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Table of Contents 

 

 

Wellcome Address by Fernando G. Fermoso  1 

Committees  3 

IMAB17 Program  5 

Plenary Lecture and Keynotes  15 

Platform   27 

Short Communications  69 

Posters  101 

 

   

 

1

WELLCOME ADDRESS

Dear colleagues and friends,

We are honored to welcome you to the 1st International congress on Metals in Anaerobic

Biotechnologies (IMAB17). IMAB17 aims to provide a unique multidisciplinary platform for

discussion of state-of-the-art research related to the role of trace metals (TMs) in anaerobic

bioprocesses, with special focus on how to manage and recover them.

Anaerobic digesters, soil reclamation processes or bioprocesses in the food industry among

others are clear examples of anaerobic bioprocesses, where addition of TM is often done. A

challenging area remains largely unchartered with respect to understanding the role of trace

metals (TMs) in enabling anaerobic bioprocesses. IMAB17 aims to create a unique platform

where this major knowledge gap and scientific challenge will be presented and discussed.

IMAB17 also provides an opportunity to stay on top of the latest developments in tools,

approaches and methods to identify, assess and manage TMs in various areas of application,

including industry, environmental management, societal needs and policy.

We hope you find here many inspiting ideas and partnerships.

Fernando G. Fermoso

Chairman of IMAB17

2

3

Organizing Committee

Fernando G. Fermoso. Instituto de la Grasa (C.S.I.C.). IMAB17 Chairman. Antonio Serrano. Instituto de la Grasa (C.S.I.C.). IMAB17 Secretary. Fernando Vidal. Universidad de Sevilla. Antonia Jimenez. Universidad Pablo de Olavide. Rafael Borja. Instituto de la Grasa (C.S.I.C.). Guillermo Rodriguez. Instituto de la Grasa (C.S.I.C.). Francisco Noe Arroyo. Instituto de la Grasa (C.S.I.C.).

Scientific Committee

Adriana Maluf Braga. Sao Paulo University, Brazil Ana Paula Mucha. CIIMAR, Portugal Ana Troncoso. Universidad de Sevilla, Spain Antonio Valero. Universidad de Córdoba, Spain Artin Chatzikiosegian. National Technical University of Athens, Greece Bariş Çallı. Marmara University. Turkey Belén Fernández. IRTA, Spain. Bernabé Alonso Fariñas. Universidad de Sevilla, Spain Blaz Stres. University of Ljubljana, Slovenia Bo Svensson. Linkoping University, Sweden Cynthia Carliel Severn Trent Water, UK David Jeison. Pontifica Universidad Catolica de Valparaiso, Chile Eduardo Medina Pradas. IG-CSIC, Spain Emmanuel Guillon, Université de Reims Champagne-Ardenne, France Eric van Hullebusch. UNESCO-IHE, The Netherlands Fátima Arroyo Torralba. Universidad de Sevilla, Spain Gavin Collins. National University of Ireland, Galway, Ireland Georg Guebitz. BOKU, Austria Gilles Guibaud. Limoges University, France Giovanni Esposito. Cassino University, Italy Ilenys Perez Díaz. United States Department of Agriculture, USA Jan Bartacek. ICT-Prague. Czech Republic Joaquín Bautista Gallego. IG-CSIC, Spain Juan Manuel González Grau. IRNASE-CSIC, Spain Katerina Stamatelatou. Democritus University of Thrace, Greece Luigi Frunzo. Naples University, Italy Marcelo Zaiat. Sao Paulo University, Brazil Marisa Almeida. Porto University, Portugal Markus Lenz. University of Applied Sciences and Arts Northwestern

Switzerland, Switzerland Mónica Rodríguez Galán. Universidad de Sevilla, Spain Nuri Azbar. Ege University. Turkey Piet Lens. UNESCO-IHE, The Netherlands

4

Raf Dewil. KU Leuven, Belgium Rocio Arias-Calderon. INIAV, Portugal Sepehr Shakeri Yekta. Linkoping University, Sweden Sergio Lopez. IG-CSIC, Spain Stefan Weiss. BOKU, Austria

Local Organization

Antonio Benitez

Ainoa Botana

Belén Caballero

Beatriz Calero

Eric Caroca

Juan Cubero

Samuel de la Cruz

Virginia Martín

Elisa Mazuelos

Verónica Romero

Ángeles Trujillo

5

IMAB17 program

6

7

4th October

Registration 9:00-9:30 Registration IMAB17 and COST meeting (Instituto de la Grasa)

COST ES1302

9:30-11:00 Final COST ES1302 meeting (open event)

11:00-11:30 Coffee

COST ES1302

11:30-13:00 Final COST ES1302 meeting (open event)

13:00-15:00 Lunch/MC Meeting

17:00-18:00

Welcome to IMAB17 (Rectorate Building of the University of Seville) Dr. Fernando G. Fermoso (IMAB17 Chairman)

Prof. L. Carlos Sanz Martínez (Director del Instituto de la Grasa, C.S.I.C.) Prof. Bruno Martínez-Haya (Vicerrector de Investigación y Transferencia de Tecnología, Universidad Pablo de Olavide)

Doctor D. José Guadix Martín (Director General de Transferencia del Conocimiento, Universidad de Sevilla)

Plenary

18:00-19:00

Trace elements as key factors to successful biogas production. B.H. Svensson

*Department of Thematic Studies, Environmental Change, Linköping University, 58183 Linköping, Sweden

Welcome reception

19:00-23:00 Reception Cocktail and social event (Hotel Doña María, C/ Don

Remondo, 19, Seville)

5th October (Instituto de la Grasa)

Keynote 9:00-9:45

Effect of reactor operating conditions on metal speciation Prof. David Stukey

Imperial College London · Department of Chemical Engineering · Bioprocessing · UK. Nanyang Technological University · Nanyang Environment and Water Research Institute

(NEWRI) · Advanced Environmental Biotechnology Centre (AEBC). Singapore.

