]. Below 40°C, it is mainly physical changes that occur inthe wood, such as the emission of water and volatile ex-tractives [10]. Industrial kiln drying at a temperature of ca.70°C is the predominant regime nowadays for Scots pine andNorway spruce [11], and at that temperature, the chemicalchanges occur mainly in the extractives and hemicelluloses.Wood dried at a temperature higher than 100°C has beenshown to have a lower carbohydrate content and lowersusceptibility to mould growth than wood dried at lowertemperatures [12, 13].

)ermal modification (TM) is a process whereby woodproperties are altered by treatment at temperatures in therange160–260°C with essentially no oxygen present. DuringTM, wood undergoes changes in its chemical and physicalproperties by degradation and partial removal of polymericcomponents, leading to a loss of mass of the wood material[14]. )is modification of the chemical structure during TMresults in a darkening of the wood, and thermally modifiedtimber absorbs less moisture than unmodified timber [15].)e TM process removes extractives from the sawn timber,and this can be an advantage, for example, when TM timberis coated due to less bleed of resin through the coating [5].

)e degradation temperatures of cellulose and lignin areconsidered to be higher than those of hemicelluloses andextractives (Figure 1). )e degradation of cellulose andlignin is, however, hard to predict, and some studies showthat these components can also start to degrade at lower

temperatures such as 110°C where minor changes start in theguaiacyl lignin units [14, 16].

Extractives, both native and those formed by degrada-tion in the TM process, tend to migrate towards the surfaceof wood during drying and TM [17], and some extractivesvolatilize already at moderate temperatures [10].

In the present work, the chemical composition of sur-faces of naturally seasoned, kiln-dried, and thermallymodified sawn timber from two different processes (the)ermoD® and WTTprocesses) has been studied with NIR(the wavelengths are from 900 to 2500 nm). )e naturallyseasoned and the kiln-dried timber were processed underatmospheric condition, the )ermoD-modified timber wastreated in superheated steam at a temperature of 212°Cunder atmospheric pressure; in the WTT process, the sawntimber was processed in saturated steam at a temperature of170°C at a pressure of up to 6 bar. Under such conditions, itwas possible to achieve a modification similar to the)ermoD process, but at a considerably lower temperature[18, 19].

2. Materials and Methods

2.1. WoodMaterial. A total of 40 green (never-dried wood)flat-sawn sideboards, 22 × 100mm in cross section and witha length of at least 4.5m, from ten different Scots pine (Pinussylvetris L.) trees were collected directly after sawing froma sawmill in Northern Sweden. )e boards were cut intosamples with a length of 1metre giving four end-matchedboards. Samples containing only sapwood were selected forthe study, and these boards were divided into two groups fornatural seasoning or kiln drying.

)e naturally seasoned samples were single-stackedbetween 25mm thick stickers and dried indoors at a tem-perature of 20°C and about 10% relative humidity, withoutany additional air circulation. )e average moisture content

Extractives

Hemicelluloses

Cellulose

Lignin

Air drying

Artificial drying

300100 200High-temperature drying

Thermal treatment

50 150 250

Temperature (°C)NegligibleModerateSignificant

Figure 1: A schematic representation of changes in wood com-ponents with increasing temperature under humid conditions, butwithout regard to time [5].

2 Journal of Spectroscopy

after 30 days was 4.2 ± 0.6%. )is was a very “soft” dryingwhere the migration of extractives to the wood surfacesduring the drying was expected to be low.

)e kiln-dried samples were double-stacked between25mm thick stickers with the bark-side surfaces of each pairturned outwards in order to achieve a steered flow ofwater/moisture from the inner part of the boards to the bark-side surfaces. )e kiln drying was performed in a small-scalelaboratory kiln with air circulation (3m/s) and a maximumdry-bulb temperature of 75°C, for 45 hours. )e averagemoisture content after drying was 13.9 ± 3.8%. Compared tonatural seasoning, this drying process was expected to beharsher and to lead to more extractives and decompositioncomponents on the surfaces after drying.

Planed thermally modified Scots pine (Pinus sylvestris L.)boards were obtained from the )ermoD® and WTT® in-dustrial processes.

)e )ermoD boards were treated according to theFinish )ermowood© process at 212°C by Heatwood AB inHudiksvall, Sweden.)e process was run as it is described in)ermoWood Handbook [20] for the class )ermoD. )e)ermoD process consists of three phases:

(1) Temperature increase and high-temperature drying,using heat and steam: the temperature is raisedrapidly to 100°C. )ereafter, the temperature is in-creased steadily to 130°C. )e total time is 12 hours.

(2) )ermal modification: the kiln is increased up to212°C. When the target level has been reached, thetemperature remains constant for 3 hours. )e totaltime is 12 hours.

(3) Cooling and moisture conditioning: the temperatureis lowered by using water spray systems. When thetemperature has reached 80–90°C, the wood isremoistured to reach the wood moisture content to4–7%. )e total time is 12 hours.

)e WTT boards were thermally modified at 170°Caccording to the Danish WTT process by the Utetra com-pany in Arvidsjaur, Sweden. During the first hour, thetemperature was raised to 40°C and prior vacuum was ap-plied. During the next 45 minutes, the temperature wasraised to 60°C and held constant (60°C) for 25 minutes. )ekiln was warmed up to 140°C during 4 hours and heldconstant (140°C) for 30 minutes. )e kiln was furtherwarmed up for around 2 hours until the temperaturereached 155°C and held constant (155°C) for 15minutes.)ereafter, the kiln was warmed until 170°C and heldconstant (170°C) for 30minutes. During the process, thepressure was applied when the kiln temperature reached60°C and the pressure was raised steadily together with thetemperature up to 6 bar at 170°C. After thermal modificationat 170°C, the kiln was cooled down first to 80°C for 8 hoursand to 65°C for 2 hours. )e pressure was steadily decreasedto 0 when the kiln temperature reached 60°C.

After drying and thermal modification, all the sampleswere sawn to give specimens with dimensions of 15 × 50 ×

220mm (T × W × L), which means that the thermallymodified specimens had both planed and sawn surfaces. All

the specimens were stored under the same conditions atroom temperature for five months prior to the NIRmeasurements.

2.2. Image Acquisition. A push-broom system equippedwith high-speed hyperspectral camera (Specim SWIR 3)in the short-wave infrared (SWIR) 900–2500 nm rangewith a cryogenically cooled mercury-cadmium-telluride(HgCdTe) detector with 384 spatial pixels and 288 spec-tral band resolution was used for the NIR measurements(Figure 2). )e camera pixel size was 24 × 24 µm, anda 31mm focal length lens was used. Internal dark and whitereference (Spectrolon) standards were used, i.e., the darkadjustment was based on camera dark current and Spec-trolon was used as a white reference before eachmeasurement.

)e samples for imaging were divided in two groups: (1)naturally seasoned and kiln-dried specimens and (2) ther-mally modified specimens.

