Isolation of bacteria and yeast strains from lupin beans ...

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Isolation of bacteria and yeast strains from lupin beans

wastewater: assessing lupanine catabolization

Joana Veloso da Silva

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisors:

Prof. Frederico Castelo Alves Ferreira

Dr. Marisa Andreia Viegas dos Santos

Examination Committee Chairperson: Prof. Ana Cristina Anjinho Madeira Viegas

Supervisor: Prof. Frederico Castelo Alves Ferreira

Members of the Comittee: Dr. Cláudia Sofia Pires Godinho

Dr. Margarida Isabel Rosa Bento Palma

November 2019

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Preface

The work presented in this thesis was performed at Institute for Bioengineering and Biosciences (iBB) of Instituto Superior Técnico (Lisbon, Portugal) in BioEngineering Research Group (BERG) and Biological Sciences Research Group (BSRG), during the period February-July 2019, under the supervision of Prof. Frederico Castelo Ferreira (BERG-iBB) and guidance of Prof. Isabel Sá-Correia (BSRG-iBB) and Dr. Margarida Isabel Rosa Bento Palma (BSRG-iBB). The work performed in isolation and identification of bacteria and yeast species has been designed by and executed with supervision of Prof. Isabel Sá-Correia and Dr. Margarida Palma. Dr. Margarida Palma was crucial, not only on the design and execution of the work, but on my daily supervision, training and education. The work on isolation and identification of fungi was designed by and executed under supervision of Dr. Marisa Andreia Viegas dos Santos. The analytics in FT-IR, TLC, NMR and enantiomer excess were performed at Faculty of Pharmacy of University of Lisbon (Lisboa, Portugal), under guidance of Prof. Carlos Afonso. The work on this thesis was funded by Fundação para a Ciência e Tecnologia on the scope of the project WaterWorks 2014 ERA-NET ID 278 – Biorg4WasteWaterVal+ (FCT references WaterJPI/0001/2014, WaterJPI/0002/2014 and WaterJPI/0003/2014) and iBB (UID/BIO/04565/2013).

I declare that this document is an original work of my own authorship and that it fulfills all the requirements of the Code of Conduct and Good Practices of the Universidade de Lisboa.

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Acknowledgements

Quero agradecer ao meu orientador Frederico Ferreira por todo o apoio ao longo deste ano, pela

dedicação, experiência e orientação que me foram transmitidos durante a tese.

À professora Isabel Sá-Correia, pelo interesse no tema da tese, por me ter dado a oportunidade de

desenvolver o trabalho de investigação no seu laboratório e pelos seus sábios conselhos ao longo dos

desenvolvimentos. À Carla Coutinho que participou nos primeiros passos da investigação no laboratório

da professora Isabel, e à Margarida Palma por ter dado apoio e supervisão durante a principal

investigação do trabalho, pela sua amizade, paciência, partilha de conhecimentos, constante ajuda e

total disponibilidade, que foram fulcrais nos desenvolvimentos da tese. Aos restantes membros do

grupo de investigação da professora Isabel, que se mostrou sempre disponível em ajudar em qualquer

situação.

Á Teresa Esteves e à Marisa, pela paciência, dedicação e preocupação com o desenvolvimento da

minha tese. Ao professor Carlos Afonso e à Késsia da Faculdade de Farmácia, que se disponibilizaram

na última fase da tese para ajudar na investigação e compreensão de resultados.

À Ana, que durante o ano sempre me ouviu e me ajudou em tudo aquilo que precisei, sempre

disponível para me dar apoio e em ensinar aquilo que precisava. Ao Flávio, que da mesma forma

sempre se revelou uma pessoa disponível ajudando nos momentos críticos, sempre transmitindo

sabedoria. À Rita e ao resto do grupo de investigação do professor Frederico, que estiveram sempre

presente na resolução de problemas.

Especialmente, um obrigado à minha família que proporcionou que toda esta experiência fosse

possível de acontecer. Ao André, aos meus amigos, aos colegas que levo do mestrado para a minha

vida, e a tudo aquilo que me ensinaram. Foi sem dúvida uma experiência enriquecedora que me fez

crescer enquanto pessoa com tudo o que aprendi e pelas dificuldades ultrapassadas.

Ao projeto WaterWorks 2014 ERA-NET ID 278 – Biorg4WasteWaterVal+, e à Fundação para a

Ciência e Tecnologia (FCT) pelos projetos Water JPI0001/2014, Water JPI0002/2014 e Water

JPI0003/2014. Ao grupo IBB- Institute for Bioengineering and Biosciences (UID/BIO/04565/2019).

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Abstract

Lupinus albus is known by the commercial potential of their seeds in the food sector, given the high

protein (around 32%) and fiber content (around 16%). Lupin beans are also comprised of quinolizidine

alkaloids (QAs) responsible for a characteristic bitter taste. Hence, to become appropriate for human

consumption, lupin beans require an industrial debittering process to remove QAs, with a huge consume

and discard of water. Lupanine is a QA present in this wastewater at significant concentrations. As a

chiral compound, lupanine can be used to synthesize other added-value alkaloids. Given this, the

identification of novel microorganisms capable of catabolizing lupanine and to reduce chemical oxygen

demand (COD) from lupin beans wastewater (LBW) is a key point in this research. For this aim,

enrichment cultures using LBW or a superficial organic-rich layer formed during summer in LBW were

performed. It was possible to isolate and identify four bacterial (Ochrobactrum anthropi,

Sphingobacterium siyangense, Stenotrophomonas maltophilia, Cellulosimicrobium funkei), two yeasts

(Pichia kudriavzevii, Rhodotorula mucilaginosa) and two filamentous fungal species (Aspergillus

fumigatus, Galactomyces geotrichum). Lupanine-catabolizing capacity was studied in liquid culture

using 1.5 g/L of lupanine. Additionally, microorganisms were grown in sterilized LBW for lupanine and

COD removal studies. The results show that only O. anthropi revealed lupanine removal capacity in

synthetic growth medium (66.7% removal), as well as the highest COD consumption (58.7%) from LBW

culture. Sub-products formation can be further studied, to evaluate if any compound of interest derived

from COD consumption of LBW is formed.

Keywords

Bacteria, bioconversion, enrichment culture, fungi, HPLC, lupin beans wastewater, yeast

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Resumo

Lupinus albus é conhecido pelo valor nutricional das suas sementes - os tremoços - ricos em

proteína (cerca de 32%) e fibras (cerca 16%). Os tremoços contêm naturalmente alcaloides de

quinolizidina, que em concentrações altas são tóxicos. Assim, antes de consumidos os tremoços são

submetidos a um processo industrial que assegura a remoção destes compostos, envolvendo grande

consumo de água e descarte do efluente gerado. A lupanina é o alcaloide de quinolizidina presente

neste efluente em concentrações significativas. Sendo um composto quiral, a lupanina pode ser usada

para sintetizar outros alcaloides de valor acrescentado. O presente estudo tem como objetivo identificar

novos microrganismos capazes de catabolizar a lupanina e reduzir o conteúdo em matéria orgânica do

efluente gerado. Para este objetivo, foram realizados enriquecimentos em cultura usando como inóculo

amostras de efluente ou de uma camada superficial, rica em matéria orgânica, formada na superfície

deste efluente durante o verão. Foi possível isolar e identificar quatro bactérias (Ochrobactrum anthropi,

Sphingobacterium siyangense, Stenotrophomonas maltophilia, Cellulosimicrobium funkei), duas

leveduras (Pichia kudriavzevii, Rhodotorula mucilaginosa), e dois fungos filamentosos (Aspergillus

fumigatus, Galactomyces geotrichum). A capacidade de catabolizar a lupanina foi estudada em cultura

líquida contendo 1.5 g/L de lupanina. Adicionalmente, os microrganismos foram também cultivados em

efluente estéril. Os resultados mostram que O. anthropi tem a capacidade de catabolizar lupanina, (66.7

% decréscimo de concentração), e reduzir o teor em matéria orgânica no efluente (58.7%). A análise

de subprodutos derivados deste efluente pode ser futuramente estudada, de forma a identificar

compostos de interesse.

Palavras-chave

Água residual de tremoço, bactéria, bioconversão, enriquecimento, fungo, levedura

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Table of contents

Acknowledgements ............................................................................................................................ 3

Abstract .............................................................................................................................................. 4

Resumo .............................................................................................................................................. 5

List of Tables ...................................................................................................................................... 9

List of Figures ................................................................................................................................... 11

List of Abbreviations ......................................................................................................................... 16

1. Aim of thesis & Research strategy ........................................................................................ 17

1.1. Aim of thesis ...................................................................................................................... 17

1.2. Research strategy.............................................................................................................. 17

2. Introduction ............................................................................................................................ 20

2.1. Lupinus albus ..................................................................................................................... 20

2.1.1. Quinolizidine alkaloids ............................................................................................... 21

2.1.2. Biosynthetic Pathway ................................................................................................. 22

2.1.3. Industrial Debittering Process .................................................................................... 23

2.1.4. Lupanine industrial interest ........................................................................................ 24

2.2 Membrane processes: a focus on Nanofiltration ............................................................... 25

2.2.1. Lupanine Recovery Processes .................................................................................. 27

2.3. Quinolizidine Alkaloid Quantification Methods .................................................................. 28

2.4. Quinolizidine alkaloid conversion processes ..................................................................... 30

2.4.1. Chemical conversion of lupanine in sparteine ........................................................... 31

2.4.2. Biological conversion of lupanine .............................................................................. 32

2.5. Bioreactors for bioconversion processes .......................................................................... 34

2.6. Biorefinery concept ............................................................................................................ 35

3. Materials and Methods .......................................................................................................... 37

3.1. Nanofiltration .................................................................................................................. 37

3.2. Bacteria .............................................................................................................................. 37

3.2.1. Culture enrichments and isolation of pure strains ..................................................... 37

3.2.2. Gram staining, DNA extraction and strain identification by 16S rDNA amplification . 39

3.2.3. Bacteria cultivation in synthetic growth medium ........................................................ 40

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3.2.4. Bacteria cultivation in lupin beans wastewater .......................................................... 40

3.3. Yeast .................................................................................................................................. 41

3.3.1. Culture enrichment and isolation of pure strains ....................................................... 41

3.3.2. DNA extraction and strain identification by 26S rDNA amplification ......................... 42

3.3.3. Yeast cultivation in synthetic culture medium ............................................................ 43

3.3.4. Yeast cultivation in lupin beans wastewater .............................................................. 44

3.4. Filamentous fungi: a complementary approach ................................................................ 44

3.4.1. Isolation and identification ......................................................................................... 44

3.4.2. Filamentous fungi culture: synthetic culture medium ................................................ 45

3.4.3. Filamentous fungi culture: assessment of using filamentous fungi for lupin beans

wastewater treatment .................................................................................................................... 45

3.5. Determination of lupanine derived-products ...................................................................... 45

3.6. Lupanine quantification by HPLC ...................................................................................... 46

3.7. Chemical Oxygen Demand (COD) measurements ........................................................... 46

3.8. Total reducing sugars determination by colorimetric method (DNS) ................................. 47

4. Results ................................................................................................................................... 48

4.1. Nanofiltration ...................................................................................................................... 48

4.2. Bacteria .............................................................................................................................. 49

4.2.1. Culture enrichments and isolation of pure strains ..................................................... 49

4.2.2. Gram staining, DNA extraction and strain identification by 16S rDNA amplification . 51

4.2.3. Bacteria cultivation in synthetic growth medium ........................................................ 53

4.2.4. Bacteria cultivation in lupin beans wastewater (LBW)............................................... 57

4.3. Yeast .................................................................................................................................. 60

4.3.1. Culture enrichment and isolation of pure strains ....................................................... 60

4.3.2. DNA extraction and strain identification by 26S rDNA amplification ......................... 61

4.3.3. Yeast cultivation in synthetic culture medium ............................................................ 61

4.3.4. Yeast cultivation in lupin beans wastewater (LBW) ................................................... 63

4.4. Filamentous fungi: a complementary approach ................................................................ 65

4.4.1. Isolation and identification ......................................................................................... 65

4.4.2. Growth of isolates in rich mediums............................................................................ 66

4.4.3. Growth of isolates: assessment of using filamentous fungi for lupin beans

wastewater (LBW) treatment ......................................................................................................... 67

4.5. Determination of lupanine derived-products ...................................................................... 69

5. Discussion ............................................................................................................................. 75

5.1. Nanofiltration ...................................................................................................................... 75

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5.2. Bacteria .............................................................................................................................. 75

5.3. Yeast .................................................................................................................................. 83

5.4. Filamentous fungi .............................................................................................................. 86

6. Conclusion ............................................................................................................................. 89

7. References ................................................................................................................................... 90

Annex ............................................................................................................................................. 104

A. Microbial cultures ................................................................................................................. 104

A1. Filter interference in culture ..................................................................................................... 104

A2. OD values of microbial cultures ............................................................................................... 104

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List of Tables

Table 1 - White lupin (L. albus) composition [6]. .............................................................................. 21

Table 2 - Main quinolizidine alkaloids (QAs) identified in seeds of lupin species [16].................... 21

Table 3 – Types of fouling and their main causes. .......................................................................... 27

Table 4 – Limit of detection (LOD) and limit of quantification (LOQ) values of lupanine and sparteine

in lupin grain and lupin-based foods for quinolizidine alkaloid quantification by gas-chromatography

mass-spectrometry [72]. ........................................................................................................................ 30

Table 5 - PCR conditions of 16S rDNA gene amplification. ............................................................ 39

Table 6 - PCR conditions of D1/D2 region amplification. ................................................................ 43

Table 7 – Concentration of lupanine and COD in the feed of wastewater solution, permeate and

retentate of the nanofiltration. ................................................................................................................ 48

Table 8 – Values of pH of the supernatant of the four bacteria isolates: O. anthropi, S. siyangense,

S. maltophilia and C. funkei, measured at different time points during cultivation. ............................... 57

Table 9 – Percentages of COD removal from LBW for the four bacterial strain isolates: O. anthropi,

S. siyangense, S. maltophilia and C. funkei. ......................................................................................... 59

Table 10 – Reducing sugar concentration determined through DNS method for the initial LBW

medium and after 7 days of cultivation of the four bacteria isolates: O. anthropi, S. siyangense, S.

maltophilia and C. funkei. ...................................................................................................................... 59

Table 11 – pH values of LBW + H2O measured in supernatant at different time points, during

cultivation of the four bacteria isolates: O. anthropi, S. siyangense, S. maltophilia and C. funkei. ...... 59

Table 12 - pH values of YNB medium measured in the supernatant at different time points, during

cultivation of the two yeast isolates: R. mucilaginosa and P. kudriavzevii yeast strain isolates. .......... 63

Table 13 – Percentages of COD removal from LBW for the two yeast strain isolates: R. mucilaginosa

and P. kudriavzevii. ............................................................................................................................... 64

Table 14 – Reducing sugars concentration determined through DNS method for the initial LBW

medium and after 7 days of cultivation of the two yeast isolates: R. mucilaginosa and P. kudriavzevii.

............................................................................................................................................................... 65

Table 15 – pH values of LBW medium measured in the supernatant at different time points, during

cultivation of the two yeast isolates: R. mucilaginosa and P. kudriavzevii. .......................................... 65

Table 16 – Lupanine concentration in LBW medium during cultivation of P. kudriavzevii, A. fumigatus

and G. geotrichum ................................................................................................................................. 68

Table 17 – Percentages of COD removal from LBW for the fungi strain isolates. n.a. - COD increased

from 30.83 to 44.71 g O2/L.................................................................................................................... 68

Table 18 – Specific growth rates (µ) of O. anthropi cultures in each growth condition. .................. 78

Table 19 – Some of the carbon and nitrogen sources used by the four bacterial species isolated. (*)

indicates two carbon sources that may be present in LBW. ................................................................. 83

Table 20 – Specific growth rates for both yeasts and culture conditions in LBW. ........................... 85

Table 21 - Some of the carbon sources used by the yeast species isolated. (*) indicates two carbon

sources that may be present in LBW. ................................................................................................... 86

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Table 22 – OD values measured during O. anthropi culture in M9 synthetic medium. (1) And (2) are

the biological replicates. ...................................................................................................................... 105

Table 23 – OD values measured during S. siyangense culture in M9 synthetic medium. (1) And (2)

are the biological replicates. ................................................................................................................ 105

Table 24 – OD values measured during S. maltophilia culture in M9 synthetic medium. (1) And (2)


Table 25 – OD values measured during C. funkei culture in M9 synthetic medium. (1) And (2) are


Table 26 – OD values measured during O. anthropi culture in LBW diluted in water. (1), (2) and (3)


Table 27 – OD values measured during S. siyangense culture in LBW diluted in water. (1), (2) and

(3) are the biological replicates. .......................................................................................................... 107

Table 28 – OD values measured during S. maltophilia culture in LBW diluted in water. (1), (2) and


Table 29 – OD values measured during C. funkei culture in LBW diluted in water. (1), (2) and (3) are


Table 30 – OD values measured during R. mucilaginosa culture in YNB synthetic medium. (1) And


Table 31 - OD values measured during P. kudriavzevii culture in YNB synthetic medium. (1) And (2)


Table 32 - OD values measured during R. mucilaginosa culture in LBW diluted in water and in YNB

medium. (1), (2) and (3) are the biological replicates. ......................................................................... 108

Table 33 - OD values measured during P. kudriavzevii culture in LBW diluted in water and in YNB

medium. (1), (2) and (3) are the biological replicates. ......................................................................... 109

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List of Figures

Figure 1 – Schematic representation of the work strategy followed in this master thesis............... 19

Figure 2 - Chemical structures of lupanine, sparteine, angustifoline and multiflorine, the main QAs

present in lupinus species. Adapted from [2]. ....................................................................................... 22

Figure 3 - Quinolizidine alkaloids biosynthetic pathway. Acyltransferase enzymes involved:

Lysine/Ornithine decarboxylase (L/ODC); Copper amine oxidase (CuAO); (+)-epilupinine/(−)-lupinine O-

coumaroyl/feruloyltransferase (ECT/EFT-LCT/LFT); and (−)-13α-hydroxymultiflorine/(+)-13α-

hydroxylupanine O-tigloyltransferase (HMT-HLT). Dotted lines stand for enzyme reactions not

characterized. Adapted from [225]. ....................................................................................................... 23

Figure 4 - Lupanine concentration of lupin beans wastewater in g/L for the different phases of the

industrial debittering process, according to published research work, in relation with the strategy of this

thesis [29]. .............................................................................................................................................. 24

Figure 5 - Lupanine enantiomers: (+) and (-) [10]. .......................................................................... 25

Figure 6 – Membrane processes with correspondent pore sizes and molecules retained. Adapted

from [36]................................................................................................................................................. 25

Figure 7 - Representation of the interaction between the molecules and the pores. Some will adhere

to the membrane surface (1); others will block the pore (2, 4); and the smaller ones will pass through

the pore (3) [44] ..................................................................................................................................... 26

Figure 8 – Conversion of lupanine into sparteine, through NaBH4 and I2 reducing agents and THF

organic solvent [84] ............................................................................................................................... 32

Figure 9 – Lupanine chemical structure (on the left) and chemical structures of novel end products

A and B, resulting from lupanine bioconversion [87] ............................................................................. 34

Figure 10 - illustration of a biorefinery concept [97]. ....................................................................... 35

Figure 11 - Representation of pyramid of biomass for a biorefinery system [107]. ......................... 36

Figure 12 - Schematic representation of the assays for bacterial isolation on (i) 2 g/L glucose; (ii)

0.75 g/L lupanine or (iii) 2 g/L glucose + 0.75 g/L lupanine, with three sequential lupanine enrichments

in 50 mL working volume. Cultures were inoculated with 5% (v/v) of the former enriched culture. The

microcentrifuge 1.5 mL tubes illustrate the key points of sample collections: day 0, 3 and 7 of each

enrichment, for HPLC analysis; and day 7 of the third enrichment for plating on M9 supplemented with

2 g/L glucose and 1.5 g/L lupanine. ...................................................................................................... 38

Figure 13 - Schematic representation of the assays for bacterial isolation on (i) 2 g/L glucose; (ii) 1.5

g/L lupanine or (iii) 2 g/L glucose + 1.5 g/L lupanine, with three sequential lupanine enrichments in 50

mL working volume. Cultures were inoculated with 5% (v/v) of the former enriched culture. The

microcentrifuge 1.5 mL tubes illustrate the key points of sample collections: day 0 and 7 of each

enrichment, for HPLC analysis; and day 7 of the third enrichment for plating on M9 supplemented with

2 g/L glucose and 1.5 g/L lupanine. ...................................................................................................... 38

Figure 14 - Schematic representation of bacterial isolates growth assays on (i) 2 g/L glucose (G); (ii)

1.5 g/L lupanine (L) or (iii) 2 g/L glucose + 1.5 g/L lupanine (G+L). Pre-cultures were inoculated with

isolates and used for the inoculation of main cultures. Pre-culture flasks contained 25 mL working

file:///C:/Users/joana_000/Documents/Mestrado/2º%20ano/Tese/TESE/FINAL/Isolation%20of%20bacteria%20and%20yeast%20strains%20from%20lupin%20beans%20wastewater-%20assessing%20lupanine%20catabolization_Joana%20Silva.docx%23_Toc26996379











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volume in 50 mL flasks, and culture flasks 50 mL working volume in 100 mL flasks. Cultivations were

performed during 7 days at 30°C and 250 rpm. .................................................................................... 40

Figure 15 - Schematic representation of bacterial isolates growth assays on lupin beans wastewater

(LBW) diluted at 1/2 in sterilized water. Pre-cultures were inoculated with isolates and used for

inoculation of main cultures. Pre-culture flasks contained 25 mL working volume in 50 mL flasks, and

culture flasks 50 mL working volume in 100 mL flasks. Cultivations were performed during 7 days at

30°C and 250 rpm. ................................................................................................................................ 41

Figure 16 - Schematic representation of the assays for yeast isolation on (i) 2 g/L glucose; (ii) 1.5

g/L lupanine or (iii) 2 g/L glucose + 1.5 g/L lupanine, with three sequential lupanine enrichments in 50

mL working volume. Cultures were inoculated with 5% (v/v) of LBW in the first enrichment. Inoculations

of the second and third enrichments were performed with 5% (v/v) of the former enriched culture. The

eppendorf’s illustrate the key points of sample collections: day 0 and 7 of each enrichment, for HPLC

analysis; and day 7 of the third enrichment for plating on YNB supplemented with 1.5 g/L lupanine and

30 µg/mL CAM. ...................................................................................................................................... 42

Figure 17 - Schematic representation of yeast isolates growth assays on (i) 2 g/L glucose (G); (ii)

1.5 g/L lupanine (L) or (iii) 2 g/L glucose + 1.5 g/L lupanine (G+L). Pre-cultures were inoculated with

isolates and used for the inoculation of main cultures. Pre-culture flasks contained 25 mL working

volume in 50 mL flasks, and culture flasks 50 mL working volume in 100 mL flasks. Cultivations

performed during 7 days at 30°C and 250 rpm. .................................................................................... 44

Figure 18 - Schematic representation of yeast isolates growth assays on lupin beans wastewater

(LBW) diluted at 1/2 in sterilized water and 1/2 in YNB medium. Pre-cultures were inoculated with

isolates and used for inoculation of main cultures. Pre-culture flasks contained 25 mL working volume

in 50 mL flasks, and culture flasks 50 mL working volume in 100 mL flasks. Cultivations performed during

7 days at 30°C and 250 rpm. ................................................................................................................. 44

Figure 19 – Values of flux through the membrane during the nanofiltration in function of the

percentage of concentration. Nanofiltration was operated in a concentration mode. The cross in the

curve represent the overnight time when the nanofiltration was stopped. ............................................ 48

Figure 20 – Cultivation assays for bacteria isolation with three sequential lupanine or glucose +

lupanine mixture enrichments. Cultures were inoculated with 5% (v/v) of lupin beans wastewater (LBW)

superficial layer in the first enrichment. Inoculations of the second and third enrichments performed with

5% (v/v) of the former enriched culture. (A) 0.75 g/L lupanine; (B) 2 g/L glucose + 0.75 g/L lupanine. 49

Figure 21 – M9 solid medium supplemented with 1.5 g/L lupanine plated with a sample from culture

with lupanine as carbon source, at the end of third enrichment, representing the enriched population

provenient from lupin beans wastewater (LBW) superficial organic-rich layer. .................................... 50

Figure 22 – Cultivation assays for bacteria isolation with three sequential lupanine enrichments.

Cultures were inoculated with 5% (v/v) of lupin beans wastewater (LBW) in the first enrichment.

Inoculations of the second and third enrichments performed with 5% (v/v) of the former enriched culture.