Platform

9:45-10:05

Effect of trace elements supplementation in agricultural biogas plants Mirco Garuti*, Michela Langone**, Claudio Fabbri*, Sergio Piccinini*

*CRPA – Research Center on Animal Production, Viale Timavo, 43/2 - Reggio Emilia, Italy **University of Trento, Via Mesiano 77 - Trento, Italy

10:05-10:25

Optimization of biogas production from anaerobic digestion of Laminaria sp. through manipulation of trace element availability

J Roussel*, L Austin*, C Carliell-Marquet* * School of Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

Short

10:25-10:35

Use of trace elements addition for anaerobic digestion of brewer’s spent grains

C. Bougrier*, D. Dognin*, C. Laroche* and J.A. Cacho Rivero* *Veolia Recherche & Innovation, zone portuaire de Limay, 291 avenue Dreyfous Ducas,

Limay F-78520, France

10:35-10:45

Is there trace metal deficiency in anaerobic membrane bioreactor (AnMBR) for municipal wastewater treatment? A.Ilic*, P.Dolejs*, M.Polaskova**, J.Bartacek*,

*Department of Water Technology and Environmental Engineering, Technicka 5, 166 28 Prague 6, Czech Republic

**Research and development division of ASIO, spol. s r.o, Kšírova 552/45, 619 00 Brno, Czech Republic

10:45-10:55

Improvement of anaerobic digestion systems co-digesting food waste and pig manure with addition of trace metals

Yan Jiang*, Conor Dennehy*, Peadar G. Lawlor**, Gillian E. Gardiner***, Xinmin Zhan*

*Civil Engineering, College of Engineering & Informatics, National University of Ireland, Galway, Ireland

**Teagasc, Pig Development Department, Animal and Grassland Research and Innovation Centre, Moorepark, Fermoy, Co. Cork, Ireland

***Department of Science, Waterford Institute of Technology, Waterford, Ireland

8

11:00-11:30 Coffee

Platform

11:30-11:50

Competing demands for trace elements in anaerobic digesters Y Zhang*, H Song*, N Sriprasert*, CJ Banks*, S Heaven*

*Bioenergy and Organic Resources Research Group, Faculty of Engineering and the Environment, University of Southampton, UK

11:50-12:10

Addition of fly ash from thermal power plant to anaerobic digestion of sewage: Effect of ash particle size.

S. Montalvo*, I. Cahn*, R. Borja**, C. Huiliñir*, A. Barahona***, L. Guerrero***

*Universidad de Santiago de Chile, Av. Lib. Bdo. O`Higgins 3363, Santiago de Chile. Chile, **Instituto de la Grasa, Campus Universitario Pablo de Olavide - Edificio 46, Ctra. de Utrera,

Km. 1, 41013 Sevilla, Spain ***Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso, Chile

Short

12:10-12:20

Balance of Selected Trace Elements at Cup-plant Cultivation for Biogas Production Compared to Reference Maize

S. Ustak, J. Munoz Crop Research Institute, Drnovska 507/73, 16106 Prague 6 – Ruzyne, Czech Republic

12:20-12:30

Full-scale agricultural biogas plant metal content and process parameters in relation to bacterial and archaeal microbial communities

over 2.5 year span Sabina Kolbl*, Domen Zavec**, Katarina Vogel Mikuš***, Fernando

Fermoso****, Blaž Stres**,***** * University of Ljubljana, Faculty of Civil and Geodetic Engineering, Hajdrihova 28, SI-1000

Ljubljana, Slovenia ** University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Group for

Microbiology and Microbial Biotechnology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia *** University of Ljubljana, Biotechnical Faculty, Department of Biology, Chair of Botany and

Plant Physiology **** Instituto de la Grasa (C.S.I.C.). Avda. Padre García Tejero, 4. 41012-Sevilla, Spain

*****University of Ljubljana, Faculty of Medicine, Vrazov trg 2, 1000 Ljubljana, Slovenia

12:30-12:40

Influence of Trace Element Supplementation on Anaerobic Mono-Digestion of Chicken Manure

Alper Bayrakdar*,**, Rahim Molaey*, Recep Önder Sürmeli*,***, Bariş Çalli*

* Environmental Engineering Department, Marmara University, 34722 Kadikoy, Istanbul, Turkey

**Environmental Engineering Department, Necmettin Erbakan University, 42140, Meram, Konya, Turkey

*** Environmental Engineering Department, Bartın University, 74100, Merkez, Bartın, Turkey

12:40-14:00 Lunch

Keynote 14:00-14:45

Effect of Trace Element Limitations on Microbial Communities and Metabolic Pathways in Anaerobic Digesters

Prof. Sabine Kleinsteuber*, Co-Authors: Babett Wintsche*, Nico Jehmlich**, Denny Popp*

*Dept. Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, Permoserstr. 15, 04318 Leipzig, Germany

**Dept. Molecular Systems Biology, Helmholtz Centre for Environmental Research – UFZ, Permoserstr. 15, 04318 Leipzig, Germany

Platform

14:45-15:05

Metagenomics Reveals The Phylogenetic Distribution Of Selenium-Reducung Microorganisms In Soils And In Anaerobic Granular Sludge

Biofilms S. Mills*, L. Staicu** and G. Collins*

* Microbial Commnities Laboratory, Microbiology, School of Natural Sciences, National University of Ireland Galway, University Road, Galway, H91 TK33, Ireland

** University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Bucharest, Romania

15:05-15:25

High-throughput characterisation of whole-ecosystem microbial communities: anaerobic granules in ‘micro-sequencing batch reactors’

Sarah O’Sullivan, Estefanía Porca Belío and Gavin Collins Microbiology, School of Natural Sciences, National University of Ireland, Galway,

9

University Road, Galway, Ireland

15:25-15:55

Influence of Trace Elements on the Methanogenic Pathway at High Ammonium Concentrations

Rahim Molaey, Alper Bayrakdar, Recep Önder Sürmeli, Bariş Çalli Environmental Engineering Department, Marmara University, 34722 Kadikoy, Istanbul,