)ree naturally seasoned and three kiln-dried matchedspecimens were randomly selected from the sample group,and a cross-cut was prepared from each specimen. A nat-urally seasoned specimen attacked by stain-fungus growthwas added to the dataset. )e sample origin is listed inTable 1.

Six of the specimens had fine-sawn surfaces, and two hada rougher surface in order to examine the influence ofsurface conditions on light reflection. )e specimens wereplaced randomly on the conveyer belt in order to obtaina representative light-depth penetration during imaging. Allthe measurements were made on sapwood cross-sections,with camera-belt movement in the tangential direction.

For the hyperspectral image analysis, data for the nat-urally seasoned and kiln-dried specimens were combinedwith data for samples from the thermal modification pro-cesses, )ermoD, andWTT, using a “merge” function in theEvince software version 2.7.9 software, Prediktera, Umea,Sweden [21]. A mean-centring procedure was applied asa general preprocessing technique for all the data sets col-lected. Results from the merged digital images are presentedin Figure 3. )e standard RGB images of the dataset inFigure 3 show that the planed surfaces of the )ermoD(Figure 3(b)) and WTT (Figure 3(d)) had a more intensecolour than the other surfaces.

2.3. Image Cleaning and Processing. )e complete datamatrix from eight specimens of naturally seasoned and kiln-dried groups and four specimens of thermally modifiedwood contained 1,875,840 observations and 288 variables(spectral bands) as measured parameters. )e merged ab-sorbance images were exported and analysed with Evincesoftware version 2.7.9 [21].

Hyperspectral image modelling of the multiple imagedataset in Figure 3 was used to determine the useful area ofinterest to study the influence of the different factors (wooddrying and thermal treatment). Principal componentanalysis (PCA) was used to clean background (conveyer belt)

Journal of Spectroscopy 3

and edge effects by the brushing method [21]. Mean cen-tering and base-line correction were applied to obtain clearclasses in the score plots. Eight wavelengths at the beginningof the spectral range were deleted due to the darker colour ofthe TM samples [22] so that only the data between 980 and2500 nm were analysed. )e final spectral range included

measurements of 280 wavelength bands, and this was used inthe PCA model. Partial least squares discriminant analysis(PLS-DA) modelling was applied to data for the thermallymodified specimens to differentiate between thermallymodified processes and surfaces. )e final PCA model in-cluded only a base-line correction for mean-centred data.

(a) (b)

Figure 2: Test setup for wood imaging: (a) the spectral camera and (b) the conveyer belt for sample movement.

Table 1: )e random positions of specimens from naturally seasoned and kiln-dried specimens.

Cross-sectional images Samples to the left Samples to the right Type of surface

Naturally seasoned Kiln-dried Fine-sawn

Kiln-dried Naturally seasoned Fine-sawn

Kiln-dried Naturally seasoned + stain fungus Fine-sawn

Naturally seasoned Naturally seasoned Rough-sawn


3. Results and Discussion

)emerged and “background cleaned” hyperspectral datasetfrom the wood surface images in Figure 3 was processed byPCA, and the results are presented in Figure 4, which relatesspatial data from the hyperspectral images to positions ina score plot with a grouping of the data (Figure 4(a)) and alsoprovides spectral information (Figure 4(b)). In Figure 4(a),five clusters of the absorbance image can be seen (numberedarrows). )ese clusters are related to particular areas in thewood surfaces. )e PCA model used to evaluate differencesin the combined hyperspectral image, whichmade it possibleto identify main distinctions among the samples and to seethe grouping pattern in the dataset. )e PCA modelexplained the variance of the data where the index R2X_cum� 0.97 (eigenvalue 3.47) explained variance of X matrix thatwas in our case the wavelength numbers. After cleaning(background and edge effect removal), the PCA model wasimproved (R2X_cum � 0.99461, eigenvalue 1.3426) withfour components. )e first and second principal compo-nents (PC) of the PCAmodel explained 94% of the total sumof squares (%SS) and 4.34% SS of the data variability,respectively.

Six zones can be identified in the score plot in Figure 4(a)related to the specimens and surface characteristics in theRGB image of the surfaces presented in Figure 3:

(1) Colour difference in knots due to the presence andconditions of extractives and a higher density thanthe surroundings. )e area indicated by a circle inthe score plot is related to the knots and latewood“bow” close to the knot on the upper right-hand sideof the surfaces of )ermoD-modified wood (Figure3(b)).

(2) )e rip-cut surface of specimens from)ermoD andWTTprocesses represented by the area indicated bythe yellow arrow.

(3) Almost all rip-cut surfaces of the WTT process andunexpectedly the areas in air-dried samples repre-sented by the area indicated by the violet arrow.

(4) Naturally seasoned and kiln-dried specimens rep-resented by the area indicated by the red arrow.

(5) )e planed surface of the WTT modification spec-imen represented by the area indicated by the greenarrow.

(6) Planed surfaces of thermally modified specimens(thermoD and WTT processes) represented by thearea indicated by the blue arrow.

)e spectra of pixels in the image related to naturallyseasoned and kiln-dried specimens (Figure 3(a)) showspectra from TM and air/kiln-dry for comparison and

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(a) (b) (c) (d) (e)50 100 150 200 250 300 350 400 500 550 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 19001850

Figure 3: Merged digital images of the specimens: (a) air-kiln dried, (b) )ermoD planed surface, (c) )ermoD rip-cut surface, (d) WTTplaned surface, and (e) WTT rip-cut surface.


presented as an average single spectrum in Figure 4(b),indicating that there is no spectral difference.

)e spectral band assignment of the NIR spectrum inFigure 4(b) was compared to the spectra of wood and woodcomponents provided by Schwanninger [23]. )e peaks inthe average spectrum in Figure 4(b) are related to pixels inimages of naturally seasoned and kiln-dried samples anddescribed in Table 2.

PC2 of the area related to the naturally seasoned andkiln-dried specimens indicated by the red arrow in Figure 4is presented as a 2D image in Figure 5. It was confirmed that

there was no difference between the naturally seasoned andthe kiln-dried wooden surfaces.

Together, PC1 and PC2 describe 98.3% of the datavariation. )e Contour 2D image and the loading plot forPC2 are shown in Figure 6. )e PC2 is dominated by thevariation in the 1972 nm and 2135 nm wavelengths regions.)e peak at 1972 nm is linked to changes inmoisture contentin the wood surface. )e peak at 2135 nm corresponds toCar-H stretching and C�C stretching in lignin and extrac-tives and to C-H stretching and C�O stretching of acetylgroups in hemicelluloses [23]. )ese peaks can be used for

Table 2: Band assignment from average spectrum of naturally seasoned and kiln-dried wood.