(A) 1.5 g/L lupanine; (B) 2 g/L glucose + 1.5 g/L lupanine. ................................................................... 50




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Figure 23 - M9 solid medium supplemented with 1.5 g/L lupanine plated with a sample from culture

with lupanine as carbon source, at the end of third enrichment, representing the enriched population

provenient from LBW. ............................................................................................................................ 51

Figure 24 – Agarose gel electrophoresis of PCR products obtained for the three gram-negative

bacterial strains. Agarose gel (0.8%) electrophoresis was run during 40 minutes at 90 V with 50x TAE

buffer. PCR products were loaded in the gel in triplicate. (L) Molecular marker NZY DNA ladder III (1kb);

(1)-(3), (4)-(6) and (8)-(10) are the PCR product corresponding to three gram-negative isolates; (7)

negative control of the reaction. ............................................................................................................ 52

Figure 25 – Agarose gel electrophoresis of PCR products obtained for the gram-positive bacterial

strain, for sonication efficiency testing. Agarose gel (0.8%) electrophoresis was run during 40 minutes

at 90 V with 50x TAE buffer. (L) Molecular marker NZY DNA ladder III; (1) DNA with 3 minutes

sonication; (2) DNA with 5 minutes sonication; (3) positive control of the reaction with known DNA; (4)

negative control of the reaction. ............................................................................................................ 52

Figure 26 - Agarose gel electrophoresis of PCR products obtained for the gram-positive bacterial

strain, sonicated for 3 minutes. Agarose gel (0.8%) electrophoresis was run during 40 minutes at 90 V

with 50x TAE buffer. PCR products were loaded in the gel in quadriplicate. (L) Molecular marker NZY

DNA ladder III; (1)-(4) is the PCR product corresponding to gram positive isolate; (5) is the negative

control of the reaction. ........................................................................................................................... 53

Figure 27 – O. anthropi cultures in M9 liquid medium. Pre-cultures of 48 hours in 1.5 g/L of lupanine

were used as inoculum of the main cultures (A) in 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L lupanine.

Pre-cultures of 48 hours in 1.5 g/L of lupanine with 2 g/L glucose mixture were used as inoculum of the

main cultures (B) in 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L lupanine. Pre-culture of 48 hours in 2

g/L glucose were used as inoculum of the main culture (C) used as a positive control of the experiment.

Growth curves are represented in (A1), (B1) and C, and lupanine concentration in (A2), (B2),

respectively. Error bars represent the respective standard deviation of biological triplicate assays. ... 54

Figure 28 – S. siyangense culture in M9 liquid medium. Pre-cultures of 48 hours in 1.5 g/L of lupanine

with 2 g/L glucose mixture were used as inoculum of the main culture (A) in 1.5 g/L lupanine and 2 g/L

glucose + 1.5 g/L lupanine. Pre-culture of 48 hours in 2 g/L glucose were used as inoculum of the main

culture (B) used as a positive control of the experiment. Growth curves are represented in (A1) and (B),

and lupanine concentration in (A2), respectively. Error bars represent the respective standard deviation

of biological duplicate assays. ............................................................................................................... 55

Figure 29 – S. maltophilia culture in M9 liquid medium. Pre-cultures of 48 hours in 1.5 g/L of lupanine



culture (B) used as a positive control of the experiment. Growth curve is represented in (A1) and (B),



Figure 30 – C. funkei culture in M9 liquid medium. Pre-cultures of 48 hours in 1.5 g/L of lupanine



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culture (B) used as a positive control of the experiment. Growth curve is represented in (A1) and (B),



Figure 31 - Control flask with, separately, M9 culture medium supplemented with 1.5 g/L lupanine

and another flask with 2 g/L glucose + 1.5 g/L lupanine. ...................................................................... 57

Figure 32 – Growth curves (left column) and lupanine concentration (right column) for 7-day cultures

of O. anthropi, S. siyangense, S. maltophilia, C. funkei in LBW medium diluted 1/2 in water. Error bars

represent the respective standard deviation of biological triplicate assays. ......................................... 58

Figure 33 – Cultivation assays for yeast isolation with three sequential lupanine enrichments.

Cultures were inoculated with 5% (v/v) of lupin beans wastewater (LBW) in the first enrichment.

Inoculations of the second and third enrichments performed with 5% (v/v) of the former enriched culture.

(A) 0.75 g/L lupanine; (B) 2 g/L glucose + 0.75 g/L lupanine. ............................................................... 60

Figure 34 – YNB solid media supplemented with 1.5 g/L lupanine and CAM, plated with sample from

culture at the end of third enrichment. ................................................................................................... 60

Figure 35 – Agarose gel electrophoresis of PCR products obtained for the two yeast strains. Agarose

gel (0.8%) electrophoresis was run during 40 minutes at 90 V with 50x TAE buffer. PCR products were

loaded in the gel in triplicate. (M) Molecular marker NZY DNA ladder III; (1)-(3) and (4)-(6) are the PCR

products corresponding to two yeast isolates; (7) negative control of the reaction. ............................. 61

Figure 36 – P. kudriavzevii cultures in YNB liquid medium. Pre-culture of 24 hours in 2 g/L of glucose

was used as inoculum of the main cultures (A) in 2 g/L glucose, 1.5 g/L lupanine and 2 g/L glucose +

1.5 g/L lupanine. Pre-culture of 24 hours in 1.5 g/L of lupanine with 2 g/L glucose mixture was used as

inoculum of the main cultures (B) in 2 g/L glucose, 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L lupanine.

Growth curves are represented in (A1) and (B1), and lupanine concentration in (A2), (B2), respectively.

Error bars represent the respective standard deviation of biological duplicate assays. ....................... 62

Figure 37 – R. mucilaginosa cultures in YNB liquid medium. Pre-culture of 24 hours in 2 g/L of

glucose was used as inoculum of the main cultures (A) in 2 g/L glucose, 1.5 g/L lupanine and 2 g/L

glucose + 1.5 g/L lupanine. Pre-culture of 24 hours in 1.5 g/L of lupanine with 2 g/L glucose mixture was

used as inoculum of the main cultures (B) in 2 g/L glucose, 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L

lupanine. Growth curves are represented in (A1) and (B1), and lupanine concentration in (A2), (B2),

respectively. Error bars represent the respective standard deviation of biological duplicate assays. .. 62

Figure 38 – Control flask for YNB medium supplemented with 1.5 g/L lupanine or 2 g/L glucose +

1.5 g/L lupanine. .................................................................................................................................... 63

Figure 39 – Growth curves (left column) and lupanine concentration (right column) for 7-day cultures

of P. kudriavzevii and R. mucilaginosa in LBW medium diluted 1:2 in water or in YNB medium. Error

bars represent the respective standard deviation of biological triplicate assays. ................................. 64

Figure 40 - Agarose gel electrophoresis of gDNA obtained for the four filamentous fungi strains.

Agarose gel (1%) electrophoresis was run during 90 minutes at 120 V with 1x TAE buffer. (L) Molecular

marker NZY DNA ladder III; (1)-(4) gDNA corresponding to the four fungi isolates. ............................ 66

15

Figure 41 – Dry weight values (left column) and lupanine concentration (right column) for 186 hours

cultures of P. kudriavzevii, A. fumigatus and G. geotrichum in ME medium supplemented with 40 g/L

glucose, 1.5 g/L lupanine or 40 g/L glucose + 1.5 g/L lupanine. Data shown from single assays. ....... 67

Figure 42 – Growth curves in cell dry weight (A) for 192 hours cultures of P. kudriavzevii, A.

fumigatus and G. geotrichum, in LBW medium. Data shown from single assays................................. 68

Figure 43 - Spectra obtained from FT-IR ATR of O. anthropi cultures among time. Pink line

represents the pure sample of lupanine, while the other coloured lines represent the different time points

of culture (0 h, 3 h, 6 h, 12 h and 24 h) being all superimposable. ....................................................... 69

Figure 44 - Spectra obtained from FT-IR ATR of C. funkei culture among time. Violet line represents

the pure sample of lupanine, while the other coloured lines represent the different time points of culture

(0 h in yellow, 48 h in blue, and 120 h in green). .................................................................................. 70

Figure 45 - TLC plate with two elutions in MTBE/hexane (8:2) and 1% diethylamine (DEA) with

phosphomolybdic acid staining. (L) is pure lupanine; (1)-(5) are the samples corresponding to 0 h, 3 h,

6 h, 12 h and 24 h of O. anthropi culture and (6)-(8) are the samples corresponding to 0 h, 48 h and 120

h of C. funkei culture. ............................................................................................................................. 70

Figure 46 - 13C NMR spectrum of a pure lupanine sample in CDCL3 [29]. ...................................... 71

Figure 47 - 1H NMR spectrum of a pure lupanine sample in CDCL3 [29]. ...................................... 71

Figure 48 - 13C NMR spectra of the samples corresponding to O. anthropi and C. funkei cultures. (A)

is the culture of C. funkei at 48 h; (B) is the culture of C. funkei at 120 h; (C) is the culture of O. anthropi

at 3 h; (D) is the culture of O. anthropi at 24 h; (E) is the pure lupanine sample. ................................. 72

Figure 49 – 1H NMR spectra of the samples corresponding to O. anthropi and C. funkei cultures. (A)

is the culture of C. funkei at 48 h; (B) is the culture of C. funkei at 120 h; (C) is the culture of O. anthropi

at 3 h; (D) is the culture of O. anthropi at 24 h; (E) is the pure lupanine sample. ................................. 72

Figure 50 – Chromatogram obtained from 0 h of O. anthropi culture, showing racemic lupanine in a

proportion of 53% of D-(+)-lupanine and 47% of L-(-)-lupanine. .......................................................... 73

Figure 51- Chromatogram obtained from 12 h of O. anthropi culture, showing racemic lupanine in a

proportion of 43% of D-(+)-lupanine and 57% of L-(-)-lupanine. ........................................................... 73

Figure 52 – Chromatograms obtained from 24 h O. anthropi culture, showing 100% of L-(-)-lupanine.

............................................................................................................................................................... 74

Figure 53 - Chromatograms obtained from C. funkei cultures. (A) is the result obtained from 0 h of

culture, with racemic lupanine in a proportion of 53% of D-(+)-lupanine and 47% of L-(-)-lupanine; (B) is

the result obtained from 120 h of culture, with a proportion of 53% of D-(+)-lupanine and 47% of L-(-)-

lupanine. ................................................................................................................................................ 74

Figure 54 – Reaction catalysed by lupanine 17-hydroxylase. ......................................................... 80

Figure 55 – Protonated and deprotonated forms of lupanine, according with the pH of solutions.

Adapted from [29]. ................................................................................................................................. 82

Figure 56 – Lupanine concentration values obtained when filters were used in the inoculum of the

culture. The result is from the S. siyangense culture. ......................................................................... 104

16

List of Abbreviations

ACN Acetonitrile

CQO Carência química de oxigénio

COD Chemical oxygen demand

CuAO Copper amine oxidase

DNS 3,5-dinitrosalicylic acid

ECT/EFT-LCT/LFT (+)-epilupinine/(−)-lupinine O-coumaroyl/feruloyltransferase

GC Gas chromatography

GC-MS Gas liquid chromatography-mass spectrometry

HMT-HLT (−)-13α-hydroxymultiflorine/(+)-13α-hydroxylupanine O-tigloyltransferase

HPLC High-performance liquid chromatography

IER Ion exchange resins

LBW lupin beans wastewater

L/ODC Lysine/Ornithine decarboxylase

LOD Limit of detection

LOQ Limit of quantification

MEA Malt extract agar

MF Microfiltration

MIPs Molecularly imprinted polymers

MTBE Methyl tert-butyl ether

MWCO Molecular weight cut off

NF Nanofiltration

NMR Nuclear magnetic resonance

OD Optical density

PCR Polimerase chain reaction

PDA Potato dextrose agar

QAs Quinolizidine alkaloids

RO reverse osmosis

TLC Thin-layer chromatography

UF Ultrafiltration

YNB yeast nitrogen base

17

1. Aim of thesis & Research strategy

1.1. Aim of thesis

This research work aimed at the isolation and identification of living bacterial, yeast and fungal

species from lupin beans wastewater in liquid medium, following a sequential enrichment culture method

using lupanine or lupanine + glucose as carbon sources. For this, the inoculum used was either a sample

of lupin beans wastewater or an organic-rich layer formed in the surface of this wastewater during

summer time. After the identification of the microorganisms of interest, the main objective was to

evaluate lupanine catabolizing capacity using synthetic culture medium’s in three growth conditions: i)

glucose, ii) lupanine, iii) glucose mixed with lupanine. Microbial catabolization of lupanine can either be

complete leading to the complete mineralization of the substrate or result on intermediate structures,

eventually conserving the core quinolizidine structure, but allowing further molecular modifications. The

industrial debittering process of lupin beans involves discarding high volumes of wastewater rich in

lupanine and organic matter. The study of an alternative process to reduce this waste and further

valorise it would be of high interest for this industry sustainability. In this research work, the isolated

microorganism were also grown in the lupin beans wastewater to evaluate lupanine removal and

reduction of wastewater organic content.

1.2. Research strategy

The higher volumes of wastewater in lupin beans debittering process are generated on the

sweetening phase (Section 2.1.2.), therefore to have a sample representative of the total wastewater

generated in that phase a composite solution was prepared (Section 3.1.). To reduce the volume of the

water needed and wastewater generated on the sweeting phase it is envisaged the use of nanofiltration

to yield a permeate, free of organic matter and lupanine, that can be recycled back on the sweetening

phase, and a retentate rich in lupanine and organic matter, but of a smaller volume. This retentate

fraction of the nanofiltration obtained using the composite sample of the sweetening phase has a

lupanine concentration and COD on the same range of values. Therefore, cooking wastewater was used

to assess the presence of microorganisms, and also as a representative of the retentate that would be

obtained after a nanofiltration of the sweetening phase.

For bacteria isolation, a sample of LBW surface organic-rich layer and from LBW was enriched in

three sequential enrichments in three conditions using M9 liquid culture: (i) glucose; (ii) lupanine and

(iii) glucose with lupanine mixture. Dilutions of the third enrichments were plated in M9 solid medium

(with lupanine) for the bacterial isolation. Gram staining was performed to four pure cultures, to assess

the most efficient DNA extraction protocol. PCR was used to amplify 16S rDNA gene and the sequencing

step was performed by STABVIDA. After species identification, the four strains were individually grown

18

in M9 liquid medium in (i), (ii) and (iii) to measure lupanine concentration by HPLC, OD600 nm and pH

values. Additionally, the four isolates were also grown in sterilized LBW diluted 1/2 times in sterilized

water to asses COD and lupanine values, OD600 nm, and total reducing sugars.

For yeast isolation, a sample of LBW was enriched in three sequential enrichments in the three

mentioned conditions using YNB liquid culture: (i), (ii) and (iii). Dilutions of the third enrichments were

plated in YNB solid medium (with lupanine) for the isolation of yeast colonies. DNA of pure cultures was

extracted and PCR was used to amplify 26S rDNA gene, the sequencing step was performed by

STABVIDA. The two yeast isolates were grown in liquid YNB in (i), (ii) and (iii) to measure lupanine

concentration by HPLC, OD600 nm and pH values. Additionally, the two isolates were also grown in

sterilized LBW diluted 1/2 in sterilized water and in 1/2 YNB culture medium, to asses COD and lupanine

values, OD600 nm, and total reducing sugars.

Filamentous fungi isolation started with dilutions of LBW and its surface organic-rich layer in PDA

and MEA solid culture medium to obtain single colonies. DNA was extracted from pure solid cultures

and sequencing by STABVIDA. After species identification, the isolates were grown in synthetic growth

medium ME in (i), (ii) and (iii) conditions for cell dry weight assessment and lupanine concentration by

HPLC. Additionally, the isolates were also cultivated in LBW for assessment of cell dry weight, lupanine

concentration by HPLC and COD.

The schematic representation of the work strategy is shown below (Figure 1).

19

Figure 1 – Schematic representation of the work strategy followed in this master thesis.

20

2. Introduction

2.1. Lupinus albus

Plant species of the genus Lupinus belong to the Fabaceae (or Leguminosae) family. The name of

the genus “Lupinus” arises from the Latin word ‘’Lupus’’, which means wolf, since Romans thought that

lupin beans took nutrients from the soil in the same way that a wolf can capture domestic animals [1].

Among the 400 Lupinus species already known, only four are characterized by their agronomic and

commercial potential: Lupinus albus (white lupin), Lupinus angustifolius (blue lupin), Lupinus luteus

(yellow lupin), and Lupinus mutabilis (Andean lupin). Lupinus plants have been cultivated for over 3000

years, initially in the Mediterranean, Australia, North Africa and North and South America. Nowadays,

lupin beans are predominantly distributed in South and North America, with a few species in Europe and

North Africa [2], and a wide distribution of L. albus in Mediterranean regions [3], [4]

Lupin seeds are produced in pods, which are structures on the main stem of the plant. Depending

on the species of lupin, pods can vary between three and seven seeds with different sizes, colours,

appearances and compositions. Among them, Lupinus albus seeds are the larger ones, with a circular

flattened shape and a creamed colour [5]. The life cycle of lupins is composed of three phases: i)

vegetative; ii) floral and iii) pod and seed growth phase. The progression of the plant among the different

phases is dependent on temperature and day length. However, in 14 to 20 weeks, the flowering phase

halts and all nutrients of the plant are redirected from growth to seed filling [6]. The lupin beans can be

incorporated into variety of food products as supplement for human food (in bakery, pastry, crisps, yogurt

analogues) thus providing functional properties such as nutritional value, aroma and texture. Due to the

high biomass above the ground and deep roots – recurrently down to 2 meters – of Lupinus albus, the

plant is also important in the soil aeration for oxygen and water supply (supporting the growth and

survival of other plants), and can be harnessed for i) livestock forage and ii) green manure, a fertilizer

consisting of growing plants [5], [7].

Lupinus albus seeds are rich sources of protein and dietary fibre, with around 32% and 16%,

respectively percentages that can vary with genetic and environmental differences [4], [3]. They also

contain vitamins and antioxidants such as tocopherols, carotenoids, B-vitamins and phenolic

compounds [4], [5]. For these reasons, lupin seeds are suitable for the food sector, and given their

composition (See Table 1), they can be applied as a food supplement either for animal feed, or for

human diet. Although, L. albus varieties are also comprised of quinolizidine alkaloids (QAs), which

confer a characteristic bitter taste to lupin beans. To make these beans appropriate for consumption, it

is required a treatment process for their fast removal [10].

21

Table 1 - White lupin (L. albus) composition [6].

Components Mean Value (%)

Moisture

8.32±0.03

Crude protein 32.20±1.10

Crude fibre 16.20±1.51

Oil 5.95±0.09

Ash 2.65±0.18

Acidity 0.13±0.02

2.1.1. Quinolizidine alkaloids

Quinolizidine alkaloids (QAs) are toxic compounds composed of nitrogenous bases in a heterocyclic

ring, more specifically a quinolizidine ring. They are secondary metabolites produced by Leguminosae

plants to protect them against insects [7]. These compounds, in high concentrations, are also dangerous

for human consumption, causing adverse health effects like acute anticholinergic toxicity, blurry vision,

nausea, headache or weakness [11]. The symptoms indicate that alkaloids can have negative

neurological impacts, resulting in loss of muscular control and coordination, effects that are usually

reversible [2]. QAs exert a blocking effect on the nicotinic cholinergic receptor, being weak antagonists

at the muscarinic cholinergic receptor [12]. Their neurotoxicity has been assessed by intravenous

administration of sparteine and lupanine, with observed inhibition of ganglionic transmissions of the

sympathetic nervous system [13]. Previous studies indicated that QAs content of wild Lupinus species

is up to 2.7 mg/100mg dry matter [14], a value above the limit traced by the health authorities of UK,

Australia, France and New Zealand of 200 mg/kg of food product [15].

The composition and content of QAs varies among lupin species (See Table 2). According to the

genetic varieties, environmental conditions or soil characteristics, the QAs content may also differ,

nevertheless, lupanine seems to be the most abundant. Moreover, the alkaloids present and their

concentrations can vary in different parts of the plant: seeds, leaves, roots or flowers [2].

Table 2 - Main quinolizidine alkaloids (QAs) identified in seeds of lupin species [16].

Lupin species Major grain QAs (% of total alkaloids) References

L. angustifolius

Lupanine (70%), 13α-hydroxylupanine (12%), angustifoline (10%) Lupanine (50.6%), 13α-hydroxylupanine (32.6%), angustifoline

(10.4%), isolupanine (6.4%; average values) [17], [18]

L. albus

Lupanine (70%), albine (15%), 13α-hydroxylupanine (8%), multiflorine (3%)

[17]

L. luteus

Lupanine (60%), sparteine (30%), unknown alkaloids (1%) Gramine (10-89%), lupinine (3-60%), sparteine (1-20%), isosparteine

(1-5%) [17], [19]

L. mutabilis

Lupanine (46%), sparteine (16%), 3β-hydroxylupanine (12%), 13α-hydroxylupanine (7%), ammodendrine (2%), 13-angeloyloxylupanine

(2%), tetrahydrorhombifoline (2%), 11-12-dehydrosparteine (1%), angustifoline (1%), 13-tigoyloxylupanine (1%)

[17]

22

Among the QAs present in lupinus species, sparteine and lupanine are considered the two molecules

with higher toxicity [20]. The main chemical structures of these QAs are represented in Figure 2.

Some beneficial properties of L. albus seeds have also been described. In folk medicine, the seeds

are boiled and the resulting water can be used as a diuretic agent or antiparasitic drug. They may be

used for treatments of liver disorders, haemorrhoids, diabetes or eczema [21]. The QAs also present

interesting pharmacological properties such as cytotoxic, antipyretic, antibacterial, antiviral and

hypoglycemic activities, as showed in studies for in vivo pharmacological screenings [22]. Particularly,

lupin leaves extract showed antibacterial and antifungal activities against microorganism species such

as, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans [21].

2.1.2. Biosynthetic Pathway

In the past decades, biosynthetic ways to obtain the QAs have been studied. In the research works

conducted, a variety of genes, transcription factors or transporters, enzymes and intermediates

associated with alkaloid biosynthesis were discovered [23], [24]. For synthesis of QAs, the first step is

L-Lysine decarboxylation, with cadaverine formation, which is the first intermediate of the biosynthetic

pathway [2]. Afterwards, the cadaverine is deaminated, originating a piperidine Schiff base that is

involved in hydrolysis, oxidative deamination and coupling reactions. This results in the major structure

of QAs [25], the diiminium cation, which is an intermediate in the biosynthesis of tetracyclic alkaloids

(e.g., lupanine, multiflorine, sparteine) (Figure 3). Once these QAs are formed, additional modifying

steps like dehydrogenation, oxygenation, glycosylation, hydroxylation or esterification can occur,

originating a range of structurally related QAs [26], [27].

Figure 2 - Chemical structures of lupanine, sparteine, angustifoline and multiflorine, the main QAs present in

lupinus species. Adapted from [2].

23

2.1.3. Industrial Debittering Process

As referred above, alkaloid-rich lupin beans require a debittering process previously to human

consumption. This process involves the soaking, cooking, and washing of the lupin beans. Since QAs

are water-soluble, they are released from the lupin beans to the water [15]. Hundreds of tons of L. albus

beans are processed per year, this debittering process allows to reduce the contents of alkaloids in the

beans from almost 3% (27 g/kg) to less than 0.02% (0.2 g/kg) [28]. The main concern with this process

is the high volumes of wastewater generated that implies the high volume of water consumed per batch.

These wastewater effluents are rich in lupanine, the most abundant QA in L. albus beans.

The industrial debittering process applied is composed by three stages: i) hydration (phase 1); ii)

cooking (phase 2); iii) sweetening (phase 3). In the first phase, the beans are submerged in water and

they increase in volume. In the second phase, the soaked beans are baked in water at high temperature

together with water vapour, and the QAs start to be released into the boiling water. In the second phase,

the temperature of the cooked beans decreases and they stay in a fresh water tank, resulting in the

phase with a higher concentration of lupanine (Figure 4). In the third phase, the beans are sweetened,

which involves washing with high volumes of fresh water [29].

For food industry applications of lupin beans, the debittering process has the disadvantage of

removing part of the soluble protein, oligosaccharides and mineral salts present in the beans, together

with the lupanine [30].

Figure 3 - Quinolizidine alkaloids biosynthetic pathway. Acyltransferase enzymes involved: Lysine/Ornithine

decarboxylase (L/ODC); Copper amine oxidase (CuAO); (+)-epilupinine/(−)-lupinine O-

coumaroyl/feruloyltransferase (ECT/EFT-LCT/LFT); and (−)-13α-hydroxymultiflorine/(+)-13α-hydroxylupanine O-

tigloyltransferase (HMT-HLT). Dotted lines stand for enzyme reactions not characterized. Adapted from [225].

24

Figure 4 - Lupanine concentration of lupin beans wastewater in g/L for the different phases of the industrial

debittering process, according to published research work, in relation with the strategy of this thesis [29].

At the industrial scale there is interest in the recovery, purification and valorisation of lupanine, from

the enriched wastewater effluent. In this study, it is used membrane technology for the recovery and

concentration of lupanine in the retentate.

2.1.4. Lupanine industrial interest

In general, alkaloids have the potential to be used in the treatment of a variety of medical conditions,

either as anticancer agents, or analgesics and as drugs to regulate hypertension and manage central

nervous system disorders [31]. Wiedemann et al. (2015) studied the effect of lupanine on type-2

diabetes mellitus, using an animal model. [32]. In rats with hyperglycemia induced by using 15 mmol/L

glucose, the presence of 0.5 mmol/L lupanine contributed to enhance the secretion of insulin, while for

the same conditions with lower glucose concentrations no insulin secretion was observed. The effect

observed is correlated with membrane depolarization and frequency increase of Ca2+ action potentials.