Turkey

Coffee 15:55-17:00

Platform

17:00-17:20

Trace elements as pH controlling agents support microbial chain elongation

H. Sträuber*, M. Dittrich-Zechendorf**, F. Bühligen*, S. Kleinsteuber* *UFZ – Helmholtz Centre for Environmental Research, Department of Environmental

Microbiology, Permoserstr. 15, 04318 Leipzig, Germany **Deutsches Biomasseforschungszentrum gemeinnützige GmbH (DBFZ), Department

Biochemical Conversion, Torgauer Str. 116, 04347 Leipzig, Germany

17:20-17:40

ADM1-based mechanistic model for trace elements bioavailability in anaerobic digestion processes

L. Frunzo*, F.G. Fermoso**, V. Luongo*, M.R. Mattei*, and G. Esposito***

* Department of Mathematics and Applications "Renato Caccioppoli", University of Naples Federico II, Complesso Monte Sant’Angelo, 80124 Naples, Italy

** Instituto de la Grasa (C.S.I.C.), Campus Universidad Pablo de Olavide. Edificio 46. Ctra. de Utrera km. 1, 41013-Sevilla, Spain

*** Department of Civil and Mechanical Engineering, University of Cassino and Southern Lazio, via Di Biasio 43, 03043 Cassino, Italy

Short

17:40-17:50

Modelling the Effect of Trace Elements in Anaerobic Digestors A. Hatzikioseyian, P. Kousi, E. Remoundaki, M. Tsezos

Laboratory of Environmental Science and Engineering, School of Mining and Metallurgical Engineering, National Technical University of Athens (NTUA)

Heroon Polytechniou 9, 15780, Athens, Greece

17:50-18:00

Biogenic selenium particles: linking water treatment with resource recovery

L.C. Staicu University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science,

Bucharest, Romania

18:00-18:10

The impact of crude glycerol from biodiesel production on biomethane production and trace metal content in batch experiment

Bojana Danilović*, Leon Deutsch**, Urška Magerl***, Dragiša Savić*, Sabina Kolbl***, Blaž Stres**,****

* University of Niš, Facaulty of Techology, Leskovac, Serbia

** University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia

*** University of Ljubljana, Faculty of Civil Engineering, Ljubljana, Slovenia

**** University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia

6th October (Instituto de la Grasa)

Keynote 9:00-9:45

Biogeochemistry of trace elements in anaerobic digesters: speciation and bioavailability

Eric D. van Hullebusch*,** *Université Paris-Est, Laboratoire Géomatériaux et Environnement (LGE), EA 4508, UPEM,

77454 Marne-la-Vallée, France **IHE Delft Institute for Water Education, Department of Environmental Engineering and

Water Technology, P.O. Box 3015, 2601 DA Delft, The Netherlands

Platform 9:45-10:05

Stability of ZnS formed during anaerobic digestion Maureen Le Bars*, Clément Levard**, Samuel Legros***, Jean-Paul

Ambrosi**, Jérôme Rose**, Daniel Borschneck**, Emmanuel Doelsch* * UPR Recyclage et risque, CIRAD, France

** CEREGE UM34, Aix-Marseille Université, CNRS, IRD, France *** UPR Recyclage et risque, CIRAD, Sénégal

10:05-10:25 A simultaneous study of organic matter and trace elements accessibility

in substrate and digestate from an anaerobic digestion plant

10

A. Laera*, S. Shakeri Yekta**, R. Buzier ***, Mattias Hedenström****, Mårten Dario**, G. Guibaud***,G. Esposito*****, E.D. van

Hullebusch*,******

* Laboratoire Géomatériaux et Environnement, Université Paris-Est, 77454 Marne-la-Vallée, France

**The Department of Thematic Studies - Environmental Change, Linköping Universitet, 58183 Linköping, Sweden

*** GRESE, Université de Limoges, 87060 Limoges, France **** Department of chemistry, Umeå Universitet, 90187 Umeå, Sweden

***** Dipartimento di Ingegneria Civile e Meccanica, Università degli Studi di Cassino e del Lazio Meridionale, 03043 Cassino, Italy

****** Department of Environmental Engineering and Water Technology, IHE Delft Institute for Water Education, 2601 Delft, The Netherlands

Short

10:25-10:35

The impact of nano ZnO on anaerobic digestion of the organic fraction of municipal solid waste

I. Temizel, B. Demirel, N.K. Copty and T.T. Onay Institute of Environmental Sciences, Boğaziçi University, Bebek, 34342, Istanbul, Turkey

10:35-10:45

Adsorption mechanism of nanoparticles ZnO and CuO on anaerobic granular sludge EPS: Contributions of EPS fractional polarity and

nanoparticle diameters Liangliang Wei*, Ming Xin*, Sheng Wang*, Junqiu Jiang*, Qingliang Zhao*

* State Key Laboratory of Urban Water Resources and Environment (SKLUWRE); School of Environment, Harbin Institute of Technology, Harbin 150090, China.

10:45-10:55

Metals Removal from Incineration Ashes of Anaerobically Digested Sewage Sludge

F. Arroyo Torralvo*, A. Serrano**, M. Rodríguez-Galán*, B. Alonso-Fariñas*, F. Vidal*

* University of Seville, Department of Chemical and Environmental Engineering, Higher Technical School of Engineering, Camino de los Descubrimientos, s/n, Seville, Spain

**Instituto de la Grasa, Spanish National Research Council (CSIC), Campus Universitario Pablo de Olavide-Ed. 46, Ctra. de Utrera, km. 1, Seville, Spain

11:00-11:30 Coffee

Platform

11:30-11:50

Ability of anaerobic granules for metal-mediated direct interspecies electron transfer

C.-D. Dubé*, S.R. Guiot* * National Research Council Canada, 6100 Royalmount Avenue, Montreal, H4P 2R2 Canada

11:50-12:10

Strategies in Ni and Co recovery with biogenic sulphide Y.Liu*, M. Yao*, I. Yoong*, M. Peces**, J. Voughan**, G. Southam***,

D.Villa-Gomez* *School of Civil Engineering, The University of Queensland, 4072 QLD, Australia

**School of Chemical Engineering, The University of Queensland, 4072 QLD, Australia ***School of Earth and Environmental Sciences, The University of Queensland, 4072 QLD,

Australia

12:10-12:30

Barium role in the pathway of the anaerobic digestion process V.Wyman*

,**, A.Serrano***, R. Borja***, A. Jiménez*, A. Carvajal**, F. G.