Peak no. in Figure 4(b) Wavelength (nm) Bond vibrations

1 1190–1221

Linked to 2nd overtone of CH-stretching and maycorrespond to CH3 groups of wood constituents suchas lignin assigned to spectra and CH, CH2 groups of

cellulose

2 1440 Arises from 1st overtone of C-H stretching and C-Hdeformation in lignin

3 1447–1491Linked to 1st overtone of O-H stretching in phenoliclignin, glucomannan, and semicrystalline regions in

cellulose, intramolecular H-bond of cellulose

4 1922–1928Related to O-H asymmetric stretching and O-H

deformation of the water molecule; this is expected, asthe moisture content was different in the specimens

5 2085–2113 Relates to O-H and C-H stretching and deformationsin cellulose and xylan molecules

6 2277

Relates to O-H plus C-C stretching and/or C-Hstretching and deformation in cellulose; this peakoverlaps the peak at 2236 nm related to lignin via C-H

stretching and C-H2 deformations

7 2331 Related to C-H stretching and C-H deformation inhemicellulose and xylan molecules

8 2450–2475 Relates to carbohydrates in general

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Figure 4: (a) Score plot after background image cleaning, and (b) average spectrum of image related to the surfaces of the naturally seasonedand kiln-dried specimens shown in Figure 3(a) (numbers relate to description below and to Table 2).


multivariate calibration of the difference between naturallyseasoned/kiln-dried wood and thermally modified wood.)e red areas in the PC2 score image shown by a red circle inFigure 4(a) are related to the small red areas in Figure 5 dueto presence of knots.

)e average spectrum of the red area of the )ermoDsurfaces in Figure 6(a) is presented in Figure 7. )e detailedinterpretation of the spectral signal recommended bySchwanninger has been combined with suggestions fromWokman and Weyer [24] and is described in Table 3.

)e average spectrum of the surface contaminated byfungi is presented as a green line in Figure 8.)e spectrum ofthe fungal-attacked surface does not differ greatly from theaverage spectrum of the naturally seasoned and kiln-driedsamples (Figure 8).

)e regions where the greatest difference in the minimalpreprocessed spectra was observed was the 1100–1200 nmregion and at wavelengths longer than 1650 nm (Figures 7and 8). )e absorption in the regions around 1695 and1744 nm is greater in Figure 7 than in Figure 8 and may berelated to extractives.

In a previous study, it was shown that low-molecularsugars and fatty/resin acid compounds migrate to naturallyseasoned and kiln-dried surfaces [25]. It is known thatchemical compounds are degraded and that new compoundssuch as extractives/phenols are formed during thermalmodification [26]. In the present study, it was not possible todistinguish the signals of fungal-attacked wood from thoseof the nonattacked wood, due to an inability to identify thefungal species that contaminated the material and to assignbands with the polysaccharide matrix.

A discriminant analysis of the four different surfaces ofthe thermally modified specimens (b, c, d, and e in Figure 3)was performed, and the result is shown in Figure 9. )egreatest differentiation was found between the planed andrip-cut surfaces, but a rather clear discrimination was alsofound between the two thermal modification processes. )escore plot of PC1 and PC4 shows a clear differentiationbetween the surfaces (Figure 9(a)), and the model co-efficients for PC1 and PC4 indicate the wavelengths that arestrongest for the discrimination but they do not give theanswer without further analysis. )e model showed high R

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Figure 5: Contour 2D image of PC2 related to the area representing data from naturally seasoned and kiln-dried wooden surfaces indicatedby red arrows in Figure 4(a).

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981.81 1119.1 1260.7 1401.4 1541.6 1681.7 1821.7 1961.8 2101.8 2241.8 2520.22381.4

Figure 6: )e loading plot for PC2.


Table 3: Band assignment for the average spectrum of naturally seasoned and kiln-dried wood.

Peak no. in Figure 7 Wavelength (nm) Bond vibrations:

1 1452 Linked to O-H 2nd overtone (CH2-OH) fromprimary alcohols

2 1695–1725

Related to C-H 2nd overtone of symmetric stretchingin aliphatic hydrocarbons and overlaps with C-H

methyl in aromatics that may be phenols or lignin at1744 nm

3 1928 Related to O-H stretching and HOH deformationcombination from water molecule

4 2148Related to first overtone of C-H stretching inaromatics (indicated by black and red arrow in

Figure 6(b))

5 2280 Related to C-H stretching and CH2 deformations inthe polysaccharides that end by signal from C-H, C-C

6 2347 Related to C-O-C stretching combinations inhydrocarbons, cellulose, and aromatics

7 2475Related to C-H in methyl groups and C-C stretching

combinations in hydrocarbons, aromatics, andcellulose

0.45

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0975 1050 1200 1350 1500 1650 1800 1950 2100 2175 2325 2475

Figure 8: )e average spectrum of naturally seasoned and kiln-dried wood surfaces (red line) and the average spectrum of surfacescontaminated by fungi (green line).

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Figure 7: )e average spectrum for the red area (knots) on the )ermoD surface in Figure 6 (numbers related to Table 3).


(explained variation) and Q (predicted variation) values(0.99 and 0.77, respectively), which indicate a clear differ-ence in the NIR spectra for PC4 in Figure 9. )e wavelengthband at 2100 nm (arrow in Figure 9(b)) is especially in-teresting and may be related to the C�O-O of polymericstretching [23], whichmay be related to a hydrolysis of acetylgroups to volatile carboxylic acids [27].

)e studied surfaces had a heterogeneous chemicalnature due to tree growth factors and sawing patterns andbecause of drying and thermal modification. In addition tothe wood diversity and processing factors, biological con-taminants from the surroundings probably also influencedthe chemical properties of the wood surface, but this in-fluence is suggested to have a minor influence on the resultsof this study.

4. Conclusions

)e use of hyperspectral imaging and multivariate analysismakes it possible to distinguish (a) Scots pine wood surfacesfrom two different thermal modification processes, (b) a rip-cut surface from a planed surface, and (c) surfaces of woodthermally modified from surfaces of naturally seasoned andkiln-dried wood. It was not however possible to distinguishbetween naturally seasoned and kiln-dried surfaces.

Regions with a high absorbance were related to woodcomponents such as lignin, cellulose, and hemicellulose aswell as to extractives with phenolic groups and aliphatichydrocarbons. )e band difference related to the peak at2148 nm obtained from a PCA model was found to be usefulfor multivariate calibration between naturally seasoned andkiln-dried wood and thermally modified wood.

PLS-DA of thermally modified wood showed the greatestdifferentiation between a planed wood surface and a rip-cutsurface and a rather clear discrimination between the twothermal processes.)e wavelength band at 2100 nm revealed

the greatest distinction and may correspond to C�O-O ofpolymeric stretching of acetyl groups.

)e hyperspectral imaging in combination with simplyoperated software opens great opportunities for differentlyprocessed wooden surfaces. For the development of propercalibration and prediction models, however, the main woodcomponents with described variability have to be chemicallyanalysed in order to move from the laboratory scale topractical implementation.

Data Availability

)e data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

)e authors declare that they have no conflicts of interestregarding the publication of this article.