Additionally, lupanine inhibited the ATP-dependent K+ channel and triggered glucose-stimulated insulin

release through ATP-dependent K+ channels, increasing insulin gene expression. Therefore, the

antidiabetic potential of lupanine is evidenced by the improvement of glucose tolerance in rats with

induced hyperglycemia, which can be advantageous for the supportive treatment of type-2 diabetes

mellitus [32].

Lupanine is a tetracyclic QA with a chiral symmetric structure (Figure 5), with the potential of being

useful for pharmaceutical industries as a starting material for the synthesis of a variety of other added-

value alkaloids, such as sparteine [33]. Molecules that are non-superimposable mirror images of one

another are defined as enantiomers, and result from the chirality of organic compounds [34]. However,

the presence of stable amine groups in lupanine can difficult target chemical modifications [35]. 3.

25

Figure 5 - Lupanine enantiomers: (+) and (-) [10].

2.2 Membrane processes: a focus on Nanofiltration

Membrane technologies are currently applied in water and wastewater treatments due to stringent

water quality standards, with varied membrane processes available. There are four categories,

according with the membrane pore size: microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and

reverse osmosis (RO). Each one of these processes require different membrane types, meaning that

for instance a reverse osmosis membrane is essentially non-porous, retaining the majority of the solutes

including ions, and operating at high pressure: 20-100 bar. The NF membranes are capable of retaining

ions and organic molecules with low molecular weight. These membranes have a considerably higher

water permeability, and lower operating pressure, usually from 7 to 30 bar, in comparison with the RO

membranes. UF membranes retain colloids, macromolecules and microorganisms, with operating

pressures ranging from 1 to 10 bar [36]. Figure 6 illustrates the different filtration categories and

associates them with the corresponding applications and pore sizes.

Figure 6 – Membrane processes with correspondent pore sizes and molecules retained. Adapted from [36]

Potable water can be obtained through RO processes in sufficient amount to be used for domestic

or industrial applications [37]. NF, a pressure-driven membrane process commonly used for liquid-phase

separations and presenting higher flow rates with lower energy requirements [36], [38]. The membranes

used in this separation process have properties between RO membranes (where the separations are

conducted by a mechanism of solution-diffusion), and UF membranes (in which separation occurs

26

according to size exclusion and charge effects). The NF membranes commercially available are usually

made of polymers exhibiting surface charge, according with the matrix or surface groups e.g. amide,

sulphonated or carboxylic acid groups. Thus, NF membranes are able to separate ions through a

combination of molecule size and ion interaction mechanisms [39]. For these reasons, they are useful

in the selection and fractionation of solutes from complex process streams, which makes them a viable

option for different industries, such as: pharmaceutical, biotechnological and food engineering [40].

Additionally, nanofiltration proved to be a promising technique for the treatment of water with natural

organic matter and pollutants, being a process used in wastewater treatment [36]. In the case of natural

organic compounds removal from water surfaces, sieving mechanisms occur, due to the larger size of

the molecules present when compared to the membrane pore size (Figure 7) [41], [42]. Therefore, it is

important to select the membrane considering its molecular weight cut off (MWCO), and according to

the desired application. The MWCO, defined as the minimal molecular weight at which a solute of known

molecular weight is 90% rejected by the membrane, can be obtained by determining the membrane

rejection for different molecular weight solutes [43].

Figure 7 - Representation of the interaction between the molecules and the pores. Some will adhere to the

membrane surface (1); others will block the pore (2, 4); and the smaller ones will pass through the pore (3) [44]

Like other membrane separation processes, NF main problem associated is the fouling. The fouling

is related with the size of the particles and membrane pores, occurring when the accumulation of these

particles on the membrane surface blocks the passage of other molecules [45]. Although, other factors

besides the pore size can affect the fouling. Some studies state that the fouling may also be related with

membrane properties such as hydrophobicity and permeability [46] or roughness, where particles are

favourably transported to the membrane valleys, resulting in “valley clogging” [47]. There are four

different forms of fouling: i) adsorption – interaction between membrane and solutes on the membrane

surface or in the pores; ii) pore blocking – obstruction of membrane pores with solute particles, due to

their size; iii) cake layer formation – sedimentation on the membrane surface of particles larger than the

pores; and iv) gel layer formation – formation of a gel layer in the membrane surface [48]. It is also

possible to characterize fouling in function of the type of molecules that participate in this phenomenon,

as summarized in Table 3. Fouling has a direct influence in the NF process, leading to flux decline (with

decreased productivity), consequently increasing the process costs due to higher operation time, energy

demand and membrane degradation and replacement [49].

27

Table 3 – Types of fouling and their main causes.

2.2.1. Lupanine Recovery Processes

Carmali et al. (2010) have demonstrated an experimental procedure for the recovery of lupanine

from Lupinus albus leaching waters through a membrane process of osmotic evaporation [10]. The

concentration occurs through a porous hydrophobic membrane separating a diluted aqueous solution

from a concentrated osmotic one. Since the membrane is hydrophobic, it establishes vapour-liquid

interfaces within the pores. A water flux that crosses this interface of air in the pore is caused by water

vapour pressure differences, which is induced by the concentration between both solutions. Therefore,

evaporation occurs in one side of the membrane and condensation occurs in the other [10].

Concentrated osmotic solutions are commonly saline, creating high osmotic pressure differences. In

general, sodium chloride (NaCl) is used, but other electrolytes such as magnesium chloride (MgCl2),

calcium chlorie (CaCl2) or magnesium sulfate (MgSO4) can be applied [54]. Although the low water

fluxes obtained represent a process limitation, the use of mass transfer equipment with enhanced

surface area may minimize this issue [55]. Carmali et al. (2010), successfully obtained a 10-fold

concentrated wastewater, using a calcium chloride solution with lupanine recovery from the

concentrated effluent through ethyl ether extraction. A fraction of 740 g of lupanine was recovered from

3 tons of a leaching batch of L. albus seeds, corresponding to a recovery of 18.5 % of the lupanine

present in the concentrate. Taking into account that a case study where a specific operation would

process 280 tons of L. albus per year through debittering process, this implies that 70 kg of lupanine

could be obtained with 90% purity, representing only 18.5% recovery [10].

Type of fouling Definition References

Organic Fouling

Particles like proteins, polysaccharides or organic

matter deposition on the membrane surface.

[52], [50]

Colloidal Fouling

Minerals, metal oxides, salt precipitates accumulation

on the membrane surface.

[51]

Scaling

Precipitation of ionic products like calcium acetate or

calcium carbonate, among others.

[52]

Biofouling

Adhesion or growth of microorganisms (bacteria, fungi) on the membrane surface, with possible agglomeration

of extracellular products.

[53]

28

Previous studies from BERG-IBB group at IST demonstrated other approaches for the water

treatment, such as: ultrafiltration and subsequent nanofiltration (previously described) with lupanine

recovery of around 99% [29], followed by purification using resins or liquid-liquid extractions. Another

approach tested the use of molecularly imprinted polymers (MIPs), using lupanine as the template and

enabling its isolation [29], [56].

Resins can be classified as either ion exchangers resins (IER) or polymeric adsorbents, and can be

used for the removal of impurities from mixtures, leading to increased selectivity of the desired product

[57]. IER selection occurs through an exchange of ions that can be cationic or anionic, according with

the ionic form of the product, thus being attached to the resin [58]. Previous studies demonstrated that

IER presents promising results for an efficient removal of metal ions from wastewaters, and their

recovery without toxic sludge production [59]. IER already proved to be effective in the treatment of

textile dye wastewaters, leading to increased quality of the treated wastewater effluent and enabling its

reuse in the textile industry [60]. Adsorbent resins are useful for pharmaceutical industries in the

purification of amino-acids, vitamins, antibiotics or peptides [61]. In general, these resins are cheap with

simple steps of binding and regeneration. For lupanine binding, both IER and adsorbent resins were

tested with the cooking phase of the industrial debittering process resulted in similar results for both

resins, around 70% of lupanine recovery from the wastewater [56].

In the case of liquid-liquid extractions, it involves the separation through transfer of a solute from one

solvent to another, where both are immiscible and form two different phases. This type of extraction is

composed by a mixing step, followed by separation of the two liquid phases. Downstream recovery of

fermentation products or wastewater treatments are examples of applications that use these extractions

[62], [63]. For the case of lupin beans wastewater, values close to 100% of lupanine can be extracted

with solvents like dichloromethane (DCM), toluene, 1-octanol, 1-butanol and methyl tert-butyl ether

(MTBE) [56].

The molecular imprinting approach aims to create specific binding sites for a given target molecule.

There are three key components in this technique: a template, a monomer and a cross-linking agent.

The target molecule that acts as a template interacts with cross-linked monomers that together form a

unique structure. The stability of this structure is dependent on the link between the template and

functional groups of the monomer. Therefore, the basic principle of this process is the ability of the

receptors to recognize their targets and bind to them [64]. The study of the most appropriate monomer

to obtain a MIP using racemic lupanine as template as already been studied. This experimental

approach enabled around 60% recovery [56].

As seen, it is possible to conclude that in comparison with Carmali et al. (2010) process for lupanine

recovery, the studies from BERG-IBB group at IST using either resins, liquid-liquid extractions or MIPs

revealed higher lupanine recovery percentages.

2.3. Quinolizidine Alkaloid Quantification Methods

Nowadays, a wide variety of known alkaloid determination methods is kwown. Examples of

qualitative tests are the taste testing, the Dragenforff test, fluorescence assays, thin-layer

chromatography (TLC) and nuclear magnetic resonance (NMR). While others such as, high-

29

performance liquid chromatography (HPLC), gas chromatography (GC), gas liquid chromatography-

mass spectrometry (GC-MS), are quantitative [65].

The taste testing was first used in lupin beans and relies on the bitter taste characteristic of QAs,

where tasters have to taste lupin beans seeds [65].

Dragendorff’s test involves the use of a reagent that is able to detect alkaloids, heterocyclic nitrogen

compounds and quaternary amines, in vegetal samples of Lupinus species like seeds, leaves or pods.

The Dragendorff reagent reacts with high QAs patterns (higher than 0.5%) present in lupins [66],

enabling visualization of the existing alkaloids.

Thin-layer chromatography is a qualitative and semi-quantitative analysis widely adopted for analysis

of alkaloids. The extracted metabolite samples are applied to a TLC plate and migrate with the help of

an eluent system, allowing the separation of different compounds. Here, the main problem is to separate

alkaloids with varying polarity, being necessary specific TLC systems [67].

Fourier Transform Infrared Spectrometer (FT-IR) is commonly used in a variety of fields such as

organic synthesis, polymer science, petrochemical engineering and pharmaceutical industries. The

mechanism of FTIR technique is in relation with transitions between vibrational energy states. The

absorption of IR radiation occurs when a proton transfers to a molecule and excites it to a higher energy

state, resulting in the vibrations of molecular bonds at different wavelengths in the IR region of the

spectrum [68]. Each IR absorbance peak has a specific wavenumber determined by the

physicochemical properties of the respective molecule, being like a fingerprint of that functional group

(e.g. C=O, O-H, etc). FITR techniques are divided in potassium bromide (KBr)-pellet FTIR, attenuated

total reflection (ATR)-FTIR and diffuse reflection infrared Fourier Transform (DRIFT) spectroscopies.

ATR-FTIR provides chemical information’s on functional groups without the need of preparing a pellet

[69]. Previous studies have already demonstrated the possibility to detect QAs using ATR-FTIR [70].

Nuclear magnetic resonance is mainly a qualitative technique that focus on the interaction between

matter and electromagnetic forces, subjecting a sample to two magnetic fields: one stationary and the

other in variation with a specific radio frequency. Energy is absorbed by the sample at specific

combinations of fields. In QAs identification, there are two NMR spectra: one for the carbon, 13C isotope,

and the other for the hydrogen, the proton. Other technique for QA characterization is X-ray, in which

the three-dimensional structure of a molecule is obtained in this technique, giving a precise

characterization of the compound. Here, X-rays are absorbed by the sample and an absorption spectra

is given, contributing to the identification of the alkaloid [65].

In terms of quantitative techniques, high-performance liquid chromatography is highly sensitive and

is used for alkaloid analysis, being especially useful when pure standards are available. It is composed

of a stationary phase that can have surface absorption or ion exchange interactions [71], and a mobile

phase that transports the sample.

Nowadays, the most common and accurate method for QAs quantification, is gas-chromatography,

many times combined with mass spectrometry [72]. Gas chromatography, similarly to HPLC, is a quick

technique for the determination of alkaloids in a mixture. This technique requires temperature stability

for maintaining the sample in the gaseous state [73]. Depending on the nature of the stationary phase,

gas chromatography is divided into two subclasses: gas-solid chromatography or gas-liquid

30

chromatography. Mass spectrometer is capable of doing the mass spectrum identification from the

alkaloid analysis through molecular fragmentation [65]. QAs quantification is often based on standard

curves of major alkaloids (for instance: lupanine, gramine or sparteine) or internal standards (caffeine),

which are applied to estimate concentrations of several other QAs [74]. For gas-chromatography mass

spectrometry, there are a limit of detection (LOD) and a limit of quantification (LOQ) stated for lupin grain

and lupin-based foods, represented for lupanine and sparteine in Table 4.

Table 4 – Limit of detection (LOD) and limit of quantification (LOQ) values of lupanine and sparteine in lupin

grain and lupin-based foods for quinolizidine alkaloid quantification by gas-chromatography mass-spectrometry

[72].

Compound Source LOD (µg/mL) LOQ (µg/mL) Reference

Lupanine Lupin grain

Lupin-based foods 2

7.9 3

18.2 [75] [74]

Sparteine Lupin grain

Lupin-based foods

1 30.5

3 87

[76] [75]

As we can see, there is a wide variety of methods for QAs analysis. The results of measurements of

each technique are not comparable, which can be a problem when plant materials are being compared.

Other problems are based in the presence of impurities on the alkaloid extracted sample through

different processes. For these reasons, it is possible to state that there is not an infallible detection

and/or quantification method [65].

2.4. Quinolizidine alkaloid conversion processes

Alkaloid transformation is an interesting topic, since many valuable compounds (for instance: drugs)

are derived from these natural molecules, providing new or already known intermediates for the

synthesis of improved or new drugs. Additionally, since they can also act as drugs by themselves, there

is a great interest in the potential of alkaloids. For these reasons, alkaloids represent new targets for

modern analyses, challenging organic chemists to develop innovative methodologies [77]. Therefore,

the synthesis of these complex natural compounds has been extensively studied in the beginning of the

20th century, where the synthesis of complex alkaloids such as quinine, served as a base for the modern

heterocyclic chemistry [78]. However, the complex characteristics of many alkaloids and the functional

groups associated, make chemical modifications a time-consuming and difficult process, frequently

resulting in low yields [31].

Scientists also face the challenge of comprehending the biochemical mechanisms involved in the

formation and biotransformation of alkaloids in living systems. Alkaloid substrates require enzymes

capable of catalysing chemical transformations, and that mimics those existing in microorganisms,

plants and mammalians. Relevant and interesting enzyme reactions with alkaloids include oxidations,

reductions and hydrolysis [77]. Enzyme modifications are an interesting and attractive alternative to

chemical synthesis, since they are generally able to operate at non-extreme pH values and temperatures

with decreased levels of toxic waste products [31]. There are a few cases of microbial transformation

of alkaloids already studied. Pseudomonas putida M10 has been isolated from industrial waste liquors

31

at an opiate-processing plant and apparently, this strain is able to use morphine to support its growth

[79]. In this study, hydroxylation of morphine alkaloids occurred, representing an industrially important

conversion since a small change in the alkaloid structure can have a large impact in its activity. The

hydroxylation resulted in significantly enhanced analgesic capacity that is chemically hard to obtain, due

to an unreactive tertiary carbon [79]. Another example is based in ergot alkaloids, in which conversion

by the bacterial strain Rhodococcus equi A4 yields lysergic acid, with a large spectrum of

pharmacological activities, commonly produced by alkaline hydrolysis of peptide ergots. From these

cases, it is possible to state that despite the complex structure of some alkaloids, microorganism

systems are able to transform or degrade them, giving rise to interesting products and applications [31].

In the context of wastewater treatments, the complete biological degradation of the alkaloid content is

also interesting.

The identification of new biological catalysts, able to transform alkaloids, has been a topic of interest

for research. In this field, microbial transformation systems are advantageous over other systems, since

biomass production is fast. These systems may lead to the production of interesting intermediates or

metabolites in sufficient quantities for their identification and further assessment as drugs concerning

their clinical potential. Additionally, in microbial systems, it is possible to express enzymes involved in

alkaloid biosynthesis in heterologous hosts, thus enabling the study of unknown catalytic mechanisms

beyond this process. Boonstra et al., (2001) have developed a recombinant bacterial morphine alkaloid

transformation system using genes from Pseudomonas putida M10 to clone and express in E. coli [79].

In this study, a whole-cell system was comprised of constitutively expressed NADP+-dependent

morphine dehydrogenase and NADH-dependent morphinone reductase. The recombinant system

generated is able to transform morphine or codeine into hydromorphone or hydrocone, respectively.

2.4.1. Chemical conversion of lupanine in sparteine

Sparteine is an alkaloid that can activate a muscarinergic acetylcholine receptor and inhibit N+

channels and Na+ ion flux. When used in small dosages, this alkaloid acts as a stimulant, but high

dosages lead to a paralyzing effect on the autonomic ganglia. An example of a sparteine medical

application is the correction of cardiac arrhythmias [80]. The enantiomer L-(-)-sparteine is widely used

as a ligand or inducer for several asymmetric reactions [81].

Lupanine and sparteine are both tetracyclic quinolizidine alkaloids (Figure 1), and two important

pharmaceutical compounds. The chemical preparation of pure lupanine and the enantiomer D-

(+)sparteine has been reported, however, since it requires multi steps, these approaches are not

practical. In 1931, the resolution of lupanine with a chiral acid (L-camphorsuphonic acid and D-

camphorsulphonic acid) was demonstrated, yielding 9.4 % of L-(-)-lupanine and 13% D-(+)-lupanine,

with subsequent reduction with red phosphorus, giving rise to both D-(+)-sparteine and L-(-)-sparteine

[82]. Przybył and Kubicki (2011), performed an optimization of the previous process using

dibenzoyltartaric acid and a nontoxic reductant [83]. Nevertheless, safety and post treatment issues

regarding the high solvent manipulation and the large amount of reductant used are the major

32

drawbacks in this process [83]. Thus, the need for a practical and economic process to obtain

enantiopure lupanine and sparteine remains an important issue.

More recently, a process for converting lupanine into sparteine was patented by Nuno Maulide, Bo

Peng, Carlos Afonso and Raquel Frade [84]. In this process, raw lupin seeds were subjected to an

alkaline solution, with a lupanine extraction step and treatment with pure enantiomers of tartaric acid,

which is a simple chiral acid. Through this process, it was possible to achieve pure lupanine

enantiomers. The enantiopure sparteine was obtained by reduction of lupanine with the catalytic agent

NaBH4. The advantage of this process is the reduced amount of organic solvents and halogenates used.

The resolution procedure with tartaric acid yielded 29% D-(+)-lupanine and 30% L-(-)-lupanine. The

highest percentage of conversion of lupanine enantiomer (88%) in sparteine was obtained using NaBH4

and I2 as reducing agents and THF as organic solvent, as illustrated in Figure 8.

In this reaction, if the temperature is further enhanced to 80 °C, only 16 hours are required to obtain

a yield of 88%. Therefore, this is considered a convenient and economic process to obtain the two

alkaloids, particularly sparteine [84].

2.4.2. Biological conversion of lupanine

The Industrial debittering process described in section 2.1.2, showed that the removal of alkaloids

from lupin beans imply a huge amount of water use and industrial wastewater being discarded. This

wastewater, rich in lupanine, has a negative environmental impact, calling for an economically viable

wastewater detoxification process successfully implemented. Bitter lupin extracts can be used either as

biological growth stimulator in agriculture or for pharmacological purposes [85], [86].

As seen in Section 2.1.3., due to stable groups in its structure, lupanine is not easily chemically

modified to originate other added-value alkaloids. For this reason, bioconversion of lupanine using

different microorganism has been studied in the last years [30], [87], [88].

In 1996, Santana et al. isolated bacterial strains capable of using lupanine as a carbon and energy

source [30]. These strains were isolated from the soil where L. albus and L. luteus species were

cultivated. Among the fifteen different strains obtained, IST20B and IST40D (from L. luteus) were the

ones with higher maximum specific growth rates when tested in the presence of medium supplemented

with lupanine (2 g/L), with a value of 0.13 h-1. In this growth media, the strains were able to remove

around 99% of the lupanine in the stationary phase, according to the gas-liquid chromatography results.

However, IST40D had slightly lower biomass yield (0.33 g dry biomass/g lupanine removed) in

comparison with IST20B (0.4 g dry biomass/g lupanine removed), indicating that the strains must

Figure 8 – Conversion of lupanine into sparteine, through NaBH4 and I2 reducing agents and THF organic

solvent [84]

33

catabolize lupanine in a different manner. Metabolic fingerprints of the bacterial isolates were performed,

demonstrating that both species catabolized the majority of the amino acids, amines, alcohols and a

considerable number of carboxylic acids. For species identification, Microlog and BIOLOG systems

(metabolic profile systems) were applied, but the results showed that none of the species was

successfully identified in the study, despite the similar metabolic fingerprint among them. IST20B strain

was also grown in a lupin aqueous extract obtained from batch leaching of portions of 50 g og L. albus

flour in 200 mL of water for 4 h (45°C) with stirring, having 7.9% total alkaloids (lupanine equivalents)

and 4.3% total nitrogen. The authors assessed the content of alkaloids and proteins during IST20B

growth, concluding that when stationary phase was reached (after 32h at 27°C) the concentration of

soluble proteins was only reduced in 8%, while alkaloids were removed in 85% [30].

Later, in 2002, Santana et al published another research article regarding bacterial removal of

quinolizidine alkaloids either in synthetic medium as well as in a Lupinus albus aqueous extract [88].

When grown in synthetic medium, both strains IST40D and IST20B showed 99% of lupanine removal,

similar to the previous study. Additionally, distinct QA as intermediate metabolites were detected during

exponential growth by gas chromatography and gas chromatography-mass spectrometry. The results

evidenced the presence of 3-hydroxylupanine, 13-hydroxylupanine and 17-oxosparteine as the major

lupanine intermediate metabolites in IST20B culture. For IST40D growth, 3,4-dehydrolupanine and α-

isolupanine were identified. It was also observed that the concentrations of the intermediate metabolites

has increased during first 8h of exponential growth and further decreases to undetectable

concentrations, with the exception of α-isolupanine that reached maximal concentrations in the early

stationary phase. Additionally, the behaviour of the two bacterial strains previously studied was tested

with lupin seeds extract complex medium (LPX), prepared from the dilution of 1:10 of an aqueous L.

albus seed extract provided by Mittex Anlagenbau GmbH in a buffered solution with pH 7. Lupanine and

total alkaloid final concentrations of LPX was 2 g/L and 3 g/L, respectively. During bacterial growth, the

determination of soluble protein, amino acids, total carbohydrates, total QA and lupanine concentrations

were performed. The total alkaloid and lupanine concentrations decreased after the inoculation of

bacteria in LPX medium, with a a complete metabolization of amino acids, decrease of 77% in the case

of lupanine, maintaining the levels of soluble proteins. From these results, the authors stated that both

bacterial strains are capable of reducing alkaloids and other organic compounds from the LPX medium.

However, further investigations are needed for the process optimization at an industrial scale [88].

Recently, Parmaki et al. (2018) studied the bioconversion of alkaloids into added-value chemicals by

comparative analysis of newly isolated lupanine degrading strains [87]. The authors isolated microbial

strains capable of using lupanine as carbon source from the wastewater of a L. albus processing plant

in Portugal, among other sources. Samples were enriched with 1.5 g/L of lupanine as the carbon source,

with three further sequential enrichments performed. One bacterial strain that revealed the ability to use

lupanine was isolated from L. albus processing wastewater and identified and classified as

Pseudomonas putida LPK411 [87]. When inoculated with growth media supplemented with 1.5 g/L of

lupanine, this microorganism was able to degrade 80% of the lupanine following 36h of cultivation. At

the end of the experiment, the resulting molecules were studied through NMR, to search for different

molecular structures. If microorganisms can convert lupanine into modified structures that are more

34

favourable to chemical modifications, conversion processes for the production of pharmaceutical drugs

can be more effective. In this study, two novel structures of end products resulting from lupanine

bioconversion, were observed for the first time (Figure 9) [87].

This study demonstrated that lupanine based structures can be obtained through P. putida LPK411

bioconversion, and can serve as a base for valorisation of lupanine in L. albus wastewater [87].

2.5. Bioreactors for bioconversion processes

In general, bioconversion processes are characterized by lower yields, when compared to chemical

synthesis reactions. The design, characterization and optimization of a bioconversion process, is crucial

for an effective process. There are some restrictive factors that make these processes less efficient,

such as, the low solubility of several organic substrates in aqueous solution and consequent inhibitory

effects of the reaction [89]. To overcome these difficulties and enhance bioconversion, it is important to

evaluate which is the best concentration of substrates to feed the reaction to reach optimal

microorganism activity in a certain period of time. For this, a continuous addition of substrates and a

selective removal of the desirable product can be applied [90].