Fermoso***

* Universidad Pablo de Olavide, Carretera de Utrera, 1, 41013 Seville, Spain

** Universidad Técnica Federico Santa María, Avenida Vicuña Mackenna 3939 San Joaquín,

Santiago, Chile. *** Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Avenida

Padre García Tejero, 4. 41012 Seville, Spain

12:30-14:00 Lunch

Platform

14:00-14:20

Fate of heavy metals over the anammox process treating the liquid fraction digestate of OFMSW effluents: A case study

Pichel, A.*, Val del Río, A.*, Méndez, R.*, Mosquera-Corral, A.* * Department of Chemical Engineering, Institute of Technology, Universidade de Santiago de

Compostela, E-15705 Santiago de Compostela, Spain

14:20-14:40

Copper plant uptake from liquid digestate – influence of the presence of enrofloxacin

S. Sayen*, E.Vulliet**, E. Guillon*, C. M. R. Almeida*** * Institut de Chimie Moléculaire de Reims (ICMR), UMR CNRS 7312, Université de Reims

Champagne-Ardenne, BP 1039, Reims Cedex 2, France.

11

** Université de Lyon, Institut des Sciences Analytiques, UMR 5280 CNRS, Université Lyon 1, ENS-Lyon, 5 rue de la Doua, Villeurbanne, France.

*** Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de

Matos s/n, Matosinhos, Portugal.

14:40-15:00

Bioaugmentation with autochthonous microbial consortia to potentiate phytoremediation of cadmium contaminated sediments

Ana P. Mucha *, Catarina Teixeira*, C. Marisa R. Almeida * *Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR),

Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal

Short

15:00-15:10

Improving Gas Counter for Measurement of BioMethane Potential and Methanogenic Activity

O K Bekmezci*,**, E Piro*, D Ucar*** * Department of Environmental Engineering, Bitlis Eren University, Bitlis, Turkey

** Department of Environmental Engineering, Marmara University, Istanbul, Turkey *** GAP Renewable Energy and Energy Efficiency Center, Harran University, Sanliurfa,

Turkey

15:10-15:20

Thermal treatment for olive oil wastes utilization: bioactive compounds and substrate for soil contaminated remediation.

G. Rodríguez Gutiérrez, J. Fernández-Bolaños Guzmán, A. Lama-Muñoz. F. Rubio-Senent, A. Fernández-Prior, E. María Rodríguez Juan, A. García

Borrego, A. Bermúdez Oria, B. Vioque-Cubero. Instituto de la Grasa, (CSIC), ctra de Utrera Km 1, Campus Pablo de Olavide, Edif. 46. CP

41013, Seville, Spain.

15:20-16:00 Coffee

16:00-17:00 IMAB17 Closing Ceremony.

20:00 IMAB17 Dinner (Abades Triana)

12

Poster session 1 Use of trace elements addition for anaerobic digestion of brewer’s spent grains

C. Bougrier*, D. Dognin*, C. Laroche* and J.A. Cacho Rivero* *Veolia Recherche & Innovation, zone portuaire de Limay, 291 avenue Dreyfous Ducas, Limay F-78520, France

2 Is there trace metal deficiency in anaerobic membrane bioreactor (AnMBR) for municipal wastewater treatment?

A.Ilic*, P.Dolejs*, M.Polaskova**, J.Bartacek*, *Department of Water Technology and Environmental Engineering, Technicka 5, 166 28 Prague 6, Czech Republic

**Research and development division of ASIO, spol. s r.o, Kšírova 552/45, 619 00 Brno, Czech Republic 3 Improvement of anaerobic digestion systems co-digesting food waste and pig manure with

addition of trace metals Yan Jiang*, Conor Dennehy*, Peadar G. Lawlor**, Gillian E. Gardiner***, Xinmin Zhan*

*Civil Engineering, College of Engineering & Informatics, National University of Ireland, Galway, Ireland **Teagasc, Pig Development Department, Animal and Grassland Research and Innovation Centre, Moorepark, Fermoy, Co.

Cork, Ireland ***Department of Science, Waterford Institute of Technology, Waterford, Ireland

4 Balance of Selected Trace Elements at Cup-plant Cultivation for Biogas Production Compared to Reference Maize

S. Ustak, J. Munoz Crop Research Institute, Drnovska 507/73, 16106 Prague 6 – Ruzyne, Czech Republic

5 Full-scale agricultural biogas plant metal content and process parameters in relation to bacterial and archaeal microbial communities over 2.5 year span

Sabina Kolbl*, Domen Zavec**, Katarina Vogel Mikuš***, Fernando Fermoso****, Blaž Stres**,*****

* University of Ljubljana, Faculty of Civil and Geodetic Engineering, Hajdrihova 28, SI-1000 Ljubljana, Slovenia ** University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Group for Microbiology and Microbial

Biotechnology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia *** University of Ljubljana, Biotechnical Faculty, Department of Biology, Chair of Botany and Plant Physiology

**** Instituto de la Grasa (C.S.I.C.). Avda. Padre García Tejero, 4. 41012-Sevilla, Spain *****University of Ljubljana, Faculty of Medicine, Vrazov trg 2, 1000 Ljubljana, Slovenia

6 Influence of Trace Element Supplementation on Anaerobic Mono-Digestion of Chicken Manure Alper Bayrakdar*,**, Rahim Molaey*, Recep Önder Sürmeli*,***, Bariş Çalli*

* Environmental Engineering Department, Marmara University, 34722 Kadikoy, Istanbul, Turkey **Environmental Engineering Department, Necmettin Erbakan University, 42140, Meram, Konya, Turkey

*** Environmental Engineering Department, Bartın University, 74100, Merkez, Bartın, Turkey

7 Modelling the Effect of Trace Elements in Anaerobic Digestors A. Hatzikioseyian, P. Kousi, E. Remoundaki, M. Tsezos

Laboratory of Environmental Science and Engineering, School of Mining and Metallurgical Engineering, National Technical University of Athens (NTUA)

Heroon Polytechniou 9, 15780, Athens, Greece

8 Understanding the impact of Fe, Ni and Co on the hydrolytic and acidogenic stage of anaerobic digestion

DK Villa-Gomez*, A Lomate*, N Islam*, M Peces* * School of Civil Engineering, The University of Queensland, 4072 QLD, Australia.