Acknowledgments

)e authors wish to thank NORRA Timber, Skelleftea,Sweden; HeatWood AB, Hudiksvall, Sweden; and Carl-JohanStenvall, Arvidsjaur, Sweden, for providing the test material.Support from the European Regional Development Fund, theCounty Administration of Vasterbotten, the Municipality ofSkelleftea, and the Wood Centre North is greatly appreciatedand acknowledged. In addition, the authors wish to expresstheir gratitude for the support of COST Action FP1407. )eauthors thank Prof. Paul Geladi, SLU, Umea, Sweden, forexperimental assistance and fruitful discussions about thehyperspectral imaging technique.)is work was supported bythe Swedish Research Council for the Environment Agri-cultural Sciences and Spatial Planning (FORMAS) (Project942-2016-64, 2016, and Project 419, 2017).

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Figure 9: (a) Score plot of PC4 versus PC1 for the two thermal modification processes and (b) loadings for PC4 in PLC-DA model. )earrow shows the greatest difference in the spectral dataset.


References

[1] K. Candelier, M.-F. )evenon, A. Petrissans, S. Dumarcay,P. Gerardin, and M. Petrissans, “Control of wood thermaltreatment and its effects on decay resistance: a review,”Annalsof Forest Science, vol. 73, no. 3, pp. 571–583, 2016.

[2] S. Tsuchikawa, “A review of recent near infrared research forwood and paper,” Applied Spectroscopy Reviews, vol. 42, no. 1,pp. 43–71, 2007.

[3] B. G. Osborne, T. Fearn, P. T. Hindle, and B. G. Osborne,Practical NIR Spectroscopy with Applications in Food andBeverage Analysis, Longman Scientific and Technical, Wiley,New York, NY, USA, 2nd edition, 1993.

[4] A. Sandak, J. Sandak, and R. Meder, “Assessing trees, woodand derived products with NIR spectroscopy: hints and tips,”Journal of Near Infrared Spectroscopy, vol. 24, no. 6,pp. 485–505, 2016.

[5] P. Navi and D. Sandberg, 5ermo-Hydro-Mechanical Pro-cessing of Wood, Presses Polytechniques et UniversitairesRomandes, Lausanne, Switzerland, 2012.

[6] W. E. Hillis, “High temperature and chemical effects on woodstability,” Wood Science and Technology, vol. 18, no. 4,pp. 281–93, 1984.

[7] Forest Products Laboratory, “Air drying of lumber,” GeneralTechnical Report FPL-GTR-117, U.S. Department of Agri-culture, Forest Service, Forest Products Laboratory, Madison,WI, USA, 1999.

[8] N. Terziev, “Industrial kiln drying and its effect on micro-structure, impregnation and properties of Scots pine timberimpregnated for above ground use. Part 1. Effects of initial,final dryings and preservative on impregnation and timberquality,” Holzforschung, vol. 56, no. 4, pp. 428–433, 2005.

[9] D. Sandberg, “Primary wood processing in Swedish sawmill1850-2010,” in Sawn Timber for Prosperity. 5e SwedishSawmill Industry History 1850-2010, R. Pettersson, Ed.,pp. 326–363, )e Royal Swedish Academy of Agriculture andForestry, Stockholm, Sweden, 2015.

[10] A.-M. Manninen, P. Pasanen, and J. Holopainen, “Comparingthe VOC emissions between air-dried and heat-treated Scotspine wood,” Atmospheric Environment, vol. 36, no. 11,pp. 1763–68, 2002.

[11] J. Tomminen and M. Nuorteva, “Pinewood nematode, Bur-saphelenchus xylophilus in commercial sawn wood and itscontrol by kiln-heating,” Scandinavian Journal of Forest Re-search, vol. 7, no. 1–4, pp. 113–120, 1992.

[12] M. Sehlstedt-Persson, “High-temperature drying of Scotspine. A comparison between HT- and LT-drying,” Holz alsRoh-und Werkstoff, vol. 53, no. 1, pp. 95–99, 1995.

[13] F. Shafizadesh and P. Chin, “)ermal deterioration of wood,”ACS Symposium Series 43, pp. 57–81, American ChemicalSociety, Washington, DC, USA, 1977.

[14] C. Hill, Wood Modification: Chemical, 5ermal and OtherProcesses. Wiley Series in Renewable Resources, Wiley, Chi-chester, UK, 2006.

[15] P. M. Kekkonen, A. Ylisassi, and V.-V. Telkki, “Absorption ofwater in thermally modified pine wood as studied by nuclearmagnetic resonance,” Journal of Physical Chemistry C,vol. 118, no. 4, pp. 2146–2153, 2014.

[16] D. Fengel and G. Wegener, Wood: Chemistry, Ultrastructure,Reactions, W. de Gruyter, New York, NY, USA, 1984.

[17] M. Nuopponen, H. Wikberg, T. Vuorinen, S. Maunu,S. Jamsa, and P. Viitaniemi, “Heat-treated softwood exposedto weathering,” Journal of Applied Polymer Science, vol. 91,no. 4, pp. 2128–2134, 2004.

[18] O. Dagbro, P. Torniainen, and T. Moren, “)ermal modifi-cation of birch using saturated and superheated steam,” inProceedings of Nordic-Baltic Network in Wood MaterialsScience and Engineering, Oslo, Norway, October 2011.

[19] O. Dagbro, P. Torniainen, O. Karlsson, and T.Moren, “Colourresponses from wood, thermally modified in superheatedsteam and pressurized steam atmospheres,” Wood MaterialScience and Engineering, vol. 5, no. 3-4, pp. 211–219, 2010.

[20] Finnish )ermowood Association, 5ermoWood® Handbook,)ermoWood, Teuva, Finland, 2003, http://www.thermowood.fi.

[21] Prediktera, Evince–Tutorials and Documentation, 2018, Pre-diktera, Umea, Sweden, 2018.

[22] H. Bachle, B. Zimmer, E. Windeisen, and G. Wegener,“Evaluation of thermally modified beech and spruce woodand their properties by FT-NIR spectroscopy,” Wood Scienceand Technology, vol. 44, no. 3, pp. 421–433, 2010.

[23] M. Schwanninger, J. C. Rodrigues, and FacklerK, “A review ofband assignments in near infrared spectra of wood and woodcomponents,” Journal of Near Infrared Spectroscopy, vol. 19,no. 5, pp. 287–308, 2011.

[24] J. Workman and L. Weyer, Practical Guide and Spectral Atlasfor Interpretive Near-Infrared Spectroscopy, CRC Press,London, UK, 2nd edition, 2012.

[25] O. Karlsson, O. Myronycheva, M. Sehlstedt-Persson,M. Ohman, and D. Sandberg, “Multivariate modeling ofmould growth in relation to extractives in dried Scots pinesapwood,” in Proceedings of IRG47 Annual Meeting, Ghent,Belgium, December 2017.