One experimental approach to obtain an effective recovery of the product of interest is the

combination of liquid-liquid extractions and membrane permeation processes. Here, the microorganism

and the product to be extracted are immiscible, and between them there is a membrane that is

responsible for the reduction in the contact [91]. Specific membrane-based extractive bioreactors have

been applied in fermentations yielding end products that are continuously extracted to avoid their

accumulation and their toxicity to the microorganism [92]. These two-phase partitioning bioreactors

seem to have great potential in bioconversion processes. This type of process has been widely explored,

for the recovery of low-molecular weight volatile products like ethanol or acetone-butanol, and organic

compounds such as, acetic, lactic, propionic or butyric acids [93]. Xudong et al. (2004) performed a

bioconversion process of acrylonitrile to acrylamide using a hollow-fiber membrane bioreactor system

[94]. The authors designed a poly-sulfone hollow-fiber membrane bioreactor system to test the

bioconversion process with a new strategy of membrane separation. Before this approach, acrylamine

was bio-converted in fed-batch mode with immobilized cells. The fed-batch process presented several

disadvantages, as the presence of impurities, instability of product quality and relatively low productivity.

The hollow-fiber membrane bioreactor alternative, allowed to overcome these barriers and replace the

conventional process, with a continuous process using free cells as the biocatalyst and separation of

Figure 9 – Lupanine chemical structure (on the left) and chemical structures of novel end

products A and B, resulting from lupanine bioconversion [87]

35

the acrylamide product from the cells. This approach was tested in laboratory and at industrial scale,

with enhanced productivity yields in comparison with the previous approach [94].

Previous studies state that the use of small-scale vessels (with a maximum of 100 mL) are adequate

to investigate the biotransformation processes, providing cost savings, due to lower reagent (solid

media, for instance) and space requirements, compared with conventional bioreactors [95]. However,

independently of the system applied, the experimental set-up (mass transfer features, mixing conditions,

oxygen demand) should be designed in a way that enables the scale-up [96]. In addition to the low

costs associated, they facilitate the processing of different parallel test conditions, to evaluate their

effects at the same time on a given bioprocess [95].

Waste materials bioconversion into economically or ecologically beneficial materials would solve the

environmental concerns related to discarded untreated waters. A proper bioreactor for these

bioconversion processes should be studied, in order to use living systems with bacteria, yeasts or fungi

to specifically exploit their capability to achieve bioconversion of waste materials.

2.6. Biorefinery concept

Inspired in traditional oil refineries for petrol production, biorefineries enable the production of fuels,

power, chemicals and products from biomass with valorisation of a natural resource through biomass

conversion [97]. Here, renewable biomass or waste materials are used as the raw material feedstock,

where subsequent conversion processes are applied for treatment and processing of these raw

materials [98]. Biorefinery concept is in conformity with the notion of sustainable development, since the

biomass used is treated and valorised with the help of different unit operations to create added-value

compounds in an economic and eco-friendly strategy [97]. A unique raw material source can generate

a wide range of products, as illustrated in the Figure 10. To guarantee the sustainable use of biomass,

the process should promote first the development of the highest added-value product, followed by the

second highest and so on [99]. Assuming that all bioresources in a biorefinery are used, including

biological leftovers, zero waste production should be achieved.

Figure 10 - illustration of a biorefinery concept [97].

36

In the last decades, this concept of biorefinery has been applied in some industries such as the corn

wet milling, the pulp, paper and the paper industries [100], [101]. Currently, this concept mostly includes

terrestrial biomass like plants, with a small part devoted to marine resources such as seaweeds [102].

Liquid biofuels production has been affecting the economy, due to the competition with the food

sector, since first generation biofuels are derived from sugars, starch, animal fats and vegetable oil. To

overcome this issue, the second generation biofuels based in natural lignocellulosic resources, arised

as more environmentally friendly in order to minimize the impacts on the food market [102]. Hence,

biorefineries success depend on several factors as, the physical and chemical nature of the raw material

and on the economic interest of the process [103], [104].

Biomass conversion processes and the resulting biobased products are highly important for several

industries. Nevertheless, some technical, strategic and commercial barriers need to be overcome before

the large-scale industrial commercialization occurs. Here, the adequate technologies for the processes

should be applied to the projected biorefinery, e.g. chemical conversion, gasification, fermentation and

also pre-treatment and storage [105]. In fermentation processes, microorganisms are used to convert

substrates into recoverable products like alcohols or organic acids.

In the recent past years, biorefinery optimization is becoming more significant, since it allows the

identification of bottlenecks and pathway improvement in the process, thus increasing biomass

conversion yield and reducing carbon footprint, reducing water disposal, fossil energy use and thus

increasing net economic value (Figure 11) [106].

Figure 11 - Representation of pyramid of biomass for a biorefinery system [107].

37

3. Materials and Methods

3.1. Nanofiltration

A representative sample of 1L composed of different fractions from sweetening phase of the

industrial debittering process was used for nanofiltration in concentration mode. This sample was

obtained by homogenization of volumes collected at different time points of sweetening phase from

Tremoceira Estrela da Piedade. The sample was centrifuged at 6000 rpm for 30 minutes at 20°C and

the supernatant was collected for the nanofiltration. A polyamide NF270 (Dow FILMTEC) membrane

was used in this experiment. A total volume of 640 mL of supernatant previously collected was added

to the filtration cell and the gear pump flow rate set to 420 mL/min. Two samples of the initial feed were

collected for lupanine quantification and COD measurements, representing time zero of the

nanofiltration. The system was closed and a pressure of 20 bar was applied to the system. Samples of

the permeate and retentate were collected over time to quantify lupanine and COD, and the flux values

registered.

3.2. Bacteria

3.2.1. Culture enrichments and isolation of pure strains

Cultivations with defined M9 liquid medium were performed, for the isolation and selection of

bacterial strains from the lupin beans wastewater (LBW) surface organic-rich layer and from the LBW.

For this, two assays were done using the LBW superficial layer (Figure 12) or the LBW (Figure 13) as

inoculum at 5% (v/v) for the first enrichment of three sequential lupanine enriched cultures, respectively.

The inoculations of the second and third enrichments were performed with 5% (v/v) of the former

enriched culture. Both assays were performed in 100 mL shake-flasks with 50 mL working volume. Cells

were cultured using minimal microbial growth medium (M9), composed of (g/L): Na2HPO4.2H2O 8.5,

KH2PO4 3, NaCl 0.5, NH4Cl 0.5, MgSO4 0.24. Three cultivation conditions were tested with three

sequential enrichments in 7-day cultures, and maintained at 30°C and 100 rpm. For the assay with LBW

superficial layer inoculum, the carbon sources used were (g/L): (i) glucose 2; (ii) lupanine 0.75; (iii)

glucose 2 and lupanine 0.75 mixtures (Figure 12). For the assays with LBW inoculum, were used (g/L):

(i) glucose 2; (ii) lupanine 1.5; (iii) glucose 2 and lupanine 1.5 mixtures (Figure 13). Lupanine

concentrated solutions were prepared, filtered with 0.22 µm filters at vacuum, and added to culture

medium to achieve the referred concentrations.

At day 0 and at day 7 of each enrichment, samples of 1 mL were collected for lupanine quantification

by HPLC. Samples were also collected for isolation of the microorganisms in culture, from the third

enrichment culture at day 7, and stored at -80°C with 1 mL of glycerol 85% (v/v). Several dilutions of the

final culture were done in NaCl 0.9% (w/v), and plated in M9 agar media supplemented with lupanine at

38

1.5 g/L. The colonies with distinct morphotypes were streaked onto new M9 agar plates with 1.5 g/L

lupanine and incubated at 30°C for seven days, for strain isolation.

Figure 12 - Schematic representation of the assays for bacterial isolation on (i) 2 g/L glucose; (ii) 0.75 g/L

lupanine or (iii) 2 g/L glucose + 0.75 g/L lupanine, with three sequential lupanine enrichments in 50 mL working

volume. Cultures were inoculated with 5% (v/v) of the former enriched culture. The microcentrifuge 1.5 mL tubes

illustrate the key points of sample collections: day 0, 3 and 7 of each enrichment, for HPLC analysis; and day 7 of

the third enrichment for plating on M9 supplemented with 2 g/L glucose and 1.5 g/L lupanine.

Figure 13 - Schematic representation of the assays for bacterial isolation on (i) 2 g/L glucose; (ii) 1.5 g/L

lupanine or (iii) 2 g/L glucose + 1.5 g/L lupanine, with three sequential lupanine enrichments in 50 mL working

volume. Cultures were inoculated with 5% (v/v) of the former enriched culture. The microcentrifuge 1.5 mL tubes

illustrate the key points of sample collections: day 0 and 7 of each enrichment, for HPLC analysis; and day 7 of the

third enrichment for plating on M9 supplemented with 2 g/L glucose and 1.5 g/L lupanine.

39

3.2.2. Gram staining, DNA extraction and strain identification by 16S rDNA amplification

Gram coloration was done in four bacteria isolates, according to Seeley Jr, H. W. et al. (1991) [108],

followed by microscopic observation in an Hitech Zeiss Axioplan with the camera Axiocam 503 color.

DNA extraction of three gram-negative bacteria was performed according to the instructions of

PureGene: Genomic DNA Purification kit (protocol for gram-negative). The cells were resuspended in

300 µL of Cell Lysis Solution and incubated at 80°C for 5 minutes. An RNase treatment was done to cell

lysate by adding 1.5 µL RNase A Solution, and samples were mixed by inverting the tubes 25 times,

with a subsequent incubation step at 37°C for 45 minutes. For protein precipitation, samples were cooled

to room temperature by placing tubes on ice for 1 minute. Then, 100 µL of Protein Precipitation Solution

were added to the cell lysates and vigorously vortexed for 20 seconds to homogenize the mixture.

Samples were centrifuged at 13000 rpm for 3 minutes and the pellet containing precipitated proteins

was discarded. The supernatant containing DNA was transferred to a clean Eppendorf with 300 µL of

100% Isopropanol for protein precipitation and the tubes were gently homogenized. Samples were

centrifuged at 13000 rpm for 1 minute and after this step DNA was visible as a small white pellet.

Supernatant was discarded and 300 µL of 70% ethanol were added to wash the DNA from residual

protein precipitates or other impurities. A final centrifugation was done at 13000 rpm for 1 minute.

Ethanol was carefully removed and the DNA was resuspended in 50 µL of nuclease-free water. The

purity and concentration of the DNA was measured in Nanodrop. Pure DNA was stored at -20°C for

further use in polymerase chain reaction (PCR).

The DNA extraction protocol used for gram-positive was followed according to Fykse et al. (2003).

One gram-positive bacteria was cultured overnight in LB medium at 30°C and 250 rpm. Then, 2 tubes

of 2 mL of bacteria suspension were centrifuged at 6000 rpm for 3 minutes at room temperature. The

supernatants were discarded and the cells were washed in 1 mL ice-cold 10 mM Tris-HCl pH8, and

sonicated in Branson Sonifier 250 under 50 W, 20 kHz for 3 minutes or 5 minutes to assess the efficiency

of the process. For PCR, 2 µL of lysed cells were directly used without a DNA purification step.

A master mix was prepared with Phusion Buffer (1x), MgCl2 (2.5 mM), dNTPs (200 µM), Forward

(27F 5’-AGAGTTTGATCMTGGCTCAG-3’) and Reverse (1492R 5’-CGGTTACCTTGTTACGACTT-3’)

Primers (0.5 µM), DNA (1 µL for gram-negative bacteria DNA and 2 µL from cell lysates), DMSO (3%),

Phusion DNA Polymerase (1 U/50 µL) and nuclease-free water to complete the volume up to 50 µL.

One sample without DNA was included as the negative control of the reaction. PCR conditions were set

for both gram-positive or gram-negative bacteria in the thermocycler Cleaver Scientific Ltd GTC96S as

described in Table 5:

Table 5 - PCR conditions of 16S rDNA gene amplification.

Temperature Time Number of Cycles

Initial Denaturation 98°C 30 seconds 1

Denaturation 98°C 10 seconds

Annealing 50°C 20 seconds 35

40

Extension 72°C 1 minute

Final Extension 72°C 10 minutes 1

In this reaction, the full-length 16S rDNA gene, a constituent of the small subunit of prokaryotic

ribosomes, was amplified [109]. The sequence of this gene is highly efficient for species identification,

since it is a very conserved region [110], [111]. Amplified PCR products were visualized in an agarose

gel (0.8%) with TAE buffer (1%). NZY DNA loading dye (1x) was added to DNA sample and the NZY

DNA ladder III was used as reference. Agarose gel was run in an electrophoresis with 90 V during 40

minutes with TAE buffer 50x. NZY Tech NZY Gel Pure purification kit was used for DNA extraction and

purification from the agarose gel. The concentration of pure DNA was measured by Nanodrop ND-1000

Spectrophotometer and DNA samples sequenced by STABVIDA. The identification of the species is

given by a BLAST that compares the sequence to be identified with biological sequences that are in the

database.

3.2.3. Bacteria cultivation in synthetic growth medium

All bacteria isolates were pre-grown during 48h at 30°C and 250 rpm in M9 containing either (g/L):

glucose 2; lupanine 1.5; or mixture of glucose 2 and lupanine 1.5, in triplicates for one isolate and

duplicates for the others (Figure 13). Lupanine concentrated solutions were prepared, filtered with 0.22

µm filters at vacuum, and added to M9 culture medium to achieve the referred concentrations.

The optical densities (OD) of the pre-cultures were measured at 600 nm on a UV/VIS

spectrophotometer (Hitachi V2001). Cells from pre-culture were harvested (centrifuged for 3 minutes at

6000 rpm, for cell concentration) and resuspendend in M9 growth medium for an initial OD600nm of 0.1,

in a working volume of 50 mL in 100 mL flasks (Figure 14). Cellular growth was followed by measuring

culture OD600 nm at regular intervals of 24h during seven days. Additionally, samples were collected every

day for quantification of lupanine concentration by HPLC. A control flask in the same conditions and

without the addition of cells was maintained to assess if lupanine consumption was due to microbial

action or degradation.

Figure 14 - Schematic representation of bacterial isolates growth assays on (i) 2 g/L glucose (G); (ii) 1.5 g/L

lupanine (L) or (iii) 2 g/L glucose + 1.5 g/L lupanine (G+L). Pre-cultures were inoculated with isolates and used for

the inoculation of main cultures. Pre-culture flasks contained 25 mL working volume in 50 mL flasks, and culture

flasks 50 mL working volume in 100 mL flasks. Cultivations were performed during 7 days at 30°C and 250 rpm.

3.2.4. Bacteria cultivation in lupin beans wastewater

Lupin beans wastewater (LBW) provenient from cooking phase of the industrial debittering process

was tested as a culture medium. For this, 1 L of the LBW from Tremoceira Estrela da Piedade (Charneca

da Caparica, Portugal) was centrifuged (20 min, 6000 rpm, 20°C) to remove solids in suspension. For

41

bacterial growth, the pH of LBW was adjusted to 7.0-7.5 with NaOH 1M. The obtained supernatant was

filtered three times with 0.22 µm filters (Whatman, ME24/21ST) under sterile conditions. The LBW

medium was diluted 1/2 times in sterilized water and used for bacteria cultivations without further

supplementations, with lupanine quantification by HPLC. The isolates were pre-cultured for 24h at 30°C

and 250 rpm in 25 mL of diluted LBW media, in triplicates. The OD of the pre-cultures was measured at

600 nm and cells were harvested (centrifuged for 3 minutes at 6000 rpm, for cell concentration) and

resuspendend in LBW + H2O growth medium for an initial OD600nm of 0.1, in a working volume of 50 mL

in 100 mL flasks (Figure 15). Cell growth was followed by measuring culture OD600 nm at regular intervals

of 24h during seven days. Samples were collected for lupanine quantification by HPLC, in periods of

24h. The COD level and the total amount of reducing sugars (DNS) were measured at day zero and day

seven of cultures.

Figure 15 - Schematic representation of bacterial isolates growth assays on lupin beans wastewater (LBW)

diluted at 1/2 in sterilized water. Pre-cultures were inoculated with isolates and used for inoculation of main cultures.

Pre-culture flasks contained 25 mL working volume in 50 mL flasks, and culture flasks 50 mL working volume in

100 mL flasks. Cultivations were performed during 7 days at 30°C and 250 rpm.

3.3. Yeast

3.3.1. Culture enrichment and isolation of pure strains

Cultivations with defined YNB liquid medium were performed, for the selection and isolation of yeast

strains from the LBW. LBW was used as inoculum at 5% (v/v) for the first enrichment of three sequential

lupanine enriched cultures. The inoculations of the second and third enrichments were started with 5%

(v/v) of the former enriched culture. Both assays were performed in 100 mL shake-flasks with 50 mL

working volume. Cells were cultured using complete yeast nitrogen base (YNB) (Difco) at 6.7 g/L. Three

cultivation conditions were tested with three sequential enrichments in 7-day cultures, and maintained

at 30°C and 100 rpm. The carbon sources used were (g/L): (i) glucose 2; (ii) lupanine 1.5; (iii) glucose

2 and lupanine 1.5 mixtures (Figure 15). Lupanine concentrated solutions were prepared, filtered with

0.22 µm filters at vacuum, and added to culture medium to achieve the referred concentrations.

At day 0 and day 7 of enrichment, samples of 1 mL were collected for lupanine quantification by

HPLC. Samples were also collected for isolation of the microorganisms in culture, from the third

enrichment culture at day 7, diluted and plated in YNB agar plates with lupanine at 1.5 g/L and glucose

at 2 g/L mixed with lupanine (1.5 g/L). To evaluate if other yeast species might be present, samples

were diluted and also plated in yeast peptone dextrose agar (YPDA) composed of Glucose 20 g, Agar

42

20 g, Peptone 20 g, Yeast extract 10 g, for 1L of medium, supplemented with chloramphenicol (CAM,

30 µg/mL) (Figure 16). Colonies with different morphotypes were streaked onto new YNB with lupanine

(1.5 g/L) agar plates and incubated at 30°C, for strain isolation.

Figure 16 - Schematic representation of the assays for yeast isolation on (i) 2 g/L glucose; (ii) 1.5 g/L lupanine

or (iii) 2 g/L glucose + 1.5 g/L lupanine, with three sequential lupanine enrichments in 50 mL working volume.

Cultures were inoculated with 5% (v/v) of LBW in the first enrichment. Inoculations of the second and third

enrichments were performed with 5% (v/v) of the former enriched culture. The eppendorf’s illustrate the key points

of sample collections: day 0 and 7 of each enrichment, for HPLC analysis; and day 7 of the third enrichment for

plating on YNB supplemented with 1.5 g/L lupanine and 30 µg/mL CAM.

3.3.2. DNA extraction and strain identification by 26S rDNA amplification

Fresh cells from yeast isolates in YNB with lupanine (1.5 g/L) were collected for DNA extraction

according with the instructions of HigherPurity Yeast Genomic DNA Isolation kit from Canvax. Here,

fresh cells were resuspended in 600 µL Buffer BLL and 50 µL of Lyticase Solution were added. Then,

samples were incubated at 30°C during 30 minutes and centrifuged at 6000 rpm for 5 minutes. The

supernatant was discarded and 200 µL of Buffer BLY were added, followed by the transfer of the mixture

for a beads tube for 5 minutes of vortexing. 15 µL of Proteinase K (20 mg/mL) were added and samples

were incubated at 56°C during 30 minutes with regular inversions of the tubes. Then, samples were

centrifuged at 6000 rpm for 1 minute and the supernatant was collected to a new eppendorf. 200 µL of

Absolute Ethanol were added with immediate homogenization of the mixture. This sample mixture was

transferred to a MiniSpin Column followed by a centrifugation of 6000 rpm during 2 minutes. The

MiniSpin Column was placed in a new collection tube and 500 µL of Buffer WB1 were added with a

13000 rpm centrifugation step for 30 seconds. 750 µL of Buffer WB2 were added followed by two

centrifugation steps (13000 rpm for 1 min) to dry the column. MiniSpin Column was transferred to a new

Eppendorf and 100 µL of a pre-heated Elution Buffer were directly added to the centre of the spin column

with a 5 minute incubation at room temperature. After this, a final centrifugation (13000 rpm during 1

43

minute) was done to elute purified genomic DNA. The spin column was discarded. DNA concentration

was measured by Nanodrop and was directly used for PCR, being stored at -20°C.

For PCR, a master mix was prepared with Phusion Buffer (1x), MgCl2 (2.5 mM), dNTPs (200 µM),

Forward (NL1 5’-GCA TAT CAA TAA GCG GAG GAA AAG-3’) and Reverse (NL4 5’-GGT CCG TGT

TTC AAG ACG G-3’) Primers [112] (0.5 µM), DNA (1 µL), DMSO (3%), Phusion DNA Polymerase (1

U/50 µL) and nuclease-free water to complete the volume up to 50 µL. One sample without DNA was

included as a negative control of the reaction. The conditions for the reaction were set as described in

Table 6:

Table 6 - PCR conditions of D1/D2 region amplification.

Temperature Time Number of Cycles

Initial Denaturation 98°C 30 seconds 1

Denaturation 98°C 10 seconds

Annealing 50°C 20 seconds 30

Extension 72°C 30 seconds

Final Extension 72°C 10 minutes 1

Here, D1/D2 region of the large subunit of the 26S ribosomal DNA was amplified. This region is

frequently used for species identification of yeasts, due to the variability within these regions [113], [114].

Amplified PCR products were visualized in an agarose gel (0.8%) with TAE buffer (1%). NZY DNA

loading dye (1x) was added to DNA samples and the NZY DNA ladder III was used as reference.

Agarose gel was run in an electrophoresis with 90 V during 40 minutes with TAE buffer 50x. NZY Tech

NZY Gel Pure purification kit was used for DNA extraction and purification from the agarose gel. The

concentration of pure DNA was measured by Nanodrop and DNA samples were sequenced by

STABVIDA.

3.3.3. Yeast cultivation in synthetic culture medium

Yeast isolates were pre-grown during 24h at 30°C and 250 rpm in YNB containing either (g/L):

glucose 2, lupanine 1.5 or glucose 2 mixed with lupanine 1.5, in duplicates (Figure 17). Lupanine

concentrated solutions were prepared, filtered with 0.22 µm filters at vacuum, and added to YNB culture

medium to achieve the referred concentrations. The OD of the pre-cultures were measured at 600 nm

on a UV/VIS spectrophotometer (Hitachi V2001). Cells from pre-culture were harvested (centrifuged for

3 minutes at 6000 rpm, for cell concentration) and resuspendend in YNB growth medium for an initial

OD600nm of 0.1, in a working volume of 50 mL in 100 mL flasks. Growth was followed by measuring

culture OD600 nm at regular intervals of 24h during seven days. Samples were withdrawn every day of

culture for quantification of lupanine concentration by HPLC. A control flask in the same conditions

without the addition of cells was maintained to assess if lupanine concentration consumption was due

to microbial action or degradation.

44

Figure 17 - Schematic representation of yeast isolates growth assays on (i) 2 g/L glucose (G); (ii) 1.5 g/L

lupanine (L) or (iii) 2 g/L glucose + 1.5 g/L lupanine (G+L). Pre-cultures were inoculated with isolates and used for

the inoculation of main cultures. Pre-culture flasks contained 25 mL working volume in 50 mL flasks, and culture

flasks 50 mL working volume in 100 mL flasks. Cultivations performed during 7 days at 30°C and 250 rpm.

3.3.4. Yeast cultivation in lupin beans wastewater

The previous filtered and centrifuged LBW from section 3.5.4. was used as culture medium for yeast

cultures, with pH 4.5. The LBW was diluted 1/2 times in sterilized water and 1/2 times in YNB medium.

One sample was collected to quantify lupanine concentration after dilutions. Therefore, isolates were

pre-grown during 24h at 30°C and 250 rpm in 25 mL working volume of the two diluted LBW medium,

in triplicates. Cells from pre-culture were harvested (centrifuged for 3 minutes at 6000 rpm, for cell

concentration) and resuspendend in LBW growth medium for an initial OD600nm of 0.1, in a working

volume of 50 mL in 100 mL flasks (Figure 18). Growth was followed by measuring OD600 nm at regular

intervals of 24h during seven days. Samples were collected for lupanine quantification by HPLC.

Additionally, the COD level and the total amount of reducing sugars (DNS), were measured at day zero

and day seven of cultures.

Figure 18 - Schematic representation of yeast isolates growth assays on lupin beans wastewater (LBW) diluted

at 1/2 in sterilized water and 1/2 in YNB medium. Pre-cultures were inoculated with isolates and used for inoculation

of main cultures. Pre-culture flasks contained 25 mL working volume in 50 mL flasks, and culture flasks 50 mL

working volume in 100 mL flasks. Cultivations performed during 7 days at 30°C and 250 rpm.

3.4. Filamentous fungi: a complementary approach

3.4.1. Isolation and identification

Cultivations with defined solid medium were performed, for the selection and isolation of fungi

strains from the LBW and LBW superficial layer. 10-1 to 10-6 dilutions of these samples were done in

sterile water and plated in potato dextrose agar (PDA powder 39 g/L) and malt extract agar medium

(MEA: Glucose 20 g/L, Malt Extract 20 g/L, Agar 20 g/L, Peptone 1 g/L). Both culture mediums were

45

sterilized in an autoclave for 15 min at 121°C. MEA medium was supplemented with, (i) 25 mg/L CAM

and (ii) 20 mg/L rose bengal, separately. Colonies with different morphotypes were streaked onto new

PDA and MEA agar plates for strain isolation.