9

The impact of crude glycerol from biodiesel production on biomethane production and trace metal content in batch experiment

Bojana Danilović*, Leon Deutsch**, Urška Magerl***, Dragiša Savić*, Sabina Kolbl***, Blaž Stres**,****

* University of Niš, Facaulty of Techology, Leskovac, Serbia

** University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia

*** University of Ljubljana, Faculty of Civil Engineering, Ljubljana, Slovenia

**** University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia

10 The impact of nano ZnO on anaerobic digestion of the organic fraction of municipal solid waste

I. Temizel, B. Demirel, N.K. Copty and T.T. Onay Institute of Environmental Sciences, Boğaziçi University, Bebek, 34342, Istanbul, Turkey

11 Adsorption mechanism of nanoparticles ZnO and CuO on anaerobic granular sludge EPS: Contributions of EPS fractional polarity and nanoparticle diameters

Liangliang Wei*, Ming Xin*, Sheng Wang*, Junqiu Jiang*, Qingliang Zhao*

* State Key Laboratory of Urban Water Resources and Environment (SKLUWRE); School of Environment, Harbin Institute of

13

Technology, Harbin 150090, China.

12 Lessons learnt and areas of interest for research in biological sulphate reduction for metal recovery

D. K. Villa-Gomez* * School of Civil Engineering, University of Queensland, 4067 QLD, Australia.

13 Improving gas counter for measurement of biomethane potential and methanogenic activity O K Bekmezci*,**, E Piro*, D Ucar***

* Department of Environmental Engineering, Bitlis Eren University, Bitlis, Turkey ** Department of Environmental Engineering, Marmara University, Istanbul, Turkey

*** GAP Renewable Energy and Energy Efficiency Center, Harran University, Sanliurfa, Turkey

14 Metals removal from incineration ashes of anaerobically digested sewage Sludge F. Arroyo Torralvo*, A. Serrano**, M. Rodríguez-Galán*, B. Alonso-Fariñas*, F. Vidal*

* University of Seville, Department of Chemical and Environmental Engineering, Higher Technical School of Engineering, Camino de los Descubrimientos, s/n, Seville, Spain

**Instituto de la Grasa, Spanish National Research Council (CSIC), Campus Universitario Pablo de Olavide-Ed. 46, Ctra. de Utrera, km. 1, Seville, Spain

15 Thermal treatment for olive oil wastes utilization: bioactive compounds and substrate for soil contaminated remediation.

G. Rodríguez Gutiérrez, J. Fernández-Bolaños Guzmán, A. Lama-Muñoz. F. Rubio-Senent, A. Fernández-Prior, E. María Rodríguez Juan, A. García Borrego, A. Bermúdez Oria, B. Vioque-

Cubero. Instituto de la Grasa, (CSIC), ctra de Utrera Km 1, Campus Pablo de Olavide, Edif. 46. CP 41013, Seville, Spain.

16 Ion-selective electrode for monitoring of cobalt in natural waters released into the environmental as a result of anaerobic biotechnologies

Cecylia Wardak*, Malgorzata Grabarczyk*, Joanna Lenik* *Department of Analytical Chemistry and Instrumental Analysis, Faculty of Chemistry, Maria Curie Sklodowska University,

Maria Curie Sklodowska 5 Sq., 20-031 Lublin Poland

17 Impact of Nano-ZnO on biogas generation in simulated landfills T.T. Onay*1, I. Temizel1, N.K. Copty1, B. Demirel1, T. Karanfil2

1T.T. Onay*, Boğaziçi University, Institute of Environmental Sciences, Istanbul, Turkey,

2Environmental Engineering and Earth Science, College of Engineering and Sciences, Clemson University, Clemson, South

Carolina, 29634, USA

18 Removal of trace metals from anaerobically digested sewage sludge using microwave assisted extraction with chelants

Valdas Paulauskas*, Ernestas Zaleckas*, Klaus Fischer** *Institute of Environment and Ecology, Aleksandras Stulginskis University, Lithuania

**Department of Analytical and Ecological Chemistry, Trier University, Germany

19 Modeling landfill gas generation and transport for energy Recovery Nadim K. Copty*, Didar Ergene* Turgut T. Onay*

*Institute of Environmental Sciences, Bogazici University, Istanbul, Turkey

20 Effect of sewage sludge co-disposal on waste degradation in anaerobic landfill bioreactors Merve Harmankaya, Turgut T. Onay*, Ayşen Erdinçler*

* Boğaziçi University, Institute of Environmental Sciences, Bebek 34342 Istanbul –Turkey

21 Thermoelectric fly ash, application for the improvement in anaerobic digestion: there are some effect in hydrolytic or methanogenic stages?

Guerrero L.*, Barahona A.*, Montalvo S.**, Huiliñir C.** Carvajal A.*, Toledo M.*, * Department of Chemical and Environmental Engineering, Federico Santa Marıa Technical University, Ave. España 1680,

Valparaıso, Chile. ** Department of Chemical Engineering, Santiago de Chile University, Ave. Lib. Bernardo O’Higgins 3363, Santiago, Chile.