[26] P. Niemz, T. Hofmann, and T. Retfalvi, “Investigation ofchemical changes in the structure of thermally modifiedwood,” Maderas. Ciencia y Tecnologıa, vol. 12, no. 2,pp. 69–78, 2010.

[27] B. Sundqvist, O. Karlsson, and U. Westermark, “De-termination of formic-acid and acetic acid concentrationsformed during hydrothermal treatment of birch wood and itsrelation to colour, strength and hardness,” Wood Science andTechnology, vol. 40, no. 7, pp. 549–561, 2006.


http://www.thermowood.fi

Paper XI

Proceedings of the 62nd International Convention of Society of Wood Science and Technology

October 20-25, 2019 – Tenaya Lodge, Yosemite, California USA

46

Portable microNIR sensor for the evaluation of mould contamination on wooden surfaces

Olena Myronycheva, Olov Karlsson, Margot Sehlstedt-Persson, Micael Öhman, Dick Sandberg

Luleå University of Technology, Wood Science and Engineering, Skellefteå, Sweden Keywords: mould fungi, microNIR, wood, naturally seasoned, kiln-dried, multivariate model Abstract: The traditional assessment of mould growth is sometimes subjective and can differ from person to person. By applying spectroscopic tools, it is possible to create an individual fingerprint of a wooden material and create databases for obtaining more objective information related to the chemical and biological composition. Side-boards (the flat-sawn sapwood part of the log) of Scots pine were single stacked on

stickers and naturally dried indoors at 20C to an average moisture content (MC) of 4.6%. Another ten side-boards were dried in a small-scale laboratory air-circulation kiln to an average MC of 14%. Another group of side-boards were double-stacked with the bark-side surfaces of each pair turned outwards in order to get a high extractive concentration on these surfaces, and less concentration on opposite surfaces. The different flat-side surfaces were planed according to a planing-depth scheme : 0 mm (unplanned), 0.25, 0.75, and 1.75 mm depth from the surface, and the residual wood particles were collected for further analysis. The planned surfaces were exposed to a mould test, performed by spraying a spore suspension of five mould fungi on the wood surfaces followed by incubation at the temperature of 24°C and 95±3%RH for 35 days. Thereafter, the surfaces were graded according to mould growth. A portable microNIR sensor (wave-length range 900-1670 nm, step 6 nm) was used for NIR-spectra detection on the surfaces after mould test, and a data matrix was created. Multivariate analysis of obtained spectra was performed. The results show that the principal component analysis (PCA) can describe and predict 99.7% of the spectroscopic data obtained. No influence of the drying method or planned depth was discovered during classification. Two mould-classes could, however, be clearly separated; no mould, and with mould growth respectively, and the separation could be detected on a 93.4% level. The study demonstrates that mould growth on the wooden surface could be evaluated by portable MicroNIR spectrometer, which is sensitive enough to detect chemical differences caused by fungal contamination. INTRODUCTION The transition towards a digital society pushes the wood industry to apply smart and robust methods for material properties evaluation. A hypothesis about a strong influence of drying on contamination and growth of mould fungi on the wooden surface was developed by Terziev and Boutelje [1]. They found that the movement of low-molecular sugars and nitrogenous compounds during drying were significant

http://pure.ltu.se/portal/sv/persons/margot-sehlstedtpersson(a41979b9-48a8-4856-8104-e483a14c56a1).html

http://pure.ltu.se/portal/sv/persons/micael-oehman(0215209a-f7b6-471e-a2a4-c280f44eee4f).html



47

and related to type of drying [2]. Later studies found that not only sugars but also acetone soluble extractives migrates during drying and influence the degree of mould attack on the wooden surfaces [3, 4]. The wet chemistry these studies [3, 4] are tedious and it is hard to make fast and reliable conclusion about wooden properties and its relation on mould fungi growth, especially when also the degree mould contamination must be judged by visual assessment by the human eye, which is a subjective method. Therefore, researchers are looking for different non-destructive methods that allow detecting changes in wooden surfaces with high accuracy and precision, e.g. for the detection of mould. By applying spectroscopic tools, it is possible to create an individual fingerprint of a material and create databases for obtaining more objective information related to the chemical and biological composition [5–7]. This study aimed at exploring near-infrared (NIR) portable spectrometer for the detection of differences in mould growth on the surface of the Scots pine side-boards. To study the influence of mould contamination, drying type and planning-depth variables on the multivariate-model efficiency was the objective of the study. MATERIALS AND METHODS Ten Side-boards (the flat-sawn sapwood part of the log) of Scots pine were single stacked on

stickers and naturally dried indoors at 20C to an average moisture content (MC) of 4.6%. after drying for 30 days. Another ten side-boards were dried in a small-scale laboratory air-circulation kiln. The boards were double-stacked with the bark-side surfaces (the surface of the boards oriented to the bark-side of the tree) in each pair oriented outwards in order to get a high flow of moisture from the inner part of the boards towards the bark-side surfaces. In this way, extractives could migrate with the water transport during drying and accumulate on the wood surfaces. The total drying time was 44 hours of which 1.7 hours was a heating regime, and the cycle ended with a 5-hour cooling regime giving a final moisture content of 14%. No conditioning regime was applied in order to prevent the influence of re-distribution of extractives after drying. The side-boards were preapred to specimens for further studies (Table 1).

Table 1. Test groups

No. Drying Planning depth (mm) Mould No. of specimens 1 Seasoned 0

0.25 0.75 1.75

No 10 10 10 10

2 Kiln 0 0.25 0.75 1.75

No 10 10 10 10

3 Seasoned 0 0.25 0.75 1.75

Yes 10 10 10 10

4 Kiln 0 0.25 0.75

Yes 10 10 10



48

1.75 10

The different flat-side surfaces were planed according to a planing depth scheme: 0 mm (unplanned), 0.25, 0.75, and 1.75 mm depth from the surface, and the residual wood particles were collected for further analysis. The planned surfaces were exposed to a mould test, performed by spraying of a spore suspension of five mould fungi on the wood surfaces and incubation at the temperature 24°C and 95±3%RH for 35 days, and thereafter surfaces were graded according to mould growth by human. A microNIR OnSite Spectrometer (VIAVI Solutions Inc., San Jose, CA, USA) with NIR wavelengths from 908 to 1676 nm with step 6 nm was used (Figure 1) was used for samples evaluation. The assessment classes were: No mold\Mold, Drying type, Planing depth, and NIR wavelengths.

Figure 1. MicroNIR portable spectrometer. Each measurement of mould exposure and reference (no mould) surface was taken in 5 replicates and the data matrix combined from the average value of those replicates. The multivariate analysis of obtained spectra [8] was performed by using Evince software version 2.7.9 software, Prediktera, Umea, Sweden. Data pre-processing was centring and UV scaling.