ZR Fungal/Bacterial DNA MiniPrep extraction kit was used for DNA extraction of fungi isolates. The

experimental procedure was performed according to the kit instructions. The concentration of pure DNA

was measured by Nanodrop. Extracted DNA was visualised in an agarose gel (1%) with TAE buffer (1x)

for visual assessment of DNA quality. The loading buffer (1x) was added to DNA samples, NZY Ladder

III 1000 pb was used as the molecular weight marker. Agarose gel was run in an electrophoresis with

120 V for 90 minutes. DNA samples were sent to STABVIDA for PCR and sequencing.

3.4.2. Filamentous fungi culture: synthetic culture medium

Isolated fungi strains were cultured at 27°C and 250 rpm in malt extract (ME) culture medium

composed of 17 g/L malt extract and 3 g/L mycological peptone, sterilized by autoclave for 20 min at

121ºC and supplemented with: (i) 40 g/L glucose, (ii) 40 g/L glucose and 1.5 g/L lupanine, and (ii) 1.5

g/L lupanine. Lupanine solution was sterilized by filtration with nylon 0.22 µm pore size filters. Samples

were collected during culture for quantification of cellular growth (as previously described) and lupanine

concentration.

3.4.3. Filamentous fungi culture: assessment of using filamentous fungi for lupin beans

wastewater treatment

LBW provenient from cooking phase of the industrial debittering process was tested as a culture

medium. For this, fungi isolates were inoculated in 50 mL working volume of sterile LBW in 250 mL

flasks. The cultures were incubated at 27°C and 250 rpm during 192h. Samples of 1 mL were collected

for lupanine quantification by HPLC. Cellular growth was measured by cell dry weight assessment

involving: cell centrifugation at 10000 rpm for 5 minutes followed by two washing steps of the cell pellet

in mili-Q water, and cells were dried for 48h at 30°C. The COD level was measured at day 0 (zero)

andday 7 (seven) of cultures.

3.5. Determination of lupanine derived-products

Work section performed in collaboration with researchers from Faculty of Pharmacy of University of

Lisbon). Cultures of O. anthropi were performed until 24 h, with 50 mL samples collected at time points

0 h, 3 h, 6 h, 12 h and 24 h after inoculation of cells. In the case of C. funkei, cultures were performed

at 120 h with 50 mL samples collection at 0 h, 48 h and 120 h after inoculation. Both experiments were

performed in biological duplicates in M9 liquid medium. All samples were lyophilized (Labocene Scanvac

Coolsafe) for further characterization studies.

After lyophilization, the obtained samples were directly analysed by Fourier-transform infrared

spectroscopy (FT-IR) (Bruker Alpha II Platinum – ATR), to assess the presence of functional

characteristic groups of lupanine molecule (e.g. C=O at 1600-1800 cm-1 range), as well as another ones

corresponding to eventual intermediates that might be present [115]. A pure lupanine sample was also

analysed to compare the results.

46

To complement the previous technique, thin layer chromatography (TLC) was also performed, in

silica plates. Here, the samples were dissolved in water and applied in the plates with a Pasteur pipette,

with two steps of elution in MTBE/hexane (8:2) and 1% diethylamine (DEA). The result was observed

after phosphomolybdic acid staining of the plates. Here, the compounds of the sample will migrate and

separate according with their polarity, enabling the observation of lupanine and other intermediates

present in the samples. A pure lupanine sample was also applied to compare the results.

Nuclear Magnetic Resonance (NMR) spectroscopy was also performed. Here, the samples were

pre-treated by dissolving in 10 mL of water and basifying with NaOH 1 M until pH 12-14, followed by two

extractions with 2 x 10 mL DCM. The combined organic phase was dried with MgSO4, filtered (using

paper filter) and the solvent was removed under reduced pressure at 40 °C. The residue was

reconstituted in 0.4 mL of deuterated chloroform (CDCl3) and analysed by 1H and 13C NMR.

The capacity of these bacterial species to consume preferably one lupanine enantiomer over the

other was investigated. Here, dried samples were diluted in DCM and filtered in a mini-column made of

celite (to retain impurities). The solvent was evaporated under reduced pressure and 1 mg of the dry

residue was diluted in 1 mL of a solution comprised of hexane (80%) and isopropanol (20%). Then,

samples were analysed in a HPLC system with an IC Chiralpak column (5 μm particle size, 4.6 x 250

mm). A sample volume of 20 µL was injected and the enantiomers were detected at 230 nm. The method

was isocratic for 40 min with a mobile phase composed of hexane with 0.1% diethylamine (DEA) (25%),

isopropanol (22%) and hexane (53%), and a 1 mL/min flux.

3.6. Lupanine quantification by HPLC

A calibration curve was done for lupanine, with concentrations ranging from 0.0016 g/L to 8 g/L. The

samples containing lupanine, pure or provenientprovenient from the lupin seeds debittering water, were

centrifuged at 10000 rpm for 3 minutes (Sigma 1-15P), the supernatant filtered with nylon syringe filters

(pore size 0.22 µm) and the pH adjusted to 13-13.5 with potassium hydroxide pellets (KOH, Panreac).

All samples and standards were analysed in a HPLC system from Hitachi LaChrom, equipped with a

core-shell organo-silica LC column from Kinetex (5 μm EVO C18 100 Å, 250 x 4.6 mm) and a pre-

column also from Kinetex, coupled to a UV detector (Hitachi LaChrom). A sample volume of 20 µL was

injected and lupanine was measured at 220 nm, during 24 minutes. The mobile phase used was

composed of 15% acetonitrile (ACN) and 85% sodium hydrogen phosphate (Na2HPO4) buffer (0.01 M

and pH adjusted to 10.5 with sodium hydroxide (NaOH) 1.25M).

3.7. Chemical Oxygen Demand (COD) measurements

For measurement of the chemical oxygen demand (COD), organic compounds are oxidized with

potassium dichromate (K2Cr2O7) under acidic conditions in presence of sulfuric acid (H2SO4), at 150°C.

Carbon dioxide (CO2) and water (H2O) result from oxidation reactions, and dichromate is reduced to

chromium. Ferrous ammonium sulfate [Fe(NH4)2(SO4)2] is used for titration of the remaining

dichromate. The amount of oxidant consumed is converted into grams of oxygen per litre of sample (g

O2/L) [116]. For COD quantification four solutions were used; i) potassium dichromate digestion solution.

An aqueous solution of dried potassium dichromate (K2Cr2O7) was prepared at 10.216 g/L, with further

47

addiction of 167 mL of H2SO4 and 33.3 g/L of mercuric sulphate HgSO4, under agitation in a final volume

of 1 L; ii) 10 g/L commercial solution of silver sulphate (Ag2SO4) in sulphuric acid (H2SO4); iii) ferrous

ammonium sulphate [Fe(NH4)2(SO4)2] titrant solution at 0.0125 M iv) a commercial ferroin indicator

solution (1, 10-phenanthroline and ferrous ammonium sulphate) [116].

For the analysis, 1 mL of solution (i) and 2 mL of solution (ii) were added to 1.5 mL of culture samples

after removing cells by centrifugation (5 minutes, 10000 rpm). Blanks were prepared in parallel using

the same volume of distilled water instead of sample. The samples were digested for 2 hours at 150°C,

and then transferred into 50 mL Erlenmeyer’s. One drop of solution (iv) was added and the dichromate

in excess was determined by titration with solution (iii), under agitation. When an orange colour was

reached, the titration was stopped and the volume of solution (iii) used was registered.

3.8. Total reducing sugars determination by colorimetric method (DNS)

DNS method is a colorimetric technique that enables to do an estimation of the concentration of

reducing sugars in a sample through a redox reaction between 3,5-dinitrosalicylic acid (DNS) and

reducing sugars. These sugars contain a free carbonyl group, with the reducing capacity of many

reagents. Therefore, DNS is reduced to 3-amino-5-nitrosalicylic acid with a visible orange colour

measurable by spectrophotometry at 540 nm [117]. Colour intensity is proportional to reducing sugars

concentration. DNS Solution is prepared by dissolving 25 g of DNS in 50 mL NaOH (2 N) and 125 mL

of distilled water. After this, 75 g of tartrate sodium phosphate and 250 mL of water are added and

dissolved under agitation and 50-100°C. Solution must be kept in a dark flask and used after 48h of

storage. Experimental procedure evolved the centrifugation of cells (6000 rpm, 3 minutes) with the

following subsequent steps: 400 µL of supernatant was mixed with 600 µL of DNS solution and the tubes

were placed in a water bath at 100°C for 5 minutes. A blank sample with only water was performed in

parallel. Samples were cooled to room temperature and 200 µL of the samples were transferred to 96

microplate well and absorbance measured at 540 nm.

48

4. Results

4.1. Nanofiltration

A nanofiltration was performed using a 640 mL representative sample of the sweetening phase of

lupin beans debittering process. The objective was to recover part of the water in the permeate and

concentrate lupanine in the retentate. The results of the filtration are presented in Figure 19 and

represent the relation between the flux through the membrane and the percentage of concentration.

From Figure 19, it is possible to observe that the flux was constant during the experiment. This

indicates that the membrane used is suitable for the nanofiltration of this wastewater and to be used at

an industrial scale.

The concentrations of lupanine and COD in the initial sample of wastewater and in the permeate and

in the retentate during nanofiltration are represented in Table 7.

Table 7 – Concentration of lupanine and COD in the feed of wastewater solution, permeate and retentate of the

nanofiltration.

Sample Lupanine (g/L) COD (g O2/L)

Initial 0.40 ± 0.01 5.31 ± 0.55

Permeate 0.15 ± 0.01 0.87 ± 0.00

Retentate 2.12 ± 0.13 26.67 ± 1.10

From Table 7, lupanine concentration increases in the retentate and decreases in the permeate, wich

is in concordance with the low and higher COD values obtained in these nanofiltration fractions. From

here, it is possible to conclude that lupanine contributes as organic matter for the COD measurements.

0

200

400

600

800

1000

0 20 40 60 80 100

Flu

x (

mL/m

in/m

2)

Concentration (%)

Figure 19 – Values of flux through the membrane during the nanofiltration in function of the percentage of

concentration. Nanofiltration was operated in a concentration mode. The cross in the curve represent the overnight

time when the nanofiltration was stopped.

49

4.2. Bacteria

4.2.1. Culture enrichments and isolation of pure strains

Both enrichments with defined M9 liquid medium aimed to favour the growth of a group of bacterial

organisms, under certain conditions. Here, the objective was to select bacterial strains with the ability to

specifically catabolize lupanine, using the superficial organic-rich layer formed during the summer in the

lupin beans wastewater from the debittering process. Cultures were performed with three sequential

lupanine enrichments, in M9 medium with glucose, lupanine or mixtures of glucose/lupanine. The

lupanine concentration results during the three enrichments for the two conditions with lupanine in the

medium are shown in Figure 20.

Figure 20 – Cultivation assays for bacteria isolation with three sequential lupanine or glucose + lupanine

mixture enrichments. Cultures were inoculated with 5% (v/v) of lupin beans wastewater (LBW) superficial layer in

the first enrichment. Inoculations of the second and third enrichments performed with 5% (v/v) of the former enriched

culture. (A) 0.75 g/L lupanine; (B) 2 g/L glucose + 0.75 g/L lupanine.

Additionally, it was observed that this superficial layer had, by nature, a small lupanine

concentration of 0.16 g/L, which can justify the values of around 1 g/L of lupanine obtained at the time

point 0h in the first enrichment. Figure 20 shows that lupanine decreases in both conditions with lupanine

or glucose mixed with lupanine, evidencing that the bacterial consortium that comes from LBW

superficial organic layer has the ability to consume lupanine until the end of the third enrichment.

Furthermore, when diluted samples of the third enrichments were plated in M9 solid medium with

1.5 g/L lupanine, it was possible to observe different types of colonies provenient from the condition

containing only lupanine as carbon source (Figure 21).

50

Figure 21 – M9 solid medium supplemented with 1.5 g/L lupanine plated with a sample from culture with

lupanine as carbon source, at the end of third enrichment, representing the enriched population provenient from

lupin beans wastewater (LBW) superficial organic-rich layer.

The observed colony growth at the end of the third enrichments confirms that some species

remained viable after successive enrichments with lupanine as carbon source, enabling further isolation

and identification.

For the second enrichment assay, the inoculation was performed with LBW (from cooking phase),

aiming to search different bacteria that might have the ability to catabolize lupanine (Figure 22).

Figure 22 – Cultivation assays for bacteria isolation with three sequential lupanine enrichments. Cultures

were inoculated with 5% (v/v) of lupin beans wastewater (LBW) in the first enrichment. Inoculations of the second

and third enrichments performed with 5% (v/v) of the former enriched culture. (A) 1.5 g/L lupanine; (B) 2 g/L glucose

+ 1.5 g/L lupanine.

From Figure 22, no lupanine consumption was observed in both conditions with sole lupanine and

glucose mixed with lupanine. This indicates that the bacterial consortium obtained from lupin beans

wastewater of cooking phase is apparently not able to consume lupanine as carbon source.

Inspite of lupanine consumption was not observed in liquid culture, when samples of the third

enrichment were plated onto M9 solid medium with 1.5 g/L lupanine, it was also observed the growth of

the bacterial colonies in lupanine as carbon source (Figure 23).

51

Figure 23 - M9 solid medium supplemented with 1.5 g/L lupanine plated with a sample from culture with

lupanine as carbon source, at the end of third enrichment, representing the enriched population provenient from

LBW.

This indicates that there were species maintained viable after three successive enrichments with

lupanine as carbon source, enabling further isolation and identification steps.

4.2.2. Gram staining, DNA extraction and strain identification by 16S rDNA amplification

In order to assess which protocol should be applied for DNA extraction of the isolated bacteria, a

gram staining was performed, since it is a rapid method to distinguish gram-negative from gram-positive

bacteria species [118]. While gram-negative bacteria strains have an outer membrane located above a

thin peptidoglycan layer, the gram-positive present a relatively simple structure [119]. The gram staining

results from the four bacterial strains isolated revealed that three bacteria were gram-negative and one

was gram-positive, given the pink and violet colours observed, respectively. The DNA extraction protocol

for three gram-negative bacteria yielded 3971.7 ng/µL, 1439.1 ng/µL and 1425.1 ng/µL of genomic DNA.

The DNA sample from the first bacteria was diluted 1:100 times and the other two were diluted 1:50

times for the PCR reaction. The PCR products of gram-negative bacteria are shown in Figure 24.

52

Figure 24 – Agarose gel electrophoresis of PCR products obtained for the three gram-negative bacterial strains.

Agarose gel (0.8%) electrophoresis was run during 40 minutes at 90 V with 50x TAE buffer. PCR products were

loaded in the gel in triplicate. (L) Molecular marker NZY DNA ladder III (1kb); (1)-(3), (4)-(6) and (8)-(10) are the

PCR product corresponding to three gram-negative isolates; (7) negative control of the reaction.

The DNA bands in the gel were cut under UV light and purified, resulting in products with a final DNA

concentration of 46.5 ng/µL, 37.2 ng/µL, and 29.4 ng/µL, respectively. Based on sequencing results, the

three gram-negative isolates were identified as Stenotrophomonas maltophilia (with 98.3% identity and

98.08% query coverage), Sphingobacterium siyangense (with 99.3% identity and 99.61% query

coverage) and Ochrobactrum anthropi (with 98.5% identity and 99.2% query coverage), respectively.

The protocol for DNA extraction of gram-positive bacteria involved a sonication step to lyse the cells.

To assess the efficacy of the protocol, the sonication step was tested during 3 and 5 minutes, separately.

The PCR was performed for both conditions, using directly 2 µL of lysed cells for reaction, as well as a

positive and a negative control of the reaction. The PCR results can be observed in Figure 25.

Figure 25 – Agarose gel electrophoresis of PCR products obtained for the gram-positive bacterial strain, for

sonication efficiency testing. Agarose gel (0.8%) electrophoresis was run during 40 minutes at 90 V with 50x TAE

buffer. (L) Molecular marker NZY DNA ladder III; (1) DNA with 3 minutes sonication; (2) DNA with 5 minutes

sonication; (3) positive control of the reaction with known DNA; (4) negative control of the reaction.

L 1 2 3 4 5 6 7

8 9 10

L 1 2 3 4

53

As demonstrated, both sonication times tested have successfully lysed the cells and the DNA

was released from the cells. The PCR for sequencing was performed with the DNA provenient from the

sample sonicated for 3 minutes (Figure 26).

Figure 26 - Agarose gel electrophoresis of PCR products obtained for the gram-positive bacterial strain,

sonicated for 3 minutes. Agarose gel (0.8%) electrophoresis was run during 40 minutes at 90 V with 50x TAE buffer.

PCR products were loaded in the gel in quadriplicate. (L) Molecular marker NZY DNA ladder III; (1)-(4) is the PCR

product corresponding to gram positive isolate; (5) is the negative control of the reaction.

The DNA bands were cut and purified, resulting in products with a final concentration of 28.5 ng/µL.

Based on sequencing results, this gram-positive isolate was identified as Cellulosimicrobium funkei (with

99.4% identity and 100% query coverage).

4.2.3. Bacteria cultivation in synthetic growth medium

After identification of the four bacterial strains (Ochrobactrum anthropi, Stenotrophomonas

maltophilia, Sphingobacterium siyangense and Cellulosimicrobium funkei), isolated from the LBW and

its surface layer, the main objective was the study of the ability of each one of them to catabolize

lupanine. For this, cultivations in M9 medium supplemented with 2 g/L glucose, 1.5 g/L lupanine or with

mixtures of 2 g/L glucose and 1.5 g/L of lupanine were performed. Parallel cultures in glucose were

maintained as a positive control of the experiment, to evaluate how the isolates grow in sole presence

of a preferred carbon source, in comparison to lupanine. The growth of the different isolates was

followed through OD600nm measurements at every hour during first 6 hours and every 24h until day 7.

Furthermore, lupanine was quantified at time point 0 (0h, after cell inoculation) and subsequently in

periods of 24h, as shown in Figures 27-30. Besides this, the pH was also monitored in regular times of

culture (Table 8).

L 1 2 3 4 5

54

Figure 27 – O. anthropi cultures in M9 liquid medium. Pre-cultures of 48 hours in 1.5 g/L of lupanine were used

as inoculum of the main cultures (A) in 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L lupanine. Pre-cultures of 48

hours in 1.5 g/L of lupanine with 2 g/L glucose mixture were used as inoculum of the main cultures (B) in 1.5 g/L

lupanine and 2 g/L glucose + 1.5 g/L lupanine. Pre-culture of 48 hours in 2 g/L glucose were used as inoculum of

the main culture (C) used as a positive control of the experiment. Growth curves are represented in (A1), (B1) and

C, and lupanine concentration in (A2), (B2), respectively. Error bars represent the respective standard deviation of

biological triplicate assays.

From Figure 27 it is possible to observe a decrease of 66.7% in lupanine concentration at 24h, with

constant values until 168h of culture. This result indicates that O. anthropi has the ability to catabolize

lupanine as carbon source for growth.

The results for S. maltophilia, S. siyangense and C. funkei are shown below.

55

Figure 28 – S. siyangense culture in M9 liquid medium. Pre-cultures of 48 hours in 1.5 g/L of lupanine with

2 g/L glucose mixture were used as inoculum of the main culture (A) in 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L

lupanine. Pre-culture of 48 hours in 2 g/L glucose were used as inoculum of the main culture (B) used as a positive

control of the experiment. Growth curves are represented in (A1) and (B), and lupanine concentration in (A2),

respectively. Error bars represent the respective standard deviation of biological duplicate assays.

Figure 29 – S. maltophilia culture in M9 liquid medium. Pre-cultures of 48 hours in 1.5 g/L of lupanine with 2 g/L

glucose mixture were used as inoculum of the main culture (A) in 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L


56

control of the experiment. Growth curve is represented in (A1) and (B), and lupanine concentration in (A2),


Figure 30 – C. funkei culture in M9 liquid medium. Pre-cultures of 48 hours in 1.5 g/L of lupanine with 2 g/L

glucose mixture were used as inoculum of the main culture (A) in 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L


control of the experiment. Growth curve is represented in (A1) and (B), and lupanine concentration in (A2),


These set of results indicate that among all four bacterial strains identified, O. anthropi is the most

interesting isolate concerning lupanine catabolization, since it demonstrated the ability to grow

exponentially in lupanine presence, consuming lupanine after 24h of culture. The remaining bacterial

isolates were not able to grow in the pre-culture containing lupanine as sole carbon source, which can

explain the use of glucose + lupanine condition for the inoculum of the main culture. In the main culture,

lupanine consumption was not observed.

57

Table 8 – Values of pH of the supernatant of the four bacteria isolates: O. anthropi, S. siyangense, S. maltophilia

and C. funkei, measured at different time points during cultivation.

Culture Pre-inoculum Inoculum Day 0 Day 1 Day 2 Day 5 Day 6 Day 7

O. anthropi

Glucose Glucose 7.30±0.71 7.25±0.78 7.80±0.27 7.90±0.23 7.85±0.16 7.90±0.07

Lupanine Lupanine 7.30±0.28 7.40±0.57 7.90±0.28 7.80±0.14 7.75±0.07 7.60±0.13

Glucose/lupanine 7.45±0.49 7.25±0.49 8.08±0.11 7.85±0.21 7.80±0.28 8.00±0.15

Glucose/Lupanine Lupanine 7.50±0.42 7.45±0.49 7.85±0.21 8.05±0.49 7.85±0.21 7.70±0.04


S. siyangense

Glucose Glucose 7.20±0.12 7.00±0.13 7.50±0.12 7.80±0.10 7.70±0.04 8.00±0.06



S. maltophilia

Glucose Glucose 7.20±0.13 7.30±0.11 7.80±0.05 7.85±0.07 7.90±0.14 7.90±0.10



C. funkei

Glucose Glucose 7.20±0.26 7.00±0.17 7.45±0.10 7.70±0.03 7.90±0.13 8.10±0.18



Results from control flasks prepared under the same conditions and without the addition of cells are

represented in Figure 31.

Figure 31 - Control flask with, separately, M9 culture medium supplemented with 1.5 g/L lupanine and

another flask with 2 g/L glucose + 1.5 g/L lupanine.

Control flasks demonstrated that lupanine was maintained constant during all the experiment,

indicating that when lupanine concentration decrease in culture, it may be due to microbial action and

not by degradation of lupanine itself.

4.2.4. Bacteria cultivation in lupin beans wastewater (LBW)

The four isolated bacterial strains were used to assess the catabolism of lupanine and organic matter

removal from LBW. For this, the LBW pH was adjusted to 7 for optimal bacterial growth and given the

high concentration of lupanine in this wastewater – 3.3 g/L, in comparison with previous assays using

1.5 g/L –, it was diluted 1/2 times in water. Additionally, COD levels and total reducing sugars were

measured after inoculation and after 7 days, while pH values were measured at 24h regular times

(Tables 9, 10 and 11, respectively). Growth curves and lupanine concentration results for the different

strains are represented in Figure 32.

0.0

0.4

0.8

1.2

1.6

0 24 48 72 96 120 144 168

Lupanin

e (

g/L

)

Time (h)M9 + Lupanine M9 Glucose + Lupanine

58

Figure 32 – Growth curves (left column) and lupanine concentration (right column) for 7-day cultures of O.

anthropi, S. siyangense, S. maltophilia, C. funkei in LBW medium diluted 1/2 in water. Error bars represent the

respective standard deviation of biological triplicate assays.

From the previous figure it is possible to conclude that all bacterial isolates are able to grow

exponential in LBW diluted in water. However, lupanine concentration levels were constant during 168h

of culture, which can be explained most probably due to the presence of other p referable carbon

sources in this wastewater.

59

Table 9 – Percentages of COD removal from LBW for the four bacterial strain isolates: O. anthropi, S.

siyangense, S. maltophilia and C. funkei.

Bacterial culture in LBW COD removal (%)

O. anthropi 58.7 ± 2.5

S. siyangense 51.1 ± 2.1

S. maltophilia 42.6 ± 3.5

C. funkei 15.8 ± 2.8

COD measurements demonstrated that all four isolates were capable to reduce organic matter

from lupin beans wastewater. As seen in Table 9, O. anthropi was the one with the ability to remove the

higher percentage of COD from LBW, with 58.7% removal. S. siyangense also demonstrated an

interesting result, with 51.1% removal, followed by S. maltophilia with 42.6%.

Table 10 – Reducing sugar concentration determined through DNS method for the initial LBW medium

and after 7 days of cultivation of the four bacteria isolates: O. anthropi, S. siyangense, S. maltophilia and C.

funkei.

Culture Reducing sugars (g/L)

LBW + H2O 0.62 ± 0.00

O. anthropi 0.54 ± 0.09

S. siyangense 0.47 ± 0.01 S. maltophilia 0.53 ± 0.00

C. funkei 0.52 ± 0.09

The total reducing sugar determination at day 0 and day 7 revealed that there was sugar

consumption. This confirms that the bacterial isolates are in fact using reducing sugars to support their

growth.