22 Effect of selected trace elements on methane productivity at anaerobic digestion of maize and grasses

S. Ustak, J. Munoz, J. Sinko Crop Research Institute, Drnovska 507/73, 16106 Prague 6 – Ruzyne, Czech Republic

23 Improvement of the microbal activity by cobalt suplementation on the biomethanization of olive mill solid waste

F. Pinto-Ibieta*, A. Serrano**, D. Jeison***, R. Borja**, F.G. Fermoso** * Escuela de Procesos Industriales, Facultad de Ingeniería, Universidad Católica de Temuco, Casilla 15-D, Temuco, Chile

**Instituto de la Grasa (C.S.I.C.). Seville, Spain ***Departamento de Ingeniería Química, Universidad de La Frontera, Casilla 54-D, Temuco, Chile

14

24 Zinc as additive in table olive fermentation J. Bautista-Gallego*, F. Rodríguez-Gomez, V. Romero-Gil, A. Benítez-Cabello, A., B. Calero-

Delgado, R. Jiménez Díaz, Garrido-Fernández, F.N. Arroyo-López *Food Biotechnology Department, Instituto de la Grasa, Agencia Estatal Consejo Superior de Investigaciones Científicas,

Seville, Spain

25 Monitoring of Co(II) content which can cause harm to the environment after anaerobic processes

M. Grabarczyk, C. Wardak Department of Analytical Chemistry and Instrumental Analysis, Chemical Faculty,

Maria Curie-Sklodowska University, Lublin, POLAND

26 Determination in river water samples of trace Ni(II) which can get to environment from the residue after anaerobic processes

M. Grabarczyk, C. Wardak, J. Wasąg Department of Analytical Chemistry and Instrumental Analysis, Chemical Faculty,

Maria Curie-Sklodowska University, Lublin, POLAND

27 Lessons learnt and areas of interest for research in biological sulphate reduction for metal recovery

D. K. Villa-Gomez* * School of Civil Engineering, University of Queensland, 4067 QLD, Australia.

28 Anaerobic digestion of animal wastes after rendering in a CSTR– experimental and modeling results

A. Spyridonidis, Th. Skamagkis, L. Lambropoulos and K. Stamatelatou* Democritus University of Thrace, Department of Environmental Engineering, Vas. Sofias 12, 67132 Xanthi, Greece

29 Simulation of biogas production from animal wastes after rendering in an anaerobic contact process

A. Spyridonidis and K. Stamatelatou* Democritus University of Thrace, Department of Environmental Engineering, Vas. Sofias 12, 67132 Xanthi, Greece

30 Biogenic selenium particles: linking water treatment with resource recovery L.C. Staicu

University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Bucharest, Romania

31 Effect of anaerobic digestate dosage over sunflower germination A. Serrano*, F.G. Fermoso*, R. Borja*, R. Arias-Calderón**

*Instituto de la Grasa (C.S.I.C.). Seville, Spain. **INIAV - Instituto Nacional de Investigação Agrária e Veterinária, I. P. Elvas, Portugal

15

Plenary session and keynotes

16

17

Plenary session

Bo H. Svensson is Professor Emeritus of Thematic Studies of Water in Nature and Society at Linköping University, Sweden. He has devoted his research to the ecophysiology of anaerobic microbiology related to the biogeochemistry of methane formation and emissions in wetlands and landfills and during the last 30 with emphases on the biogas process. The latter part has involved the use of trace elements for the optimization of anaerobic digestion and possibilities to utilize the waste water streams within the pulp and paper industry for biogas production.

Keynote speakers

David Stuckey is currently a Professor of Biochemical Engineering in the Department of Chemical Engineering at Imperial College London, and a Visiting Professor at Nanyang Technological University (NTU) in Singapore. His current research interests are in the development of a submerged anaerobic membrane bioreactor (SAMBR), the production and analysis of soluble microbial products (SMPs), the role of SMPs and colloids in membrane fouling, and trace metal speciation and bioavailability. He has published over 190 technical papers, including the standard technique for Biological Methane Potential (BMP) and the

Anaerobic Toxicity (ATA).

Sabine Kleinsteuber is senior scientist at the Helmholtz Centre for Environmental Research - UFZ (Leipzig, Germany) and head of the group Microbiology of Anaerobic Systems at the UFZ Department of Environmental Microbiology. Her current research interests are in the microbial ecology of anaerobic digestion, anaerobic bioreactors for the production of platform chemicals using open mixed cultures, and anaerobic degradation of benzene in contaminated groundwater ecosystems.

Eric D. van Hullebusch is Professor of Environmental Science and Technology at UNESCO-IHE (Delft, the Netherlands) and head of the chair group Pollution Prevention and Resource Recovery. He has been working mostly on the biogeochemistry of heavy metals and metalloids in engineered ecosystems. More especially he is interested in trace elements dosing for several years with a particular focus on understanding the influence of speciation and bioavailability of trace

elements on anaerobic digesters performance. https://www.unesco-ihe.org/eric-van-hullebusch

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Trace elements as key factors to successful biogas production.

B.H. Svensson

*Department of Thematic Studies, Environmental Change, Linköping University, 58183 Linköping, Sweden

Keywords: Trace element (TE) in biology; anaerobic microorganisms; biogas applications; microbial uptake mechanisms for TE

This presentation will remind on some of the history of our nowadays large interest and use of trace elements (TE) in anaerobic digestion (AD) applications. This will include the role of TE in biology in general and for the metabolism of microorganisms involved in AD including the pathways for TE assimilation. Examples of AD areas, where TE supplementation seems to be necessary for any establishment of the process will be given and backgrounds for this will be discussed. Attention will be given to the response by the microorganism to supplementation of TE and how this may be used to elucidate corresponding microbial physiology changes. The area has been reviewed by several authors and a comprehensive overview covering many aspects of TE in anaerobic environments is given by Fermoso et al. (2015).

It should be emphasised that the role of TE in biogas reactors are the same as for any living organism: TE is needed for the metabolic machinery leading to growth and survival. Thus, TE are included directly in active sites of many enzymes and as cofactors in compounds involved in metabolic reactions. The metabolic features of organisms display different requirements for TE and their proportions to enable their living. This is important to have in mind especially in the area of anaerobic degradation of organic matter to methane, which by necessity involves a hierarchy of bacteria and archaea for the process to function. These different players will have different requirements for TE all of which have to be met. This means that any AD facility will show a different need for supplementation with TE.