Results and Discussion

The exploratory PCA model was initially built from data from air and kiln-dried samples containing no mould, and the same samples after mould test for discovering the grouping pattern. The drying type and plaining depth were not efficient for classification of the spectroscopic data. Figure 2 clearly demonstrates the differentiation between samples with classes No mould (green group of dots) and with Mould (blue group of dots). Obtained PCA model (Table 2) had a good fit. Therefore, the decision was made towards using Soft Independent Modelling of Class Analogy (SIMCA) that allows identifying local models around those classes (Table 2). Table 2. Multivariate models of NIR spectra of air- and kiln-dried side-boards; No mould and Mould Classes. Data pre-processing - Centred + UV scaled.

Type Comp R2X_cum Q2X_cum Eigen value

PCA 3 0.997 0.997 1.1

SIMCA



49

Class “No mould” 3 0.999 0.999 0.1

Class “Mould” 4 0.998 0.998 0.2

Figure 2. The score plot PC2 against PC3 where green - no mould class and blue - mould class.

Two models were adopted: a local 3-component for No mould class, and a 4-component for Mould class models. As can be seen in Table 1, the performance of the SIMCA models was improved compare to PCA model and eigenvalues that represent self-projection of eigenvector was quite small due to a good data fit. Figure 3 shows a Coomans’ plot, which graphically presents the classification analysis. It is notable that data for air-dried and kiln-dried samples overlap (a green coloured group of the samples) and cannot be totally separated. Samples from Mould class (a blue group of the dots) are quite different from those in No mould class (red dots) as could be seen in Figure 3, as well as shown in the prediction table (Table 2).



50

Figure 3. Coomans’ Plot where green - class No mould, blue – class Mould and red – class No class.

The 6 observations out of totally 151 observations (total data matrix used for analysis) representing about 4% of the data the program algorithm could not be classified (Table 3). Still 141 observations were predicted correctly, that is 93.4% of accuracy whereas 10 were assigned not correctly in general. Table 3. Prediction table from SIMCA model.

Classes Total

observations

No classes Class No mould Class Mould

No classes 6 (3.97%) 2 (33.3%) 4 (66.7%)

Class No mould 70 (46.4%) 70 (100%)

Class Mould 75 (49.7%) 4 (5.33%) 71 (94.7%)

Predicted 151 (100%) 0 (0%) 76 (50.3%) 75 (49.7%)

- correctly 141 (93.4%)

-not correctly 10 (6.62%)

Conclusion This study demonstrates that mould growth on a wooden surface can be evaluated by a portable MicroNIR spectrometer. The Micro NIR was sensitive enough to detect chemical differences from biological contamination such as mould growth on the wooden surfaces. Unfortunately, the sensitivity was not enough to differentiate the drying type and planning depth at current experimental conditions. We suggests that an increased number of specimens will give a better differentiation, but that is a question for future studies. References



51

1. Terziev, N.; Boutelje, J. Effect of Felling Time and Kiln-Drying on Color and Susceptibility of Wood to Mold and Fungal Stain During an Above-Ground Field Test. Wood Fiber Sci. 2007, 30, 360–367. 2. Terziev, N. Migration of Low-Molecular Sugars and Nitrogenous Compounds in Pinus sylvestris L. During Kiln and Air Drying. Holzforsch. - Int. J. Biol. Chem. Phys. Technol. Wood 2009, 49, 565–574. 3. Myronycheva, O.; Karlsson, O.; Sehlstedt-Persson, M.; Öhman, M.; Sandberg, D. Distribution of low-molecular lipophilic extractives beneath the surface of air- and kiln-dried Scots pine sapwood boards. PLOS ONE 2018, 13, e0204212. 4. Karlsson, O.; Myronycheva, O.; Sehlstedt-Persson, M.; Öhman, M.; Sandberg, D. Multivariate modeling of mould growth in relation to extractives in dried Scots pine sapwood. In Proceedings of the 48th Conference of the International Research Group on Wood Protection, IRG48; Ghent, Belgium, 2017. 5. Thumm, A.; Riddell, M.; Nanayakkara, B.; Harrington, J.; Meder, R. Mapping Within-Stem Variation of Chemical Composition by near Infrared Hyperspectral Imaging. J. Infrared Spectrosc. 2016, 24, 605–616. 6. Williams, P.J.; Geladi, P.; Britz, T.J.; Manley, M. Near-infrared (NIR) hyperspectral imaging and multivariate image analysis to study growth characteristics and differences between species and strains of members of the genus Fusarium. Anal. Bioanal. Chem. 2012, 404, 1759–1769. 7. Via, B.K.; Eckhardt, L.G.; So, C.-L.; Shupe, T.F.; Groom, L.H.; Stine, M. The Response of Visible/Near Infrared Absorbance to Wood-Staining Fungi. Wood Fiber Sci. 2007, 38, 717–726. 8. Eriksson, L.; Byrne, T.; Johansson, E.; Trygg, J.; Vikström, C. Multi- and megavariate data analysis: basic principles and applications; 2013; ISBN 978-91-973730-5-0.

Proceedings of the

2019

Society of Wood Science and

Technology

International Convention

Convention Theme: Renewable

Materials and the Wood-based

Bioeconomy

Edited by Susan LeVan-Green

Overall General Chair: Eve Haviarova,

Purdue University, USA

October 20-25, 2019

Tenaya Lodge

Yosemite National Park, California, USA

Paper XII

Proceedings of the 2020 Society of Wood Science and Technology International Convention

“Renewable Resources for a Sustainable and Healthy Future”

Edited by Susan LeVan-Green

Overall General Chair: Andreja Kutnar, InnoRenew CoE and University of Primorska, Slovenia

July 12-15, 2020

2020 INTERNATIONAL CONVENTION, VIRTUAL CONFERENCE – (FORMERLY HOTEL BERNARDIN), PORTOROŽ, SLOVENIA


57

SPECTROSCOPIC CHARACTERISATION OF WOOD TREATED BY DIFFERENT

COATINGS AFTER WEATHERING IN SUBARCTIC CONDITIONS

Olena Myronycheva1, Alona Sekan1, Injeong Kim1, Olov Karlsson1, Peter Jacobsson2, Margot

Sehlstedt-Persson1, Dick Sandberg1

1 Wood Science and Engineering, Luleå University of Technology, Skellefteå, Sweden

2 Martinsons Byggsystem AB, Skelleftea, Sweden

Abstract

Weathering of wood is a complex natural phenomenon that is influenced by factors both in macro-

and micro-scale. The degradation by weathering constitutes of complex physical-chemical changes

influenced by environmental factors such as moisture, sunlight, temperature, chemical compounds,

wind, and biological organisms and their related agents. The aim of our study was the evaluation

of MIR and NIR spectroscopic technique for characterisation of wood treated by commercial

coatings contained different biocidal treatments after weathering in subarctic conditions.

Tangential and radial surfaces of the Scots pine and Norway spruce were treated with coatings

containing various biocides. Treatment was in the form of fairly transparent wood oil (T), while

the two others were coatings that had more of a non-transparent character (H and P). Treated

specimens and control were placed in various directions on racks outdoor for natural weathering.