Table 11 – pH values of LBW + H2O measured in supernatant at different time points, during cultivation of

the four bacteria isolates: O. anthropi, S. siyangense, S. maltophilia and C. funkei.

From Table 11, it is possible to observe that pH values are increasing during culture in the four

isolates. At day 0 the pH was around 7.4 and at day 7 the pH had increased to 9.4 approximately,

evidencing that a basification of the LBW culture medium is occurring.

Days Culture

0 1 2 3 6 7

O. anthropi 7.30±0.28 7.65±0.35 8.17±0.23 8.43±0.32 9.30±0.69 9.30±0.69

S. siyangense 7.40±0.28 8.05±0.07 8.27±0.06 8.43±0.31 9.40±0.61 9.43±0.64

S. maltophilia 7.45±0.21 7.90±0.14 8.30±0.20 8.73±0.38 9.10±0.10 9.50±0.44

C. funkei 7.45±0.21 7.90±0.14 8.33±0.06 8.47±0.15 9.23±0.40 9.23±0.50

60

4.3. Yeast

4.3.1. Culture enrichment and isolation of pure strains

As mentioned in 4.2.1. Section, culture enrichment aims to isolate strains adapted to a certain

condition. In this study, the objective was to select and isolate yeast strains with the ability to catabolize

lupanine, provenient from LBW. For this, three sequential enrichment cultures were performed in YNB

defined medium, with lupanine enrichments of 0.75 g/L lupanine or 2.0 g/L glucose and 0.75 g/L lupanine

mixture. The results from lupanine quantification are presented in Figure 33.

Figure 33 – Cultivation assays for yeast isolation with three sequential lupanine enrichments. Cultures were

inoculated with 5% (v/v) of lupin beans wastewater (LBW) in the first enrichment. Inoculations of the second and

third enrichments performed with 5% (v/v) of the former enriched culture. (A) 0.75 g/L lupanine; (B) 2 g/L glucose +

0.75 g/L lupanine.

Enrichment cultures for yeast isolation revealed that there was no lupanine comsumption. However,

when diluted samples from the third enrichment were plated in YNB solid medium supplemented with

lupanine and CAM (antibiotic to avoid bacterial growth), in the growth of several colonies of two different

morphotypes was observed (Figure 34). This indicates that the two isolates remained viable until the

end of the third enrichment under lupanine as carbon source.

Figure 34 – YNB solid media supplemented with 1.5 g/L lupanine and CAM, plated with sample from culture at

the end of third enrichment.

61

4.3.2. DNA extraction and strain identification by 26S rDNA amplification

From the yeast strain isolation from LBW, pure colonies of two different morphotypes were

obtained in YNB solid medium supplemented with 1.5 g/L lupanine. Fresh cells were collected from this

growth medium and the DNA was extracted, yielding 31.3 ng/µL and 378 ng/µL, for the two strains. PCR

product results are shown in Figure 35.

Figure 35 – Agarose gel electrophoresis of PCR products obtained for the two yeast strains. Agarose gel (0.8%)

electrophoresis was run during 40 minutes at 90 V with 50x TAE buffer. PCR products were loaded in the gel in

triplicate. (M) Molecular marker NZY DNA ladder III; (1)-(3) and (4)-(6) are the PCR products corresponding to two

yeast isolates; (7) negative control of the reaction.

DNA bands were cut under UV light and purified, with a final concentration of 39.1 ng/µL and 122.1

ng/µL obtained, respectively. Based on sequencing results, the two yeast strains isolates were identified

as Pichia kudriavzevii (with 99.5% identity and 99.65% query coverage) and Rhodotorula mucilaginosa

(with 99.7% identity and 99.3% query coverage).

4.3.3. Yeast cultivation in synthetic culture medium

Firstly, the catabolizing capacity of lupanine was studied in the two yeast isolates, Pichia kudriavzevii

and Rhodotorula mucilaginosa. For this aim, both strains were individually grown in YNB medium

supplemented with 1.5 g/L lupanine. Samples for lupanine quantification were collected at time point 0

(0h, after inoculation) and in 24h periods until the end of the culture (at day 7) (Figures 36 and 37). The

cell growth was followed by measuring OD600 nm every hour during first 6 hours of culture, and then every

24h until 168h, for cultures with 2 g/L glucose, 1.5 g/L lupanine and 2 g/L glucose mixed with 1.5 g/L

lupanine. Besides this, the pH was also monitored during cultivation time (Table 12). The culture

condition with glucose was used as a positive control of the experiment, to evaluate how the isolates

grow under a preferred carbon source, in comparison with growth in lupanine.

M 1 2 3 4 5 6 7

62

Figure 36 – P. kudriavzevii cultures in YNB liquid medium. Pre-culture of 24 hours in 2 g/L of glucose was used

as inoculum of the main cultures (A) in 2 g/L glucose, 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L lupanine. Pre-

culture of 24 hours in 1.5 g/L of lupanine with 2 g/L glucose mixture was used as inoculum of the main cultures (B)

in 2 g/L glucose, 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L lupanine. Growth curves are represented in (A1) and

(B1), and lupanine concentration in (A2), (B2), respectively. Error bars represent the respective standard deviation

of biological duplicate assays.

Figure 37 – R. mucilaginosa cultures in YNB liquid medium. Pre-culture of 24 hours in 2 g/L of glucose was

used as inoculum of the main cultures (A) in 2 g/L glucose, 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L lupanine.

63

Pre-culture of 24 hours in 1.5 g/L of lupanine with 2 g/L glucose mixture was used as inoculum of the main cultures

(B) in 2 g/L glucose, 1.5 g/L lupanine and 2 g/L glucose + 1.5 g/L lupanine. Growth curves are represented in (A1)

and (B1), and lupanine concentration in (A2), (B2), respectively. Error bars represent the respective standard

deviation of biological duplicate assays.

From these results, it is possible to observe that none of the yeast isolates was able to grow when

lupanine is the only carbon source. Additionally, when yeast isolates were cultured in presence of

glucose and lupanine mixtures, although growth was observed, no lupanine consumption was observed.

Results from control flasks prepared under the same conditions and without the addition of cells are

represented in Figure 38.

Table 12 - pH values of YNB medium measured in the supernatant at different time points, during cultivation of

the two yeast isolates: R. mucilaginosa and P. kudriavzevii yeast strain isolates.

Cultures Pre-inoculum Inoculum Day 0 Day 1 Day 2 Day 3 Day 6 Day 7

R. mucilaginosa

Glucose 5.6±0.2 3.1±0.1 3.4±0.1 3.3±0.3 3.4±0.4 3.3±0.2

Glucose Lupanine 6.3±0.1 6.3±0.1 5.5±0.3 5.7±0.2 6.0±0.4 6.7±0.5


Glucose 5.8±0.1 3.3±0.3 3.3±0.2 3.4±0.1 3.3±0.3 3.3±0.4



P. kudriavzevii

Glucose 5.6±0.3 3.6±0.2 3.3±0.4 3.4±0.5 3.5±0.2 3.3±0.3

Glucose Lupanine 6.5±0.2 5.6±0.1 5.4±0.1 6.3±0.3 6.7±0.6 7.0±0.5


Glucose 5.8±0.3 3.5±0.2 4.0±0.5 3.7±0.4 3.5±0.1 3.4±0.5



Figure 38 – Control flask for YNB medium supplemented with 1.5 g/L lupanine or 2 g/L glucose + 1.5 g/L

lupanine.

4.3.4. Yeast cultivation in lupin beans wastewater (LBW)

Since the yeast isolates identified were not able to consume lupanine, the hypothesis of COD

reduction from LBW was investigated. For this, LBW was filtered and used as culture medium in two

conditions: i) diluted in sterilized water at 1/2 and ii) diluted in YNB medium at 1/2, to assess if yeasts

need any compound of YNB medium to grow that may not be present in LBW. The dilutions to LBW

medium were performed due to the high initial concentration of lupanine in this wastewater – 3.3 g/L –,

compared to the concentration of 1.5 g/L lupanine used in previous assays. Additionally, COD levels

0.0

0.4

0.8

1.2

1.6

0 24 48 72 96 120 144 168

Lupanin

e (

g/L

)

Time (h)YNB + Lupanine YNB Glucose + Lupanine

64

and total reducing sugars were measured after inoculation and after 7 days, while pH values were

measured at 24h regular times (Tables 13, 14 and 15 respectively).

Growth curves and lupanine concentration results are represented in Figure 39.

Figure 39 – Growth curves (left column) and lupanine concentration (right column) for 7-day cultures of P.

kudriavzevii and R. mucilaginosa in LBW medium diluted 1:2 in water or in YNB medium. Error bars represent the

respective standard deviation of biological triplicate assays.

The results from Figure 39 show that both isolates were able to grow exponentially without any

significant differences between the dilutions in YNB or water. However, lupanine concentration values

were maintained approximately constant during culture, indicating that most probably these yeast

isolates are consuming other preferable carbon sources instead of lupanine.

Table 13 – Percentages of COD removal from LBW for the two yeast strain isolates: R. mucilaginosa and P.

kudriavzevii.

Bacterial culture with LBW

COD removal (%) in H2O

COD removal (%) in YNB

R. mucilaginosa 47.4 ± 1.5 14.2 ± 0.5

P. kudriavzevii 21.81 ± 2.0 33.1 ± 1.6

65

Inspite of lupanine was not consumed, positive results arised from COD removal in LBW either

diluted in water or in YNB. Rhodotorula mucilaginosa was the yeast isolate able to reduce the higher

percentage of organic matter at the end of the culture.

Table 14 – Reducing sugars concentration determined through DNS method for the initial LBW medium and

after 7 days of cultivation of the two yeast isolates: R. mucilaginosa and P. kudriavzevii.

Culture Medium Reducing

sugars (g/L)

LBW (H2O) LBW (YNB)

LBW+H2O 1.04 ± 0.00

LBW+YNB 1.00 ± 0.00

R. mucilaginosa

LBW+H2O 0.61 ± 0.12

LBW+YNB 0.50 ± 0.02

P. kudriavzevii

LBW+H2O 0.71 ± 0.22

LBW+YNB 0.54 ± 0.05

As previously mentioned, it is speculated that yeast isolates might consumed other carbon sources

instead of lupanine. The total reducing sugar values obtained are confirming this evidence. Table 14

shows that occurred reducing sugar consumption either in LBW diluted in water and in YNB.

Table 15 – pH values of LBW medium measured in the supernatant at different time points, during cultivation

of the two yeast isolates: R. mucilaginosa and P. kudriavzevii.

Culture Medium 0h 24h 48h 72h 144h 168h

R. mucilaginosa

LBW+ YNB 4.75±0.35 5.90±0.42 6.93±0.21 6.97±0.38 8.53±0.81 8.47±0.40

LBW+ H2O 4.85±0.49 6.10±0.85 7.53±0.35 7.57±0.21 8.47±0.40 8.03±0.45

P. kudriavzevii

LBW+ YNB 4.65±0.21 7.25±0.07 7.67±0.15 7.63±0.15 8.33±0.06 8.17±0.21

LBW+ H2O 4.75±0.49 7.55±0.07 7.87±0.15 7.77±0.25 8.57±0.23 8.23±0.06

The initial pH value was around 4.75, and similarly to bacteria, it was also observed a basification

of the LBW culture medium in the two tested dilutions.

4.4. Filamentous fungi: a complementary approach

4.4.1. Isolation and identification

Fungi screenings were performed by plating different dilutions of LBW and its surface organic-rich

layer, in PDA and MEA solid media, supplemented with CAM to avoid bacterial growth. It was possible

to isolate four fungi strains with apparent different morphologies. The genomic DNA extraction was

performed according to the extraction kit protocol and yielded for the different strains 102.5 ng/µL, 44

ng/µL, 59 ng/µL and 23.5 ng/µL of gDNA. The integrity of gDNA was assessed in an agarose gel

electrophoresis (Figure 40), with 200 ng of gDNA loaded for each sample.

66

Figure 40 - Agarose gel electrophoresis of gDNA obtained for the four filamentous fungi strains. Agarose gel

(1%) electrophoresis was run during 90 minutes at 120 V with 1x TAE buffer. (L) Molecular marker NZY DNA ladder

III; (1)-(4) gDNA corresponding to the four fungi isolates.

The gDNA samples obtained were amplified by PCR and sequenced, by STABVIDA. Based on the

sequencing results, the isolates were identified as Pichia kudriavzevii (99.92% identity), Aspergillus

fumigatus (99.93% identity) which represented two of the strains, and Galactomyces geotrichum

(99.64% identity).

4.4.2. Growth of isolates in rich mediums

After identification of the strains, with one of them, Pichia kudriavzevii, already identified in section

4.3.2., the main objective was to study the strains ability to catabolize lupanine. For this, cultures were

performed in ME medium supplemented with 40 g/L glucose, 1.5 g/L lupanine or 40 g/L glucose mixed

with 1.5 g/L lupanine. Glucose growth condition was used as a positive control of the experiment, to

evaluate how the isolates grow with a preferred carbon source, in comparison with growth in lupanine.

During the cultivation, cellular growth was measured through cell dry weight and the lupanine was

quantified, at time point 0 (0h, after inoculation) and subsequently in periods of 24h until 192h, as shown

in Figure 41.

L 1 2 3 4 4

67

Figure 41 – Dry weight values (left column) and lupanine concentration (right column) for 186 hours cultures of

P. kudriavzevii, A. fumigatus and G. geotrichum in ME medium supplemented with 40 g/L glucose, 1.5 g/L lupanine

or 40 g/L glucose + 1.5 g/L lupanine. Data shown from single assays.

In the results from Figure 41, none of the isolates showed lupanine catabolization capacity either

with glucose and lupanine mixtures or only with lupanine as carbon source.

4.4.3. Growth of isolates: assessment of using filamentous fungi for lupin beans wastewater

(LBW) treatment

Similar to the experiments previously performed for bacteria and yeast, the capability of fungi strains

to reduce COD from LBW was investigated. For this, LBW pH was corrected to 6 and sterilized to be

used as a culture medium. A sample was taken before cell inoculation to assess lupanine concentration

and COD level at the beginning and at the end of culture (192h). The results are shown in Table 16 and

17, respectively. Lupanine was also quantified at time point 0h and at 192h (Figure 42).

68

Figure 42 – Growth curves in cell dry weight (A) for 192 hours cultures of P. kudriavzevii, A. fumigatus and

G. geotrichum, in LBW medium. Data shown from single assays.

Figure 42 evidences that all isolates were able to grow according with their cell dry weight. A.

fumigatus cell dry weight decrease after 144h because this filamentous fungi grows in aggregates

and it was not possible to collect homogenized samples after this time point.

Table 16 – Lupanine concentration in LBW medium during cultivation of P. kudriavzevii, A. fumigatus and G.

geotrichum

Culture

Lupanine (g/L)

0h 192h

Pichia kudriavzevii 1.07 1.23

Aspergillus fumigatus 1.07 0.94

Galactomyces geotrichum 1.07 0.87

Lupanine concentrations at day 0 and day 7 did not showed significant reductions. For further

investigations, it can be suggested the use of basal culture mediums without any other carbon source

present besides lupanine, since the culture mediums used are rich mediums.

Samples were collected at the end of the assay, to measure the COD for the cultures with the

different fungi strains, and evaluate the ability of the fungi isolates to detoxify the water, as shown in

Table 15. COD level at time point 0 was 30.83 g O2/L).

Table 17 – Percentages of COD removal from LBW for the fungi strain isolates. n.a. - COD increased from

30.83 to 44.71 g O2/L.

Culture COD (% removal)

Pichia kudriavzevii 5

Aspergillus fumigatus 40

Galactomyces geotrichum n.a

COD removal percentages show that A. fumigatus was able to remove 40% of the organic matter

present. However, since LBW was sterilized by autoclave, the values are not comparable to the ones

obtained from bacterial and yeast growth where LBW was sterilized by filtration.

0

2

4

6

8

0 24 48 72 96 120 144 168 192

Dry

weig

ht

(g/L

)Time (h)

P. kudriavzevii Aspergillus fumigatus

Galactomyces geotrichum

A

69

4.5. Determination of lupanine derived-products

The bioconversion of lupanine in O. anthropi and C. funkei cultures was investigated given the

decrease of 66.7% of lupanine concentration by the action of O. anthropi (Figure 27), and the decrease

of 33.3% at 24 h observed in C. funkei (Figure 30) cultures.

FT-IR ATR was the first spectroscopy test to evaluate lupanine derived-product formation. The

obtained results from FT-IR ATR of O. anthropi cultures for the different time points of culture in

comparison with a pure sample of lupanine are shown in Figure 43.

Figure 43 - Spectra obtained from FT-IR ATR of O. anthropi cultures among time. Pink line represents the

pure sample of lupanine, while the other coloured lines represent the different time points of culture (0 h, 3 h, 6 h,

12 h and 24 h) being all superimposable.

The results from C. funkei cultures for different time points of culture in comparison with a pure

sample of lupanine are shown in Figure 44.

C=O

70

Figure 44 - Spectra obtained from FT-IR ATR of C. funkei culture among time. Violet line represents the pure

sample of lupanine, while the other coloured lines represent the different time points of culture (0 h in yellow, 48 h

in blue, and 120 h in green).

Unfortunately, given the interference of the culture medium compounds (Na2HPO4·2H2O, KH2PO4,

NaCl, NH4Cl, MgSO4), the obtained results were not conclusive, and the observation of other functional

groups was not possible.

To complement the previous technique, thin layer chromatography (TLC) was also performed, in

silica plates. The TLC silica plate obtained from application and elution of samples is illustrated in Figure

45.

Figure 45 - TLC plate with two elutions in MTBE/hexane (8:2) and 1% diethylamine (DEA) with

phosphomolybdic acid staining. (L) is pure lupanine; (1)-(5) are the samples corresponding to 0 h, 3 h, 6 h, 12 h

and 24 h of O. anthropi culture and (6)-(8) are the samples corresponding to 0 h, 48 h and 120 h of C. funkei culture.

Observing Figure 35, the results demonstrate that lupanine presented a small impurity, shown by

the bands with lower retention factor (Rf) than lupanine in the pure sample that are also present in further

culture samples. Given this, it is not possible to observe other compounds besides lupanine with different

polarities.

To confirm the obtained results, NMR spectroscopy was also performed. For demonstration, the 13C

and 1H spectra from a pure lupanine sample are shown in Figures 46 and 47, respectively. In these

figures, it is possible to observe peaks distribution according with literature [29]. In Figures 48 and 49

C=O

71

the 13C and 1H spectra obtained with the samples of O. anthropi and C. funkei cultures are presented,

in comparison with a pure lupanine sample.

Figure 46 - 13C NMR spectrum of a pure lupanine sample in CDCL3 [29].

Figure 47 - 1H NMR spectrum of a pure lupanine sample in CDCL3 [29].

72

Figure 48 - 13C NMR spectra of the samples corresponding to O. anthropi and C. funkei cultures. (A) is the

culture of C. funkei at 48 h; (B) is the culture of C. funkei at 120 h; (C) is the culture of O. anthropi at 3 h; (D) is the

culture of O. anthropi at 24 h; (E) is the pure lupanine sample.

Figure 49 – 1H NMR spectra of the samples corresponding to O. anthropi and C. funkei cultures. (A) is the

culture of C. funkei at 48 h; (B) is the culture of C. funkei at 120 h; (C) is the culture of O. anthropi at 3 h; (D) is the

culture of O. anthropi at 24 h; (E) is the pure lupanine sample.

73

Given these results, the capacity of these bacterial species to consume preferably one lupanine

enantiomer over the other was investigated by HPLC determination of enantiomeric excess. Results

from HPLC are shown in Figures 50-53.

Figure 50 – Chromatogram obtained from 0 h of O. anthropi culture, showing racemic lupanine in a proportion

of 53% of D-(+)-lupanine and 47% of L-(-)-lupanine.

Figure 51- Chromatogram obtained from 12 h of O. anthropi culture, showing racemic lupanine in a proportion

of 43% of D-(+)-lupanine and 57% of L-(-)-lupanine.

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

-10

-5

0

5

10

15

20

25

30

35

40

45

50

55

mAU240nm,4nm (1.00)

19.4

84/1

719981

21.4

42/1

550146

12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

-7.5

-5.0

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

mAU240nm,4nm (1.00)

20.0

99/7

71841

21.9

92/1

036018

D-(+)-lupanine

L-(-)-lupanine

D-(+)-lupanine

L-(-)-lupanine

74

Figure 52 – Chromatograms obtained from 24 h O. anthropi culture, showing 100% of L-(-)-lupanine.

The results show that after 12 h of culture, D-(+)-lupanine starts decreasing approximately 10 %,

and at 24 h it is only observed the enantiomer L-(-)-lupanine in culture. This suggests that between 12

h and 24 h of culture, a higher consumption occurs. With this result, it can be concluded that O. anthropi

uses D-(+)-lupanine as carbon source to produce energy for growth. C. funkei culture results revealed

that this specie is not able to consume none of the two enantiomers. Thus, the proportion of 53% D-(+)-

lupanine and 47% L-(-)-lupanine of racemic lupanine was maintained constant until 120 h of culture

(Figure 43).

Figure 53 - Chromatograms obtained from C. funkei cultures. (A) is the result obtained from 0 h of culture, with

racemic lupanine in a proportion of 53% of D-(+)-lupanine and 47% of L-(-)-lupanine; (B) is the result obtained from

120 h of culture, with a proportion of 53% of D-(+)-lupanine and 47% of L-(-)-lupanine.

12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

-10

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

mAU240nm,4nm (1.00)

21.3

98/2

163283

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

-15.0

-12.5

-10.0

-7.5

-5.0

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

mAU240nm,4nm (1.00)

19.6

74/9

96289

21.6

46/8

92180

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

-10

-5

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5

10

15

20

25

30

35

40

45

50

55

60

mAU240nm,4nm (1.00)

20.4

18/2

006809

22.4

47/1

845754

L-(-)-lupanine

A B D-(+)-lupanine

L-(-)-lupanine

D-(+)-lupanine

L-(-)-lupanine

75

5. Discussion

5.1. Nanofiltration

The nanofiltration experiment was used to obtain a retentate fraction rich in lupanine and a

permeated fraction composed of water. As observed in Figure 19, after the initial 10% concentration

where the flux decreases from 48.12 to 34.98 L/h/m2, the nanofiltration flux was constant during time,

indicating that the membrane is adequate and the nanofiltration process may be feasible at an industrial

scale. The results indicate that the nanofiltration process was successful with high rejection of lupanine

and COD by the membrane. With this process was possible to obtain a concentrated fraction of 150 mL,

of 2.12 g/L of lupanine in concentration mode. The COD values are in aggreement with the increased

lupanine concentrations on the retentate, contrary to the permeate that presents a low concentration of

lupanine and COD, as expected.

Further experiments could include permeated water characterization to ensure that it complies with

the required quality parameters to be recycled back to the industrial debittering process. These

improvements to the process would decrease the amount of fresh water consumed, enable the effluent

treatment and consequently reduce the environmental impact of the wastewaters.

5.2. Bacteria

The present work aimed at the isolation and identification of microorganisms with potential to

catabolize lupanine. For this, the working strategy was, first, to isolate bacteria, yeast and fungi strains

from the LBW and a superficial organic-rich layer formed during the summer, both rich sources in

biodiversity, using media containing lupanine as carbon source. This isolation technique called

enrichment culture consists in using particular growth conditions designed to favour growth of a

microorganism of interest. Here, culture conditions are set to increase small populations of desired

microorganisms to detectable levels, illustrating natural selection in vitro [120]. These cultures are set

in liquid media, with the inoculation of a sample containing natural material with the mixed microbial

population. This sample is collected from environmental habitats in which the desired microorganism is

expected to occur [121].

In the current study, wastewater from cooking phase of industrial debittering process of lupin beans

was used as inoculum, as well as the organic-rich layer formed on the LBW surface. These samples

were enriched using liquid cultures supplemented with lupanine, glucose and lupanine/glucose mixture

as carbon sources. Figure 20 evidences that lupanine concentration decrease after 7 days of

enrichment for glucose/lupanine mixture conditions, reaching unmeasurable values by HPLC when a

sample of the superficial layer was used as inoculum. When lupanine was the sole carbon source, a

decrease was also shown in the second and third enrichments. In the end, it was possible to distinguish

three different gram-negative bacterial morphotypes, identified as Ochrobactrum anthropi,

Stenotrophonomas maltophilia and Sphingobacterium siyangense. All isolates were identified with more

than 98% of identity. Until this date, none of these strains had been isolated from LBW surface layer.

Otherwise, when LBW was used as inoculum, lupanine concentrations remained approximately

constant (Figure 22), despite the increasing visible turbidity observed in the cultures (indicating cellular

growth). From the enrichment with LBW, a fourth bacteria was identified, the gram-positive

76

Cellulosimicrobium funkei. However, given that bacterial isolation was performed according to the

different morphotypes, it is possible that very similar colonies had not been successfully isolated.

Additionally, in culture enrichments, there is continuous variation of culture conditions, with nutrient

consumption and excretion of metabolic products according to each individual microorganism growth,

which is a disadvantage of this technique since these changes may favour the growth of certain

microorganisms that become prevalent over others. Meaning that, a culture medium with apparently no

selectivity may become highly selective depending on chemical changes promoted by the developing

organisms. In this case, natural selection depends on the growth rates of each individual from the

population for given conditions [121].