The knowledge on the importance trace elements (TE) for efficient anaerobic digestion (AD) of organic matter resulting in biogas and methane production was recognised early during the development of the AD biotechnology by the studies by e.g. Callander & Bradford (1983a,b) and Takashima & Speece (1989). These studies set the scene for the physical and chemical interactions of the AD environment for the availability of the TE for the microorganism. This was then furthered by the studies later by e.g. Jansen et al. (2007), Aquino & Stuckey (2007) and Fermoso et al. (2009). At this time the focus on AD had changed considerably. AD biotechnology now had become an industry for the production of methane rather than being a means of organic waste volume reduction, which was further developed as a result of the EU landfill directorate forbidding landfilling of compostable wastes. Thus, optimization of the AD process for the highest possible methane production for a given plant and substrate profile became the challenge for the research and development of the biogas industry issue.

All these studies to different extents address the issue of ligand interference affecting the available TE fraction in relation to e.g. carbonate, phosphate, organic compounds and especially sulphide. The methodology for fractionation and characterisation of the complexes were refined (cf. van Hullebush et al. (2003), Gustavsson et al. 2013). The mostly inherent formation of sulphide and its high reactivity with TE in biogas reactors is a strong key player in this context exhibiting very strong complexes with metals. Due to the reactions by sulphide with organic compounds by sulfurization such as thiols (Shakeri Yekta et al. 2012) will be present and although their complex constants are several orders of magnitude lower than for the inorganic sulphide compounds, they are still form very strong complexes Shakeri Yekta et al. 2014). Furthermore, the interaction between iron at different redox stages and sulphur

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compounds in biogas reactors has been shown to influence the TE availability. The microorganisms are bound to have the capacity to interfere with the bulk chemistry by the production of organic compounds able to bind TE and facilitate the TE uptake (Aquino & Stuckey, 2007, Fermoso et al. 2009, d´Abzac et al. 2013).

Concomitantly with the development of our understanding on how the physical chemical environment interfere with the TE availability for the microorganisms in AD reactors several efforts have been made to investigate the composition of the microbial communities developed under different TE conditions. Although changes in the methanogenic populations mostly have been in focus (e.g. Fermoso et al. 2008, Karlsson et al. 2012) indeed there are large differences among the bacterial populations occurring. E.g. Feng et al. (2010) observed different communities of methanogens and bacteria fed with the same substrate but exposed to different concentration levels and combinations of TE. However, more specific studies on the TE effects on the bacteria have been presented for the dynamic changes in degradation pathways of propionate in the presence or absence of Mo, Se or W (Worm et al. 2009, 2011) as was also discussed by Banks et al. (2012) for the syntrophic utilisation of propionate in digesters at high ammonia concentrations. A recent study on the difference in Ni availability for cytochrome- and none-cytochrome- based methanogenic metabolism show that these features strongly affects the hydrogen threshold levels set by methanogens (Neubeck et al. 2016). The former needed more Ni than the latter, which was able to sustain very low hydrogen levels which would outcompete the other.

Thus, during the last ten to fifteen years the importance of TE for the successful management of the biogas process has become a common feature within the AD industry. A considerable knowledge on the interactions between different microorganisms and the supply/availability of TE in the AD process has been gained both connected to the physical and chemistry as well as to the microbial physiology reactions associated. The flavours of the area given above will be developed during the presentation.

References

Aquino, S.F.; Stuckey, D.C. (2007). Bioavailability and toxicity of metal nutrients during anaerobic digestion. J Environ Eng 133, 28–35.

Banks, C.J.; Zhang, Y.; Jiang, Y.; Heaven, S. (2012). Trace element requirements for stable food waste digestion at elevated ammonia concentrations. Biores Technol 104, 127–135.

Callander, I.J.: Barford, J.P. (1983a). Precipitation, chelation, and the availability of metals as nutrients in anaerobic digestion: I. Methodology. Biotechnol Bioeng 25, 1947–1957.

Callander, I.J.; Barford, J.P. 1983b). Precipitation, chelation, and the availability of metals as nutrients in anaerobic digestion: II. Applications. Biotechnol Bioeng 25, 1959–1972.

d’Abzac, P.; Bordas, F.; Joussein, E.; van Hullebusch, E.D.; Lens, P.N.L.; Guibaud, G. (2013). Metal binding properties of extracellular polymeric substancesextracted from anaerobic granular sludges. Environ Sci

Pollut Res 20, 4509–4519. Feng, X.M.; Karlsson, A.; Svensson, B.H.; Bertilsson, S. (2010). Impact of trace metal addition on biogas

production from food industrial waste – linking process to microflora. FEMS Microbiol Ecol 74, 226–240. Fermoso F.G.; Collins, G.; Bartacek, J.; O’Flaherty, V.; Lens, P.N.L. (2008) Acidification of methanol-fed

anaerobic granular sludge bioreactors by cobalt deprivation: induction and microbial community dynamics. Biotechnol Bioeng 99, 49–58.

Fermoso, F. G.; Bartacek, J.; Jansen, S.; Lens, P. N. L. (2009). Metal supplementation to UASB 764 bioreactors: from cell-metal interactions to full-scale application. Sci Total Environ 407, 3652-3667.

Fermoso, F.E:.van Hullebusch, E.D.; Guibaud, G. Collins, G; Svensson, B.H.; Carliell-Marquet, C.; Vink, J.P.M.¸ Esposito G. Frunzo L. (2015) Fate of Trace Metals in Anaerobic Digestion. – In: Georg M. Guebitz, GM; Bauer, A.; Bochmann, G Andreas Gronauer, A.; Weiss, S. (eds) Advances in Biochemal Engineering/Biotechnology 151 pp. 174-195. Springer.

Gustavsson, J.; Shakeri Yekta, S.; Karlsson, A.; Skyllberg, U.; Svensson, B.H. (2013). Potential bioavailability and chemical forms of cobalt and nickel in biogas reactors - An evaluation based on sequential and acid volatile sulfide extractions. Engineer Life Sci 13, 572–579.

van Hullebusch, E.D.; Zandvoort, M.H.; Lens, P.N.L. (2003) Metal immobilisation by biofilms: Mechanisms and analytical tools. Rev Environ Sci Biotechnol 2, 9–33.