The measurement of the NIR spectra of specimens that were not exposed, after weathering and

dried after weathering, was done by the point-based microNIR portable sensor. Multivariate

statistical analysis (MSA) showed that data from types of exposure contributed less compared to

surface treatments in the 3D principal component analysis model (PCA). Grouping pattern around

treatments H and P could be obtained as the two individual groups more or less fully separated.

From the treatment T and non-treated specimens, no individual grouping behaviour could be

observed, however, more intense exposed seemed to be clustered in a different way than mildly

exposed ones. A more extensive weather exposure gave also a more degraded surface for T-treated

specimens. The NIR portable spectrometer may be used for assessment of the extent of weathering,

but the treatment P followed by treatment H had minimal changes. Coefficients Plot in the PLS-

DA model for different types of exposure for those treatments showed the influence of the NIR

wavelengths in classes related to exposure type. The changes caused by weathering phenomenon

was evaluated by using multivariate methods in which resulting multivariate model parameters for

prediction of the changes were determined. Analysis of the chemical structure of coatings after

weathering analysed by mid-infrared spectroscopy gave support to the lower stability of T-

treatment during weathering.

Keywords: wood, treatment, natural weathering, infrared, near-infrared, spectroscopy

INTRODUCTION

The measurement by electromagnetic radiation such as mid-infrared (MIR) and near-infrared

spectroscopy (NIR) is non-destructive analytical techniques providing an individual chemical

fingerprint of the sample. The non-destructive nature of the technique made it quite popular in

about:blank

about:blank


58

material science and widely applied to the wood (Sandak, Sandak, and Meder 2016). Industrial

digitalisation and movement towards the Industry 4.0 revolution opened new applications to

spectroscopic techniques in environmental chemistry. However, the semi-quantitative approach

and heterogeneity of wooden material limit its application in the material involved in

environmental analysis such as weathering. Weathering of wood is a complex, natural

phenomenon that is influenced by factors both in macro- and micro-scale. The surface degradation

by weathering evolves complex physical-chemical-biological changes caused by environmental

factors such as moisture, sunlight, temperature, chemical compounds, wind, and biological

organisms.

Therefore, the aim of our study was the evaluation of field NIR spectroscopic technique for

characterisation of wooden material treated by commercial coatings of three different types, in

outdoor applications in subarctic conditions.

MATERIAL AND METHODS

Kiln-dried Scots pine sapwood boards were cut into specimens with a dimension of 15x12x2 cm

(TxWxL). Kiln-dried Norway spruce boards were cut to specimens with a dimension of 15x8x1.5

cm. The end-grain surfaces of each specimen were sealed with a SikaFlex, Switzerland. The total

non-sealed surface area of the specimens was 420 cm2 and 300 cm2 for Scots pine and Norway

spruce, respectively. Three surface treatments with different biocidal composition and an uncoated

control group were evaluated, and variables in the experimental design of the current study

presented in Table 1.

Table 1. Experimental design.

Variables Code Description of Variables No. of replicates

Treatment H

Waterborne Pigmented Primer with two

biocides and alkyd binder

18

T Waterborne wood oil with a single biocide 18

P Waterborne paint for indoor use with four

biocides and acrylate binder

18

C No treatment 18

Wood specie Scots pine 36

Norway spruce 36

Angle of

exposure

0 24

45 (south) 24

90 (north)

No exposed

24

20

Type of

exposure

No exposed/After exposure for 115/Dried

after exposure for 115 days

20/72/72

The amount of applied treatment coating on each specimen was 100±10 g/m2. The specimens were

placed on the exposure site (in Skellefteå latitude: 64.744453, longitude: 20.955569) at June 5th,

and removed September 27th with a total exposure time 115 days. The specimens were inclined to

the south with an angle of 45˚, and to the north by an inclination angle of 90˚ against the horizontal

plane as well as in the horizontal position.

The moisture content of the specimens before and after outdoor exposure was measured

gravimetrically by heating in an oven at 103˚C until constant weight.


59

The NIR spectra were collected by portable microNIR OnSite Spectrometer (VIAVI Solutions

Inc., San Jose, CA, USA) with NIR wavelengths from 908 to 1676 nm with step 6 nm, every week

during the observation period. NIR spectral data collected in five replicates and dataset contained

1166 observations in total used for the prediction model. The analysis was performed using

variables Treatment, Wood Specie, Day and Angel of Exposure. The FTIR spectra in the MIR range

4000-400 cm-1 were collected from dried specimens (MC in Table 2) isolated by scraping surface

material from the treated wood after weathering of specimens in ATR mode and transformed to

the absorbance mode by FT-IR Frontier Spectrometer, PerkinElmer. The comparison of unexposed

treatment and treatments at different directions of exposure was made for chemical changes

detection. Data analysis was performed with MS Excel 2016, IBM SPSS, USA and SIMCA14,

Umetrics AB, Sweden (Eriksson et al. 2013).

RESULTS AND DISCUSSION.

In Figure 1, the multivariate modelling of the NIR spectra of the grouping of all specimens in the

study is presented. This is shown as a three-dimensional plot of principal component analysis

(PCA). In this way clear grouping pattern by treatments that are coloured differently could be seen:

treatment with P-product (red) and H-products (blue) dots where separated quite well from the

other groups whereas untreated (yellow) and treated with T-treated (green) were mixed (Figure

1a).

a) b)


60

c) d)

Figure 1. The 3D PCA model of NIR spectra overview with legends coloured by: a) coating

treatment, b) type of exposure, c) wood species, d) angle of exposure (0 – non exposure

specimens)

Already in the non-exposed specimens (red dots in Figure 1b), such separation between treatment

H and P was seen whereas for T-treatment mixing with uncoated specimens were obvious. Also,

after the weathering exposure, such separation of specimens related to the various treatments could

be observed (green dots in Figure 1b). Furthermore, the weathered specimens in treatment H and

P (green dots in Figure 1b) were clustered at somewhat different position in the plot than the

corresponding non-exposed specimens (red dots in Figure 1b). Moisture is a frequent contributor

in NIR spectra, and H- and P-dried weathered specimens (green dots in Figure 1b) were also

clustered at somewhat different positions than the dried weathered specimens (blue dots in Figure

1b). This indicated that also, other factors than moisture contributes to the separations of groups

in the PCA-plot as the treatment itself played a dominant role in the separation in the multivariate

plot of weathered specimens.

From visual inspection, T treatment was found to be more vulnerable to weathering conditions

than treatment H and especially treatment P (Table 2).

Table 2. The final pictures of the treated Scots pine boards after 115 days of exposure before drying

Angle H T P C


61

90

180

45

Furthermore, moisture content in the specimens varied depending on the exposure position as

shown in Table 3. In Figure 1d influence of exposure position on the modelled NIR-data is

presented; specimens from T-treated and uncoated ones that were more exposed (180 degree) were

clustered more or less at other position than the mildly exposed (90 degree), while a similar trend

was more difficult to observe for H- and P-treatments.