According to the literature, strains of Ochrobactrum genus are part of the family Brucellaceae and

are well adapted to a wide range of ecological niches. Ochrobactrum anthropi, formerly “Achromobacter”

was proposed as a new strain in 1988 by Holmes et al. [122]. The species in this group are potential

human pathogens, with a frequent isolation from hospital water sources, sinks, seawage and soil [123].

Ochrobactrum anthropi MP3 strain is able to produce an exopolysaccharide and degrade hydrocarbons

in a petroleum refinery wastewater, which are highly toxic to the environment and must be treated before

discharge [124]. Here, the authors concluded that this strain is an efficient degrader of diesel oil, and

that the exopolysaccharide produced increased the uptake of oil as a carbon source [124]. The ability

of this bacteria to degrade various polycyclic aromatic hydrocarbons such as phenanthrene, hexane,

heptane, hexadecane and pesticides has also been documented [125]–[127].

Recent research works demonstrated that Ochrobactrum lupini and Ochrobactrum anthropi are

strains that share high similarity in phylogenetic markers, evidencing 100% for 16S rRNA, 99.9% for

dnaK and 99.35% for rpoB [128]. RNA polymerase beta subunit (rpoB) and 70-kDa heat shock protein

(HSP70, gene dnaK) are other stable marker genes used as complementary tools for the 16S gene.

Gene rpoB typically occurs in a single copy, while most bacterial genomes have 16S rRNA gene in

multiple copies (with copy number differing with species), representing a disadvantage for species

identification since there are sequence variations between different gene copies in some genomes [129],

[130]. Single-copy genes favour an accurate measurement of diversity and phylogenetic relationships,

avoiding biases and loss of phylogenetic resolution caused by intragenomic heterogeneity [131].

Therefore, 16S rRNA may not be always suitable for distinguish closely related species. Gene HSP70

is another alternative marker for phylogenetic studies, since it is one of the most conserved proteins

found in all biota [132]. Given this, molecular microbial ecology is not limited to 16S rRNA gene, although

this is by far the most frequently used gene. O. lupini is a bacterial strain isolated from Lupinus albus

plant root nodules [133], that is closely related in phylogenies with O. anthropi strain. These two strains

do not have pronounced differences at phenotypic, phylogenetic, chemotaxonomic and genomic levels.

Given this, O. lupini may be considered a later heterotypic synonym of O. anthropi [134]. Therefore, it is

reasonable that O. anthropi appears in LBW.

Yabuuchi et al. (1983) [135], proposed the genus Sphingobacterium as a group of aerobic gram-

negative bacilli with high concentrations of cellular lipid components – sphingophospholipids – in the

cell membrane [136]. So far, about 50 species have already been identified as belonging to this genus

[137], either in natural environments: sandy soils, activated sludge, farm and forest soils, or in water

77

sources (including those in hospitals) [138]–[143]. Sphingobacterium siyangense has been isolated from

farm soil, with optimal conditions for its growth described as the following: 30-37°C, pH 6.0-8.0 and 0-

2% NaCl, in nutrient agar. From a biotechnological perspective, there are evidences that

Sphingobacterium sp. is capable of producing a biosurfactant mixture [144], which is interesting due to

the potential applications in different sectors: foods, cosmetics, agriculture and pharmaceutics. And,

since the stricter legal control and health regulations from industrial activities have been favouring the

replacement of chemical surfactants by their biodegradable substitutes [144]. There are no published

findings of this bacteria isolated from lupin beans wastewater, neither the study of its lupanine

catabolizing capacity or growth under LBW. However, due to the isolation and identification of this

species in farm soils as well as in water sources, which is in concordance with lupin beans plant

environment, its presence is foreseen.

Stenotrophomonas maltophilia was classified in 1993 [145], [146] as a gram-negative bacillus,

aerobe, considered an environmental global emerging multiple-drug-resistant organism commonly

related with respiratory infections in humans, and their incidence is particularly increasing in

immunocompromised patients [147]. This pathogen has been isolated from aqueous-associated

sources either inside or outside the clinical setting of hospitals: in soils and plant roots [148], wastewater

plants [149], biofilms in aquifers [150], bottled water [151], haemodialysis water [152] among others.

Additionally, S. maltophilia has also been associated with a few plants, such as oat, cucumber, maize

oilseed rape, potato and the rhizosphere of wheat [148], [153], [154]. One particular feature of S.

maltophilia is its potential to adhere to plastics and form bacterial films – biofilms. Therefore, this bacteria

has been isolated from surfaces of intravenous materials like prosthetic devices or dental unit waterlines

(e.g.) [155], [156]. This bacterial species has demonstrated potential in the biotechnology field, since it

is involved in several studies in: bioremediation capacity for copper removal from aqueous solution [157];

biodegradation of two petrochemical hydrocarbons: diesel oil and engine oil [158]; production of

enzymes with an important role in synthesis of compounds with medical applications e. g. anti-

inflammatory analgesics using lipase obtained from this bacteria [159]. To our best knowledge, the work

developed in this thesis is the first study describing the presence of S. maltophilia in LBW, as well as

the first study to test lupanine catabolization capacity and the growth under LBW (topics further

discussed in this thesis). Neverthless, this bacteria had previously been isolated from plant roots and

wastewater plants [148], [149], what can explain its presence in lupin beans wastewater for the first time.

Schumann et al.(2001), referred Cellulosimicrobium species as gram-positive bacteria widely

distributed in soil and water [160]. Bacteria from this species are uncommon opportunistic pathogens

that can cause infection in immunocompromised patients [161]. Cellulosimicrobium funkei is able to

grow at 35 and 45 °C, forming substrate hyphae visible in solid medium. In 2017, a novel bacterial strain

of C. funkei was identified in soil contaminated with leather industrial effluent and distinguished due to

its high tolerance to Cr(VI) (the most toxic form) and ability to reduce it to Cr(III) [162]. Besides this,

other studies have demonstrated that C. funkei has also potential to detoxify aflatoxin B1, a secondary

fungal metabolite considered a toxic mycotoxin with mutagenic and carcinogenic effects on living

species [163]. This secondary metabolite can easily contaminate maize-based food and feed around

the world, leading to significantly economic losses [164]. Until date, there are no published research

78

works concerning the isolation and identification of this bacterial species from LBW, nor lupanine

catabolization capacity studies. However, since it is commonly isolated from soil and water, it is

reasonable that this species occur in lupin beans wastewater.

Considering cultures of the characterized bacterial species in synthetic growth medium M9, it is

possible to assume that only O. anthropi revealed clear lupanine catabolizing capacity.Here, initial

lupanine concentration of around 1.2 g/L, decreases to values of around 0.4 g/L, which is a 66.7%

reduction, in both lupanine and glucose + lupanine conditions. The results from the control cultures

(Figure 31) indicate that lupanine decrease levels are due to microbial activity, since no variations were

observed. Specific growth rates (µ) calculated for the first 6h revealed the following values for the

different conditions (Table 18):

Table 18 – Specific growth rates (µ) of O. anthropi cultures in each growth condition.

Pre-culture Culture Specific growth rate (h-1)

Glucose Glucose 0.13 ± 0.05

Lupanine Lupanine 0.10 ± 0.03

Glucose + Lupanine 0.11 ± 0.03

Glucose + Lupanine

Lupanine 0.08 ± 0.03

Glucose + Lupanine 0.12 ± 0.02

It is possible to see that when lupanine is present as carbon source, the specific growth rate is

lower in comparison with glucose and glucose with lupanine mixtures.

Additional assays were performed for FT-IR, TLC and NMR of lupanine at different time points to

evaluate possible by-product formation. FT-IR is here used as a comparative and qualitative technique,

since it demonstrates the difference in functional groups and not exactly their location in the molecule.

Unfortunately, given the interference of the culture medium compounds the obtained results were not

conclusive, and the observation of other functional groups was not possible (Figure 33). In a similar way,

TLC only indicates the presence or absence of other compounds besides lupanine, without any other

information about the resulting structure of these molecules. Observing Figure 35, the Rf of the pure

sample of lupanine is higher than the culture samples, evidencing a higher lupanine concentration with

a bigger stain for the applied sample. Given this, it is not possible to observe other compounds besides

lupanine with different polarities.

NMR results (Figures 38-39) showed that only lupanine was present in the culture, indicating that

there by-product formation did not occurred by comparison with the spectra obtained for a pure lupanine

sample (Figures 36-37).

When enantiomeric excess was determined, The results show that after 12 h of culture, D-(+)-

lupanine starts decreasing approximately 10 %, and at 24 h it is only observed the enantiomer L-(-)-

lupanine in culture. This suggests that between 12 h and 24 h of culture, a higher consumption occurs,

which is in agreement with the increase in the OD600 nm value measured at 12 h (0.14) and 24 h (0.80),

respectively. With this result, it can be concluded that O. anthropi uses 100% of D-(+)-lupanine as carbon

source to produce energy for growth (Figures 40-42). Further experiments to verify this assumption

79

could include more hours of culture, to assess if the remaining enantiomer (L-(-)-lupanine) is consumed

after the tested 24 h.

The HPLC quantification of the enantiomers in C. funkei cultures revealed that this specie is not

able to consume none of the two enantiomers. Thus, the proportion of 53% D-(+)-lupanine and 47% L-

(-)-lupanine of racemic lupanine was maintained constant until 120 h of culture (Figure 43).

Until date, there are no studies with O. anthropi strain and its ability to catabolize lupanine. However,

there are former studies related with gram-negative bacterial strains capable of using lupanine as sole

carbon and energy source [30], [87], [88]. In 1996, Santana et al., isolated seven strains from soil of L.

albus and L. luteus growth, with specific growth rates ranging from 0.05 to 0.13 h-1 in the presence of 2

g/L lupanine, which are similar values to the ones obtained for the O. anthropi strain isolated in the

present study. In 2018, Parmaki et al., published results from four isolates with ability to use lupanine

as sole carbon source, grown in M9 with 1.5 g/L of lupanine, in conditions identical to those used in this

work. The four isolates were identified as Rhodococcus rhodochrous LPK211, Rhodococcus ruber

LPK222, Rhodococcus sp. LPK311 and Pseudomonas putida LPK411, with 81% (in 36h), 66% (42h),

71% (36h) and 80% (36h) of lupanine removal, respectively. Final OD600 nm measurements of sample

cultures from these isolates were 1.2, 1.3, 1.2 and 0.6, respectively. In the present work for O. anthropi

show a similar lupanine removal percentage was obtained (66.7%), and a higher final value of OD600 nm

of 1.73 for the condition with lupanine as sole carbon source. There is no published information

regarding O. anthropi ability to degrade lupanine. However, related studies reported Ochrobactrum

intermedium DN2 as a strain with 99.5% homology with O. anthropi and with nicotine-degrading

capacity, which is another alkaloid. This strain has totally degraded nicotine in basal medium containing

between 500-4000 mgnicotine/L. Biomass produced was affected by initial concentration of nicotine,

meaning that high concentrations of nicotine (5000 mg/L) restrict cellular growth, evidencing substrate

inhibition. The authors state that this behaviour was similar to the growth of Pseudomonas putida when

nicotine is used as carbon source [165]. Additionally, it was concluded that enzymes involved in the

degradation of nicotine by O. intermedium were allocated in the cell-wall membrane, enabling the direct

transferring of electrons from the substrate to components of the respiratory chain during enzymatic

reaction [166]. However, nicotine catabolic pathway of O. intermedium is likely to differ from other

bacterial strains like Artrobacter nicotinovorans that produces a blue pigment in result of nicotine

metabolization, which is not observed in O. intermedium [167].

To investigate the lupanine catabolizing capacity of P. putida, Detheridge et al. (2018), analysed

the genome sequence of one strain capable of growing in lupanine named Psp-LUP. Previous

investigations on the lupanine degradation mechanism by Psp-LUP demonstrated that the first step in

degradation of the heterocyclic ring containing tertiary nitrogen atoms comprise the hydroxylation in the

17- position to give rise to 17-hydroxylupanine (Figure 43) [168].

80

Figure 54 – Reaction catalysed by lupanine 17-hydroxylase.

It is already known that this step is catalysed by lupanine 17-hydroxylase, an inducible enzyme

characterized as PQQ (pyrroloquinoline quinone)-containing haemoprotein [169], [170]. This enzyme

operates in lupanine as a dehydrogenase, to introduce the hydroxyl group. In 2002, Hopper et al.

sequenced the gene encoding for this enzyme, and the enzyme was characterized, following its

heterologous expression in E. coli [171].

Since the gene encoding for lupanine hydroxylase is known (AJ318095) in P. putida strain, the

genome sequence of O. anthropi strain used in this research work could be studied in order to better

understand if there is an identical gene that can further clarify the catabolic pathway for lupanine

assimilation in this bacteria. In the future, different lupanine concentrations could also be tested, to

assess if there is a relation between biomass produced and lupanine concentration and if it has an

inhibitory effect on the culture at some point. Furthermore, to assess the possibility of the lupanine-

degrading enzyme being in the cell-wall membrane of O. anthropi (similarly to O. intermedium), an

identical protocol to the one described in Yuan et al. (2005) for cell-free preparations could have been

performed. Here, cells were disrupted and the intracellular fraction corresponding to cell debris was

collected and solubilized with 0.1% (w/v) of SDS in 0.1 M phosphate buffer (pH 7.0). This solution was

transferred (10% v/v) into a phosphate buffer solution (pH 7.0) with 100 mgnicotine/L and incubated at

30°C and 120 rpm with regular nicotine concentration analysis. The same steps can be performed using

lupanine, with regular collections of samples for lupanine quantification by HPLC, to assess if decrease.

Considering the results obtained for the remaining bacteria isolates in synthetic medium, none of

them revealed growth or lupanine-degrading capacity when lupanine was used as sole carbon source.

However, growth was observed in sole presence of glucose and in glucose/lupanine mixtures, indicating

that lupanine also does not inhibit bacterial growth, otherwise they would not be able to grow in glucose

+ lupanine condition. Figure 28 (A2) and 30 (A2), corresponding to S. siyangense and C. funkei show

a little decay in lupanine concentration at 48h and 24h, respectively, with a slight increase after this time

points. The initial decrease in lupanine concentration may be, in fact, due to a small consumption of the

molecule, but the increase in the values are questionable. These results can be considered false positive

results, due to experimental errors in sampling withdraw or in HPLC quantification of lupanine (in the

case of a sub-product detected at lupanine retention time). Other approaches to investigate this result

can be further tested: an inoculum with higher biomass, or cultures with different initial lupanine

concentration in a range of different values.

The main objective of having glucose mixed with lupanine in a growth condition was to hypothesise

it the isolates could have lupanine-degrading capacity after using glucose as main substrate and

81

reaching stationary phase. In this case, glucose would be used as substrate and lupanine as a reagent

to give rise to sub-products. The metabolism of a growing bacteria is divided in two categories: primary

and secondary. Primary metabolites are intracellular molecules that enable growth and proliferation of

the bacteria and participate in exponential growth phase, while secondary metabolites are mainly

extracellular molecules that facilitate the interaction of the bacteria with its environment, and are

produced throughout late-exponential and stationary phase growths [172]. However, the research

developed during the past century revealed a detailed, quantitative and holistic characterization of

primary metabolism [173], [174], and the same is not yet applied for secondary metabolism. The

biochemical processes behind metabolism can be classified into catabolism (break down of complex

molecules into simpler ones, releasing energy) or anabolism (synthesis of complex molecules from

simpler ones, requiring energy). Metabolites are the end-products of these reactions, and are used for

the formation of intermediates and substrates for other metabolic pathways. These metabolites can have

several biological properties of pharmaceutical, nutritional or agricultural importance. Secondary

metabolites usually serve as a survival strategy for the microorganism during adverse conditions, and

its production is triggered during exhaustion of nutrients, environmental stress or limited growth

conditions [175]. For these reasons, consumption of lupanine after glucose depletion was studied, but

the results evidence that during 168h of culture and only with 2 g/L of glucose, lupanine concentrations

remained constant.

The pH homeostasis of the culture is important for cell metabolism, since the function of biological

macromolecules (especially proteins, enzymes), as well as chemical reactions, depend on these values.

Bacteria can live at different ranges of pH values, being classified as acidophiles (pH 1-3), alkalophiles

(pH 10-13) or neutrophils (pH 5.5-9). Bacterial strains found in this work are classified as neutrophiles,

with optimum pH values comprehended in an interval of 6-8 (pH 7 for O. anthropi; pH 6.0 - 8.0 for S.

siyangense; pH 6.0 for S. maltophilia) [141], [166], [176], [177]. Extracellular pH can be changed during

culture due to the metabolism itself, medium composition, the growth phase, and the physiology of the

bacteria [178]. Table 8 demonstrates an initial pH around 7.3-7.5 and a final pH of 7.7-8.2, indicating a

small increase in these values. Usually, microorganisms can change the environment through the

uptake of resources and the excretion of metabolites, which can affect the own growth. Variations in

environmental pH is common during cultures. This parameter can decrease or increase, leading to

beneficial or deleterious effects on its growth [179]. In the results obtained from the bacterial cultures,

since the pH during the culture did not vary significantly, the growth of each bacteria was not apparently

affected by this parameter. Different initial pH values of the culture could have been tested, to evaluate

if lupanine consumption by O. anthropi might be affected. Additionally, the optimum pH values for each

bacteria could have been used to evaluate if a different behaviour would be observed.

Lupanine charge can vary according to the pH of the culture. Since it has a pKa of 9.1, bellow this

pH it has a positive charge, being protonated, and above this pH it is deprotonated (Figure 44). The pH

changes during the culture may influence lupanine transporter, which has not yet been identified.

82

Figure 55 – Protonated and deprotonated forms of lupanine, according with the pH of solutions. Adapted from

[29].

In 2002, Santana et al. investigated the potential of two lupanine-catabolizing strains (IST20B and

IST40D) to remove quinolizidine alkaloids (at 20°C, 27°C and 34°C), from a lupin seeds extract [88].

This extract was diluted in a buffered solution with a final lupanine concentration of 2 g/L and final total

alkaloid concentration of 3 g/L. Here, the authors verified the levels of soluble protein, amino acids, total

carbohydrates, total QA and lupanine concentrations after removing cells by centrifugation. They verified

that the initial concentration of QA, especially lupanine, decreased after cell inoculation, as well as the

initial concentration of amino acids. However, the concentration of carbohydrates remained stable

during the active catabolism of amino acids and QAs. This first period of bacterial growth corresponded

to a first phase of exponential growth, followed by a second slower phase of growth, evidencing a diauxic

profile. The second phase of growth apparently corresponded to partial utilization of the carbohydrates

present. IST20B and IST40D presented specific growth rates of 0.37 h-1 and 0.26 h-1 at 27°C, which were

higher values compared with the µ obtained in synthetic medium for the same temperature (0.14 h-1 for

IST20B and 0.17 h-1 for IST40D). The three temperatures tested have demonstrated that this

environmental parameter can significantly affect growth kinetics and by consequence the bacterial

catabolic performance. The metabolic fingerprints of these two strains revealed that they are able to

grow using the following common amino acids: glutamic acid, alanine, leucine, phenylalanine, proline,

serine and threonine. All of these amino acids are present in lupin seeds [30], [180], [181].

In the present research work, none of the isolates demonstrated lupanine-catabolizing capacity with

LBW as growth medium, despite the high growth observed (Figure 32). O. anthropi showed lupanine-

catabolizing capacity in synthetic medium M9. However, when using LBW as medium, lupanine

concentrations were maintained during the culture, despite the high specific growth rate of 0.31 h-1 ±0.03

observed (a similar result to the one obtained for IST40D), higher than µ obtained in synthetic medium

with lupanine as sole carbon source. LBW lupanine concentrations remained constant probably because

of other available carbon sources used preferably over lupanine. Although, besides the unobserved

variation of lupanine, Table 9 results show that all isolates were able to reduce the COD, and that O.

anthropi was the strain presenting higher removal percentage (58.7%), enabling partial organic matter

removal of LBW. It would be interesting to assess if the isolates are able to remove the total amount of

COD from LBW extending the culture time or will eventually start consuming lupanine as other carbon

sources are depleted. Additionally, the analysis of sub-products during these fermentations can be

further studied, to understand if the isolates produce any compound of interest derived from the COD

consumed of LBW. From the pH values obtained in Table 11, it is clear an increase in the pH, which

can be due to protein or amino acid decomposition, since ammonia is formed by many bacteria as an

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end product of this decomposition. Bacteria can cleave ammonia from the amino acids and since reacts

with water recruiting a proton, ammonium is formed in aqueous solution, increasing the pH [182].

In this work, total reducing sugars were measured by the DNS method. This method can only be

used to quantify the presence of a free carbonyl group (C=O), having as basis the oxidation of the

aldehyde or ketone functional groups from reducing sugars like glucose or fructose. However, this

method has some limitations, since it is not very suitable for determination of sugar contents in complex

mixtures (as it is the case of LBW) and can be either preferably used for accurate measurements of

pure solutions. Non-reducing sugars like sucrose or others cannot be determined by DNS since they do

not participate in the carbonyl reactions used in DNS. Here, HPLC-MS would be the more accurate,

sensitive and reliable method for quantification of the different sugars present in LBW [117].

There are a few common carbon and nitrogen sources used by the four bacterial species, as

illustrated in Table 19.

Table 19 – Some of the carbon and nitrogen sources used by the four bacterial species isolated. (*) indicates

two carbon sources that may be present in LBW.

Species Threonine Proline Trehalose Arabinose Glucose Fructose Galactose*

Maltose Sucrose*

O. anthropi [122]

S. siyangense [141]

- -

S. maltophilia [151]

- - - -

C. funkei [183]

- -

In 2015, Fritsch et al., evaluated the fermentation performance of lactic acid bacteria in different

lupin substrates [184]. One of the substrates tested was the flour of the alkaloid-rich Lupinus

angustifolius, in which carbohydrate composition determination by high performance anion exchange

chromatography gave rise to the following results (g of carbohydrates/kg bitter lupin flour): galactose

0.5, sucrose 37.1, raffinose 8.0, stachyose 34.4 and verbascose 17.3. Given this information, it is

possible to do a speculation about some compounds that may be present in LBW.

Further investigations in this field could include the study of different growth temperatures, to asses

if temperature can affect substrate consumption, as well as different LBW dilutions in sterilized water

and the measurement of initial and final soluble proteins, amino acids and total quinolizidine alkaloids.

This way, it would be possible to clarify which substrates are preferred for bacterial consumption.

5.3. Yeast

Considering enrichment cultures for yeast isolation, a sample of LBW was enriched in lupanine (1.5

g/L), glucose (2 g/L) and lupanine (1.5 g/L) mixed with glucose (2 g/L) aiming to isolate yeast species

that are more adapted to lupanine in culture. As observed in Figure 33, lupanine concentration levels

did not provide a constant concentration, indicating an experimental error in the collection of the sample

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for HPLC, or in the preparation of lupanine solution. Still, at the end of the third enrichment, yeast

colonies from two different morphotypes were observed in YNB solid media supplemented with lupanine

(1.5 g/L). Both yeast strains (Pichia kudriavzevii and Rhodotorula mucilaginosa) were identified with

more than 99% of identity, and until date none of them had been isolated from LBW. For this case,

enrichment assays were not performed using the superficial layer of the LBW, however, it was done an

isolation step with dilutions of this sample and the identification revealed the presence of Pichia

kudriavzevii and Diutina (candida) rugosa. The work with the last yeast isolate was not followed, since

was observed very low population density and low growth (1 colony in a 10-5 dilution of the LBW

superficial organic layer) in YNB supplemented with lupanine (1.5 g/L).

According to the literature, Pichia kudriavzevii was initially described as Issatchenkia orientalis by

Kudryavtsev (1960), and was reclassified as Pichia kudriavzevii some years later by Boidin et al. (1965)

[185]. This yeast has been isolated from a variety of sources: cocoa bean fermentation, mango pulp

peel compost, cereal-based beverage, fermented butter, sugar cane juice, sweet sorghum stalk and rice

straw [186]–[189]. P. kudriavzevii is characterized by their robust physiology due to the tolerance to

fermentation inhibitory compounds that are relevant to second-generation bioethanol production, e. g.

acetic acid, formic acid or vanillin [186], [188], [190]. Additionally, P. kudriavzevii is able to grow at

extremely low pH conditions (around pH 2) and it has more potential for ethanol production at

temperatures higher than 35°C compared with S. cerevisiae, being able to ferment at up to 45°C [190].

To our best knowledge, the work developed in this thesis is the first study involving the isolation and

identification of this yeast from LBW. Additionally, there are no published results relating P. kudriavzevii

with lupanine catabolizing consumption. However, since this species had already been isolated from

different types of plants or foods (as mentioned above), is likely that it can be found in lupin beans plant.

The first systematic study on the genus Rhodotorula was published in 1922 by Saito [191], where

red and yellow-colored Torulas were classified. A few years later, these coloured yeasts in Rhodotorula

and Chromotorula, in which carotenoid pigments producers where part of Rhodotorula genus [192].

Rhodotorula yeasts are pink basidiomycetes forming rudimentary hyphae and small capsules [193].

These yeasts were considered non-pathogenic, widespread in nature and isolated from environmental

sources [194]. However, those organisms had emerged as opportunistic pathogens, particularly

infecting immunocompromised patients, causing fungemia associated with catheters, endocarditis,

peritonitis, meningitis as the most common infections in the literature [194], [195]. R. mucilaginosa is the

most common species isolated from clinical patients, with a high physiological variability among strains

[196], [197]. Until date, there are no published results regarding R. mucilaginosa isolation and

identification from LBW nor lupanine catabolizing capacity.

Considering microbial cultures (Figures 36 and 37), both yeast strains, Pichia kudriavzevii and

Rhodotorula mucilaginosa, were uncapable of grow in lupanine as sole carbon source. When glucose

is present in the medium, exponential curves are followed, indicating that lupanine also does not inhibit

yeasts growth, otherwise they would not be able to grow in glucose + lupanine condition. Lupanine

concentration levels remained constant, with small oscillations that can be due to experimental errors in

the collection and preparing of samples for HPLC. Until date, there are no published works with both P.

kudriavzevii and R. mucilaginosa yeasts involved in lupanine conversion or consumption for energy, nor

85

other alkaloid. It can be concluded that R. mucilaginosa and P. kudriavzevii do not have the ability to

grow when lupanine is the sole carbon source.

The variation of pH in yeast cultures is presented in Table 12. It is clear that here yeasts grow at

pH values lower than bacteria. This is because yeasts grow better in a range of pH 4.5 – 7.0, preferring

acid environments [198]. Fermenting yeasts, usually acidify their growth environment by a combination

of proton secretion during nutrient transport (in this case glucose transport) by the action of the plasma

membrane proton-pumping ATPase. Yeasts can directly secrete organic acids (succinic or acetic acids),

which leads to a decrease in the pH [199]. In Table 12, it is possible to observe a decrease in pH under

glucose as carbon source, while in lupanine growth conditions, the pH values remain approximately

constant.

These two yeast species were cultured in LBW as medium to evaluate possible COD or lupanine

removal, as well as total reducing sugar consumption. Two growth conditions for each yeast isolate were

tested: i) growth in LBW diluted in water, ii) growth in LBW diluted in YNB medium, to assess if LBW

could lack essential nutrients for yeast growth. In Figure 39, the growth curves presented show that

both yeasts grown without any difference between exponential phases in both LBW+H2O and

LBW+YNB, which mean that LBW comprises all necessary nutrients for yeast growth. This can also be

confirmed by the results of specific growth rates (Table 20).

Table 20 – Specific growth rates for both yeasts and culture conditions in LBW.

Yeast strain LBW + H2O LBW + YNB

P. kudriavzevii 0.41 h-1 ± 0.03 0.40 h-1 ± 0.01

R. mucilaginosa 0.17 h-1 ± 0.01 0.17 h-1 ± 0.01

P. kudriavzevii has grown with higher specific growth rate than R. mucilaginosa, however, both

strains reached values of around 7.5 in the OD600 nm measurements at 24 hours.

Lupanine concentration graphs revealed that lupanine was not consumed during the culture of P.

kudriavzevii and R. mucilaginosa. However, in Table 13 the COD results demonstrate that both yeasts

were able to remove part of the COD from LBW diluted in water, with a higher removal of 47.1% ± 1.5

by R. mucilaginosa than the 21.81% ± 2.0 removed by P. kudriavzevii. Previous studies revealed the

ability of R. mucilaginosa to degrade six phenolic compounds (protocatechuic, vanilic, p-coumaric acids,

tyrosol, gallic acid and catechol) present in olive mill wastewater [200]. These compounds are highly

toxic and characterized by their stability and bioaccumulation capacity, thus remaining in the

environment for a long time [201]. Additionally, in this study, it was verified that using olive mill

wastewater as culture medium, R. mucilaginosa was able to reduce 38.38%, 47.69% and 56.91% of

COD and 5.84%, 27.89% and 34.81% of phenol content, respectively, for initial COD concentrations of

26700, 14400 and 6500 mg/L. Here, yeast biomass was influenced by the COD concentration, and the

biodegradation reaction was intense in the first 3 days, meaning that this yeast can easily metabolize

olive mill wastewater components (sugars, proteins and phenolic compounds). The authors have

verified that an increase in the pH occurred during the culture, and mention that this can be indicative

of the deamination of amino acids leading to subsequent ammonia production, similarly to what was

86

explained previously for the case of the increase in bacterial pH in LBW. This hypothesis was also

mentioned by Welthagen et al. (1999) [202]. Other studies state that R. rubra (which is a synonym of

mucilaginosa) was able to reduce 50% and 90% of COD and biological oxygen demand, respectively,

as well as to degrade some aromatic compounds of hydrocarbon refinery wastewater [203]. The authors

were able to conclude that this yeast has potential to produce a red pigment and an antioxidant while it

purifies olive mill wastewater [200]. Curiously, P. kudriavzevii has also been involved in a study with

olive mill wastewater [204]. Here, the authors explored the feasibility of producing bioethanol during a

bioremediation process that reduces the polluting charge of this wastewater through aerobic yeast

fermentation. Biological treatment of olive mill wastewater originated bioethanol as a co-product using

selected yeasts, such as P. kudriavzevii. Polyphenolic compounds from olive mill wastewater were used

for ethanol production during the bioremediation process. In a biotechnological perspective, Koutinas et

al (2015) has exploited a citrus peel based biorefinary with this yeast, showing favourable technological

features for ethanol production by means of utilization of this food waste [205]. Here, the isolated strain

revealed to be highly thermotolerant and used hexoses (glucose, sucrose, fructose and galactose) and

pentoses (xylose) for ethanol production. Besides bioethanol production, P. kudriavzevii strain M12 has

also demonstrated to have a potential to produce valuable enzymes in food processing and agriculture,

named phytases [206].

There are a few common carbon sources used by the two yeast species, as illustrated in Table 21.

Table 21 - Some of the carbon sources used by the yeast species isolated. (*) indicates two carbon sources

that may be present in LBW.

Glucose Ethanol Glycerol Succinate Glucosamine Sucrose* Trehalose Raffinose*

P. kudriavzevii [207]

- - - -

R. mucilaginosa [198]

-

Given this information, it is possible to conclude that R. mucilaginosa might have used sucrose or

raffinose for the growth under LBW.

Similar to the LBW cultures with bacterial species, further investigations in this field could include

different growth temperatures testing, to assess its effect on substrate consumption, as well as different

dilutions in sterilized water and the measurement of initial and final soluble proteins, amino acids and

total quinolizidine alkaloids. In this way, it would be possible to clarify which carbon sources are yeast

strains consuming.

5.4. Filamentous fungi

Given the difficulties in working with liquid microbial culture with filamentous fungi strains (due to

the high aggregates formed), this was a complementary approach and an additional characterization of

biodiversity in LBW. Here, three different morphotypes of isolates were identified as Pichia kudriavzevii,

Aspergillus fumigatus and Galactomyces geotrichum. As seen in this research, Pichia kudriavzevii was

87

identified by the second time (similarly to Yeast section), which confirms the prevalence of this yeast in

LBW.

According with the literature, Aspergillus fumigatus is considered a saprophytic fungus (gets energy

from dead and decaying organic matter) that plays a key role in recycling environmental carbon and

nitrogen [208]. It is commonly found in the soil, where it survives and grows on organic debris, playing

an important role in carbon and nitrogen recycling in nature. This fungus sporulates abundantly with

every conidial head producing thousands of conidia. This fungal strain has become the most prevalent

airborne fungal pathogen, causing severe and sometimes fatal invasive infections in

immunocompromised patients from developed countries [209]. A. fumigatus is a thermotolerant, with

efficient growth and germination at 37°C with nutritional versatility and the ability to grow under various

carbon and nitrogen sources [210]. This species can grow under a variety of carbon and nitrogen

sources like glucose, mannose, maltose, fructose, sucrose, arabinose, trehalose, lactose, peptone,

alanine, proline as examples [211]. In the present work, cultures either in synthetic growth medium or

LBW revealed that this fungus is not able to catabolize lupanine (Figure 41, Table 16).

The growth results, quantified in terms of cell dry weight are presented in Figure 41, showing that at

some point there is no measurement in glucose growth condition, due to the aggregation state of

biomass that was observed after 48hours of culture. It was also observed growth in lupanine condition

around 1.5 g/L, which can be because of the composition of the ME culture medium, once it has 17 g/L

of malt extract and 3 g/L mycological peptone, and these compounds were probably used as energy

sources. To better evaluate lupanine consumption levels, further experiments need to readjust culture

medium concentrations, by reducing glucose and malt extract concentrations. When grown in LBW, A.

fumigatus was able to remove 40% of COD, which indicates a positive result. However, similarly to

previous experiments, the concentrations of total amount of amino acids, proteins and total QA could

have been measured to better investigate the growth of the isolate in LBW medium. Cell dry weight

results from Figure 42 are not representative from real cell growth, since this fungus grown in

aggregates as well. There are some studies with A. fumigatus that demonstrated the ability of this strain

to efficiently treat wastewater effluent from dyeing industry [212]. Here, the authors observed that this

fungus had decolorized a dye from an industry effluent, by degrading azo dyes that have a complex

structure and synthetic nature, which makes them difficult to remove [213]. Additionally, other studies

also state the decolourization of molasses wastewater accompanied by 51% of COD removal in a

molasses melanoidin solution and 75% of decolourization [214].

Galactomyces geotrichum is a mould widely used in biotechnology, since it has been isolated from

several types of cheeses, milk and alcoholic beverage [215]–[217]. Literature information state that this

species is able to grow on glucose, galactose, sucrose, and xylose. However, no growth was observed

on cellobiose, maltose, raffinose, mannitol and erythritol [217]. Microbial cultures revealed no lupanine-

degrading catabolization ability, and the percentage of COD obtained for the final culture was higher

than the initial, which indicates release of organic compounds into the LBW, meaning that it is not an

adequate species for purification of this wastewater. From the biotechnology perspective, this species

is used in cheese production since it is able to reduce the bitterness in the cheese due to the breakdown

of bitter peptides (by proteolysis) [218]. Additionally, G. geotrichum has also been found as the dominant

88

fungus among the identified ones from a Chinese liquor preparation. Here, the authors state that this

species can affect the flavour of the liquor due to their protein and lipid-degrading capacity into

precursors of volatile compounds [219]. One of the harmful substances that this species is able to

degrade is an organochlorine pollutant that contaminates the environment affecting humans and

animals, since it exists in the soil, sediment, water or air and it has been used since World War II in pest

control for agriculture [220]. Other example is the case of the azo dyes, similarly to A. fumigatus, in

which G. geotrichum revealed the ability to decolorize the toxic Methyl red azo dye [221]. Given this

information, it is possible to conclude that although this species can have potential applications in other

fields, it was not that relevant in lupanine and COD removal.

In 2013, the degradation of quinolizidine alkaloids of lupin by Rhizopus oligosporus was

investigated [222]. Here, the authors state that this species demonstrated positive results for

detoxification of lupin seeds. They investigated that the fermentation process was mainly affected by

initial pH values in growth medium, in which pH of 5.5 was directly related with detoxification and fungal

development. Lupin flour was used in cultures of lupin agar, and the results showed that detoxification

levels achieved were 16.58 and 62.23% at pH 3.5 and 5.5, respectively. At pH 5.5, total alkaloid content

changed from 0.228 to 0.084 mg/cm2 of agar after 48 h. Lupanine, specifically, was degraded up to

70.82% with a reduction of 0.160 to 0.047 mg/cm2 of agar, corresponding to a degradation of 16.46%.

Here, alkaloids were possibly used to produce energy with the carbon atoms and/or to obtain nitrogen

for protein synthesis. Hydroxylation reactions are the first reactions commonly performed in

biodegradation processes of cyclic molecules, like aromatic rings, pesticides or others [223]. Hydroxyl

groups addiction increase the reactivity of stable molecules and enables the opening of the cyclic

structure until destruction. Rhizopus produces a glucoamylase enzyme that is used in food industry that

can perform the first stage of degradation reaction of lupanine [168], [224].

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6. Conclusion

The identification of novel microorganisms capable of catabolizing lupanine is an opportunity for

the development of an alternative microbial debittering process and/or to further treat the wastewater

for re-use purposes in the industry. The need of this process development is expected to attenuate the

environmental problem associated with the alkaloid concentration in lupine beans wastewater. For this

purpose, in the present work, four bacteria, two yeasts and two filamentous fungi species were isolated

and identified for the first time, from lupin beans wastewater. Among the identified microorganisms, the

Ochrobactrum anthropi bacteria revealed to be the most promising and interesting, due to its lupanine-

catabolizing capacity and at the same time highest COD removal from the LBW, with more than 50%

removal. Further investigations on the catabolism of this bacteria should be performed, since the

catabolism in natural processes of degradation is specific of each organism. However, it is possible that

lupanine was used to produce energy with the carbon atoms and/or to obtain nitrogen for the synthesis

of proteins. In general, alkaloid degradation is performed by enzymes capable of producing changes in

molecules. Hydroxylation is the main step for lupanine degradation, and the intermediate stages may

be used as carbon and nitrogen sources for growth and energy support.

The other remaining microorganisms isolated, did not showed lupanine removal. However, most

strains were able to remove part of the COD from liquid culture medium of LBW, which is a positive and

promising result if other valuable compounds may result from this removal capacity. Moreover, once the

gene encoding lupanine hydroxylase is known and sequenced, these species can be used as basis for

genetic engineering assays, for the artificial manipulation or modification of the DNA to alter a certain

characteristic, in this case lupanine catabolizing capacity.

The results presented in this work provide important information for the design and development of

future works in the bioremediation field, with further valorisation of lupin beans wastewater.

90

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Annex

A. Microbial cultures

A1. Filter interference in culture

In bacterial and yeast cultures, it was observed that the filters Membrane filters, 0.2 µm, Whatman,

ME24/21ST affected lupanine concentration in solution. This was observed when bacterial and yeast

isolates were pre-grown during 48 and 24 h in the same conditions shown in Figures 14 and 17,

respectively, and the inoculum of the main culture was performed by re-inoculating cells by filtration

under sterile conditions. In this experiment, the filter remained in culture during 168h of the experiment.

Lupanine measurements were not consistent, with concentration levels oscillating in higher and lower

values as shown in e.g. in Figure 56.

Figure 56 – Lupanine concentration values obtained when filters were used in the inoculum of the culture. The

result is from the S. siyangense culture.

Given the high range of values measured, a different methodology was tested. Lupanine was

prepared in a concentration solution and filtered with the same filters has above. After filtration, the filter

was collected and submerged in 30 mL of distilled water for 24 h and lupanine concentration was

measured at 0 h and 24 h. The results indicated that around 100 mg of lupanine remained in the filter.

When the inoculum of the main culture was performed by centrifuging pre-grown cells and

resuspending in culture medium, lupanine results were constant, as shown from the results in this

research.

A2. OD values of microbial cultures

- Bacterial cultures

The OD values in M9 synthetic medium and LBW are shown in Tables 22-25 and Tables 26-29,

respectively.

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0 24 48 72 96 120 144 168

Lupanin

e

(g/L

)

Time (h)Lupanine Glucose + Lupanine

105

Table 22 – OD values measured during O. anthropi culture in M9 synthetic medium. (1) And (2) are the biological

replicates.

Pre-culture: Glucose Lupanine Glucose + Lupanine

Culture: Glucose Lupanine Glucose + Lupanine Lupanine Glucose + Lupanine

Time (h) (1) (2) (1) (2) (3) (1) (2) (3) (1) (2) (3) (1) (2) (3)

0 0.09 0.10 0.08 0.13 0.10 0.08 0.11 0.09 0.09 0.11 0.12 0.09 0.10 0.15

2 0.11 0.12 0.06 0.07 0.11 0.07 0.09 0.13 0.06 0.07 0.10 0.06 0.06 0.12

4 0.20 0.18 0.10 0.07 0.14 0.11 0.07 0.16 0.11 0.06 0.11 0.11 0.05 0.15

6 0.13 0.25 0.07 0.09 0.20 0.07 0.09 0.25 0.08 0.05 0.15 0.08 0.07 0.22

24 1.04 1.06 1.08 1.42 1.66 1.90 1.68 2.28 1.08 0.64 1.22 2.62 1.24 1.20

48 1.28 1.10 1.56 2.22 1.70 2.64 2.38 3.52 1.80 1.46 1.66 1.76 1.24 1.82

120 1.02 1.12 1.10 1.76 1.46 4.60 2.92 3.94 1.74 1.64 1.52 3.84 1.10 3.12

144 0.92 1.06 1.68 1.82 1.44 5.08 3.08 3.96 1.98 1.54 1.56 3.96 1.02 3.40

168 1.02 0.90 1.58 2.10 1.50 4.80 2.96 3.92 2.40 1.90 1.58 3.80 1.24 3.24

Table 23 – OD values measured during S. siyangense culture in M9 synthetic medium. (1) And (2) are the

biological replicates.

Pre-Culture: Glucose Glucose + Lupanine

Culture: Glucose Lupanine Glucose + Lupanine

Time (h) (1) (2) (1) (2) (1) (2)

0 0.12 0.09 0.09 0.11 0.10 0.09

2 0.09 0.10 0.09 0.11 0.11 0.11

4 0.12 0.12 0.09 0.10 0.14 0.16

6 0.08 0.15 0.05 0.11 0.20 0.23

24 0.72 0.54 0.09 0.09 1.04 1.12

48 1.22 1.12 0.09 0.09 1.48 1.04

120 0.96 1.12 0.07 0.09 1.24 0.84

144 1.02 1.00 0.07 0.08 2.34 0.88

168 0.96 1.14 0.07 0.09 1.38 0.80

106

Table 24 – OD values measured during S. maltophilia culture in M9 synthetic medium. (1) And (2) are the




Time (h) (1) (2) (1) (2) (1) (2)

0 0.08 0.08 0.09 0.12 0.09 0.11

2 0.09 0.09 0.09 0.11 0.11 0.12

4 0.11 0.08 0.10 0.11 0.15 0.14

6 0.14 0.08 0.05 0.11 0.10 0.17

24 0.40 0.10 0.09 0.10 0.90 1.16

48 0.47 0.99 0.09 0.09 1.24 1.04

120 0.61 0.10 0.08 0.09 1.22 0.96

144 0.66 0.95 0.08 0.09 1.20 0.92

168 0.65 0.10 0.08 0.09 1.30 0.98

Table 25 – OD values measured during C. funkei culture in M9 synthetic medium. (1) And (2) are the biological

replicates.



Time (h) (1) (2) (1) (2) (1) (2)

0 0.09 0.09 0.11 0.12 0.12 0.11

2 0.09 0.11 0.10 0.12 0.13 0.13

4 0.14 0.16 0.09 0.11 0.16 0.15

6 0.11 0.23 0.05 0.11 0.11 0.18

24 1.04 1.36 0.10 0.10 1.24 1.88

48 1.32 1.76 0.09 0.10 2.54 2.46

120 1.12 1.44 0.09 0.10 3.06 2.58

144 1.00 1.98 0.09 0.09 2.76 2.54

168 1.14 1.70 0.10 0.10 3.06 2.42

Table 26 – OD values measured during O. anthropi culture in LBW diluted in water. (1), (2) and (3) are the


Culture LBW+H2O

Time (h) (1) (2) (3)

0 0.11 0.14 0.15

2 0.21 0.27 0.25

4 0.42 0.47 0.29

6 0.84 0.82 0.43

24 2.54 2.66 4.46

48 3.80 2.48 5.4

120 3.36 2.08 5.4

144 2.96 1.08 3.76

168 3.16 2.32 3.4

107

Table 27 – OD values measured during S. siyangense culture in LBW diluted in water. (1), (2) and (3) are the


Culture LBW+H2O

Time (h) (1) (2) (3)

0 0.12 0.15 0.18

2 0.27 0.27 0.44

4 0.58 0.58 0.72

6 1.24 1.05 1.14

24 2.00 3.24 4.42

48 2.56 4.16 5.12

120 2.84 3.64 4.52

144 2.52 2.80 4.6

168 2.40 3.2 4.36

Table 28 – OD values measured during S. maltophilia culture in LBW diluted in water. (1), (2) and (3) are

the biological replicates.

Culture LBW+H2O

Time (h) (1) (2) (3)

0 0.13 0.12 0.15

2 0.21 0.21 0.34

4 0.40 0.38 0.51

6 1.00 0.07 0.94

24 2.20 1.86 2.88

48 2.28 2.78 3.28

120 2.40 3.28 2.72

144 1.40 3.00 2.36

168 2.00 4.52 2.32

Table 29 – OD values measured during C. funkei culture in LBW diluted in water. (1), (2) and (3) are the


Culture LBW+H2O

Time (h) (1) (2) (3)

0 0.12 0.13 0.15

2 0.20 0.21 0.29

4 0.38 0.35 0.40

6 0.60 0.48 0.56

24 2.10 1.64 2.00

48 2.74 2.76 3.08

120 3.04 2.8 4.24

144 2.92 2.16 4.24

168 3.00 5.72 3.88

- Yeast cultures

The OD values in YNB synthetic medium and LBW are shown in Tables 30-31 and Tables 32-33.

108

Table 30 – OD values measured during R. mucilaginosa culture in YNB synthetic medium. (1) And (2) are the



Culture: Glucose Lupanine Glucose + Lupanine Glucose Lupanine

Glucose + Lupanine

Time (h) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2)

0 0.10 0.11 0.13 0.08 0.12 0.10 0.11 0.09 0.12 0.09 0.13 0.11

2 0.07 0.11 0.04 0.03 0.06 0.06 0.14 0.05 0.05 0.04 0.13 0.05

4 0.27 0.23 0.04 0.03 0.20 0.13 0.16 0.12 0.07 0.05 0.14 0.08

6 0.60 0.41 0.11 0.05 0.23 0.31 0.38 0.26 0.06 0.06 0.34 0.18

24 3.48 3.92 0.05 0.03 4.04 4.62 3.44 3.70 0.07 0.05 3.96 4.70

48 3.24 4.06 0.05 0.04 3.86 4.66 3.56 4.00 0.08 0.05 3.94 4.74

120 2.86 3.56 0.06 0.04 3.28 4.52 2.80 3.78 0.06 0.04 3.28 4.28

144 3.20 3.48 0.06 0.04 3.64 4.42 3.24 3.54 0.08 0.04 3.54 4.42

168 2.68 3.28 0.04 0.04 3.22 4.08 2.48 3.32 0.06 0.04 3.08 4.10

Table 31 - OD values measured during P. kudriavzevii culture in YNB synthetic medium. (1) And (2) are the



Culture: Glucose Lupanine Glucose + Lupanine Glucose Lupanine

Glucose + Lupanine

Time (h) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2)

0 0.13 0.08 0.14 0.08 0.13 0.09 0.11 0.10 0.13 0.10 0.13 0.10

2 0.10 0.05 0.04 0.03 0.07 0.05 0.06 0.07 0.07 0.05 0.07 0.09

4 0.11 0.07 0.05 0.03 0.13 0.08 0.13 0.14 0.08 0.05 0.38 0.24

6 0.40 0.21 0.04 0.07 0.36 0.14 0.39 0.44 0.05 0.08 0.54 0.40

24 3.30 2.30 0.03 0.03 2.84 2.38 3.02 2.68 0.04 0.06 3.00 2.04

48 2.78 2.76 0.02 0.03 2.70 2.52 3.02 2.84 0.06 0.07 2.52 2.40

120 2.40 2.82 0.03 0.05 2.04 3.32 2.24 3.04 0.05 0.07 2.32 2.50

144 2.98 2.96 0.02 0.05 2.92 2.44 3.04 3.34 0.06 0.06 2.60 2.54

168 3.08 2.78 0.02 0.05 2.60 2.02 3.00 2.88 0.05 0.06 2.70 2.40

Table 32 - OD values measured during R. mucilaginosa culture in LBW diluted in water and in YNB medium.

(1), (2) and (3) are the biological replicates.

Culture LBW+YNB LBW+H2O Time (h) (1) (2) (3) (1) (2) (3)

0 0.12 0.17 0.16 0.14 0.18 0.13

2 0.16 0.23 0.21 0.19 0.23 0.13

4 0.24 0.34 0.24 0.32 0.33 0.14

6 0.44 0.46 0.31 0.48 0.51 0.15

24 8.10 7.32 7.92 9.60 7.02 6.64

48 12.96 11.00 9.00 10.44 10.08 9.04

120 12.60 12.08 7.92 9.36 10.04 8.88

144 11.88 11.16 9.88 11.40 12.00 12.52

168 12.36 14.60 10.12 11.80 10.96 10.16

109

Table 33 - OD values measured during P. kudriavzevii culture in LBW diluted in water and in YNB medium. (1),

(2) and (3) are the biological replicates.

Culture LBW+YNB LBW+H2O

Time (h) (1) (2) (3) (1) (2) (3)

0 0.18 0.18 0.19 0.12 0.16 0.19

2 0.31 0.28 0.51 0.13 0.21 0.45

4 0.92 0.82 1.10 0.40 0.51 0.76

6 2.58 1.84 2.34 1.58 1.71 2.21

24 8.56 9.20 8.46 6.12 9.32 7.32

48 9.52 8.56 9.20 7.60 6.52 6.88

120 10.48 7.64 9.08 9.12 8.04 7.08

144 9.80 5.36 11.32 8.80 7.56 7.20

168 11.28 6.44 12.08 9.48 7.52 7.08

Isolation of bacteria and yeast strains from lupin beans ...

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