Table 3. Moisture content of weather exposed specimen

Treatment Angle of

Exposure

Wood Specie

Pine Spruce

H 45 (south)

0

90 (north)

16.77±0.16

19.75±0.56

16.38±1.25

15.03±0.13

17.70±0.02

16.59±0.32

T 45 (south)

0

90 (north)

16.78±0.31

17.75±0.34

17.78±0.13

17.04±0.46

18.42±0.26

16.54±0.75

P 45 (south)

0

90 (north)

16.84±0.48

18.79±2.36

17.20±1.06

15.42±0.70

18.81±0.11

17.01±2.83


62

C 45 (south)

0

90 (north)

15.99±0.13

18.07±0.30

16.75±0.36

16.97±2.62

18.32±0.15

16.99±0.62

Alkyd and acrylate binder were the dominant organic components in H- and P-coating treatment

and biocides contributed only to low extent. Functional groups in these two binders also

contributed to absorptions when analysing the surface materials using FTIR in which carbonyl

absorptions at 1720 cm-1 was found to be fairly stable towards weathering, while changes in

hydrocarbon absorptions were more difficult to evaluate. Preliminary data for T-treatment

indicated changes in the carbonyl absorptions that might reflect its less stable appearance. Thus,

the importance of degradation of coating in the clustering within each coating is still difficult to

verify and needs further studies.

Validation of multivariate models

The PCA model of NIR spectra which provided the data for the presentations in Figure 1 gave a

description of data (R2) 99.9% and prediction (Q2) 99.9% that is quite good for NIR spectra (Table

4). The partial least squares regression (PLS) model with Y variables (Treatment, Wood Specie,

Day, Angel of Exposure, Type of Exposure) contained 21 principal components and less prediction

value (Q2 in Table 4) was used to find the best-modelled variable. The variable Type of Exposure

contributed mostly to the models' performance compared to other variables after the diagnostic of

the PLS model (data not shown here). Therefore, that variable (Type of Exposure) was used as a

response in the PLS-DA model since that model provides maximum separation between such

amounts of classes (Eriksson et al. 2013). The parameters of all models developed are described

in Table 4.

Table 4. The multivariate models' parameters

№ Name Component Observations R2X(cum) R2Y(cum) Q2(cum)

1 PCA 3 1166 0.999 0.999

2 PLS 21 1166 1 0.799 0.788

3 PLS-DA 8 1166 1 0.845 0.844

The Coefficients plot in Figure 2, displays regression coefficients correlated to scaled and centred

X-variables (NIR-spectra) from all specimens in class After exposure. As the model is based on all

samples and their composition varies this means that correlations could not be related to a specific

treatments structure. However, some general conclusions can be drawn. Negative and positive

correlations were found indicating decreases and increases in presence of absorptions upon

weathering exposure, respectively. The negative high coefficients at the beginning of the NIR

spectra (lower wavelength) could be related to the VIS region and visible changes of colour on the

surface (Schwanninger et al. 2011). Although moisture was found in the specimen (Table 3),

typical strong water signal (around 1450 nm) have not been generated.

The observation of Coefficients plot for all classes in the PLS-DA model showed the strongest

correlation for wavelength around 1620 nm which may be related to C-H hydrocarbon chains

possibly in alkene groups (Workman and Weyer 2012; 2008). Class Dried after exposure had the

strongest positive correlation around wavelength 1403 nm that is close to absorptions originated

from methyl C-H absorptions associated with branched aliphatic hydrocarbon (Workman and

Weyer 2012; 2008).


63

Figure 2. The Coefficients plot of PLS-DA model from all specimens.

The misclassification table in the PLS-DA model shows the proportion of correctly classified

observations in the prediction set. The NIR spectra in different treatments could be predicted to

the extent of around 80% in total, as presented in Table 4.

Table 4. Misclassification table PLS-DA

(green - correct prediction, yellow -misclassification)

Variable Number Correct After

exposure

Dried

after

exposure

No

exposure

No class

(YPred≤)

After exposure 599 100% 599 0 0 0

Dried after exposure 367 95.91% 0 352 15 0

Non exposure 200 100% 0 0 200 0

No class 0 0 0 0 0

Total 1166 98.71% 599 352 215 0

The classes represented types of exposure regarding no exposure and after exposure could be

predicted with 100%. When in dried after exposure type 15 specimens were misclassified. The

explanation of such consequences could be due to the nature of the treatment P that was less

degraded during exposure than other treatments-product and homogeneity of the weathered and

the non-weathered wood

CONCLUSION

The evaluation of three commercial wood coating by spectroscopic techniques revealed that NIR

spectra might be used for the detection of weathered or non-weathered specimens. From our data

from NIR and MIR, the most variability in the model was probably from changes in hydrocarbons

as well as carbonyl compounds related to the chemical composition of the treatment T. Despite

complicated for interpretation spectra and step 6 nm in the portable microNIR spectrometer, the

PLS-DA model gave almost 99% of the prediction. That indicates portable microNIR as a

promising technique for the assessment of the weathered coated wood in outdoor exposure.


64

ACKNOWLEDGEMENT

The acknowledgement by authors provided to the Swedish Research Council for the Environment

Agricultural Sciences and Spatial Planning, FORMAS (project number 419, 2017) and IPOS DP2

Vinnova for funding our research. Kempe foundation is gratefully acknowledged for financial

support.

REFERENCE

Eriksson, L, T Byrne, E Johansson, J Trygg, and C Vikström. 2013. Multi- and Megavariate

Data Analysis: Basic Principles and Applications.

Sandak, Anna, Jakub Sandak, and Roger Meder. 2016. 'Tutorial: Assessing Trees, Wood and

Derived Products with near Infrared Spectroscopy: Hints and Tips'. Journal of Near Infrared

Spectroscopy 24 (6): 485. https://doi.org/10.1255/jnirs.1255.

Schwanninger, Manfrred, Jos&#x00E9 Rodrigues, Carlos, and Karin Fackler. 2011. 'A Review

of Band Assignments in near Infrared Spectra of Wood and Wood Components'. Journal of Near

Infrared Spectroscopy 19 (5): 287–308. https://doi.org/10.1255/jnirs.955.

Workman, Jerry, and Lois Weyer. 2008. Practical Guide to Interpretive Near-Infrared

Spectroscopy. Boca Raton: Taylor & Francis.

———. 2012. Practical Guide and Spectral Atlas for Interpretive Near-Infrared Spectroscopy.

2nd ed. Boca Raton ; London: CRC Press.

Biography

Department of Engineering Science and MathematicsDivision of Wood Science and Engineering

ISSN 1402-1544ISBN 978-91-7790-960-6 (print)ISBN 978-91-7790-961-3 (pdf )

Luleå University of Technology 2021SV

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