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Jansen, S.; Wim. F.S.; Threels. F.; van Leuwen, H.P. (2005). Speciation of Co(II) and Ni(II) in anaerobic bioreactors measured by competitive ligand exchange–adsorptive stripping voltammetry. Environ Sci Technol 39, 94939-9499.

Jansen, S., Gonzalez-Gil, G., van Leeuwen, H. P. 2007. The impact of Co and Ni speciation on methanogenesis in sulfidic media-biouptake versus metal dissolution. Enz Microb Technol 40, 823-830.

Karlsson, A.; Einarsson, P.; Schnürer, A.; Sundberg, C.; Ejlertsson, J.; Svensson, B. H. (2012). Impact of trace element addition on degradation efficiency of volatile fatty acids, oleic acid and phenyl acetate and on microbial populations in a biogas digester. J Biosci Bioengineer 114, 446-452.

Neubeck, A.; Sjöberg, S.; Price, A.; Callac, N.; Schnürer, A. (2016) Effect of Nickel Levels on Hydrogen Partial Pressure and Methane Production in Methanogen. Plos One 11, Article Number: e0168357.

Shakeri Yekta, S., Gonsior, M., Schmitt-Kopplin, P., Svensson, B. H. (2012). Characterization of dissolved organic matter in full scale continuous stirred tank biogas reactors using ultrahigh resolution mass spectrometry: A qualitative overview. Environ Sci Technol 46, 12711-12719.

Shakeri Yekta, S.; Svensson, B.H.; Björn, A.; Skyllberg, U. (2014) Thermodynamic modeling of iron and trace metal solubility and speciation under sulfidic and ferruginous conditions in full scale continuous stirred tank biogas reactors. Appl Geochem. 47, 61–73.

Takashima, M.; Speece, R.E. (1989) Mineral nutrient requirements for high-rate methanefermentation of acetate at low SRT. Res J Water Pollut Control Fed 61, 1645-1650.

Worm, P.; Fermoso, F.G.; Lens, P.N.L.; Plugge, C.M. (2009) Decreased activity of a propionate degrading community in a UASB reactor fed with synthetic medium without molybdenum, tungsten and selenium. Enz Microb Technol 45,139–145.

Worm, P.; Fermoso, F.G.; Stams, A.J.M.; Len,; P.N.L.; Plugge, C.M. (2011).Transcription of fdh and hyd in Syntrophobacter spp. and Methanospirillum spp. as a diagnostic tool for monitoring anaerobic sludge deprived of molybdenum, tungsten and selenium. Environ Microbiol 13, 1228–1235.

van der Veen, A.; Fermoso, F. G.; Lens, P. N. L. (2007). Bonding form analysis of metals and sulfur

fractionation in methanol-grown anaerobic granular sludge. Engineer Life Sci 7, 480-489.

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Effect of complexation and reactor operating conditions on trace

metal partitioning/speciation and bioavailability

Prof. David C. Stuckey

Department of Chemical Engineering, Imperial College London, UK, and;

Visiting Professor, Nanyang Environment and Water Research Institute (NEWRI) (AEBC), Nanyang

Technological University, Singapore.

Anaerobic digestion (AD) is a complex process where in order to function well it needs both electron donors (substrates) and acceptors such as carbon dioxide. In addition, it needs a variety of both macronutrients such as N and P, and micronutrients such as trace metals (TM) in order to function optimally. These TMs are involved in a variety in biological functions from structure (Ca, Mg) to enabling very specific reactions in anaerobes such as Fe, Ni and Co. Traditionally, operators have either assumed TMs are present in the feed in sufficient concentration for optimal operation, or have added excess in the hope that the culture has sufficient. However, the question of bioavailability of TMs has never really been understood well, especially with complexation, and the effect of changing reactor operating parameters, eg. HRT, pH, SRT, on the partitioning of TMs between the liquid phase (where it may be washed out of the reactor), adsorption to the solid phase (sludge), and uptake into the cell. Our work in Singapore involved looking at complexing metals to enhance their bioavailability, and then at how shock loads influences partitioning and bioavailability. We used the BCR method throughout our studies to measure partitioning and fractionation.

Anaerobic digestion requires a variety of TMs in the feed, and the dominant ones are Fe, Ni, Co, Zn, Mn, Mo, Se (not strictly a TM). Since Fe plays a key role in AD, and is influenced by the presence of sulphide (low KSP), we examined its effects and its bioavailability for controlling VFAs during organic shock loads in batch reactors and a submerged anaerobic membrane bioreactor (SAMBR). When seed grown under Fe-sufficient conditions, an organic shock resulted in leaching of Fe from the residual to organically bound and soluble forms. Under Fe-deficient seed conditions, Fe2+ supplementation with an acetate feed resulted in a 2.1-3.9 fold increase in the rate of methane production, while with propionate it increased by 1.2-1.5 fold compared to non-Fe2+ supplemented reactors. Precipitation of Fe2+ as sulphides, and organically bound Fe were bioavailable to methanogens for acetate assimilation. The results confirmed that the transitory/long term limitations of Fe play a significant role in controlling the degradation of VFAs during organic shock loads due to their varying physical/chemical states, and bioavailability.

We then looked at the effects of a biodegradable/environmentally benign chelating agent, Ethylenediamine-N,N'-disuccinic acid (EDDS), on the bioavailability of Fe2+. Supplementation at 10 mg/L (0.18 mM) increased methane yield, but the presence of 0.25 mM sulfide led to the precipitation of Fe2+ as FeS, which limited its bioavailability. The results confirmed that EDDS could replace common chelating agents with low biodegradability (EDTA and NTA), and improve bioavailability by forming a Fe-EDDS complex, thereby protecting Fe2+ from sulfide precipitation. BCR measurements, and quantification of free EDDS and Fe-EDDS complex using UHPLC, confirmed that 30% of Fe2+ was present in bioavailable forms, i.e. soluble and exchangeable, when EDDS was added at a 1:1 molar ratio to Fe2+. As a result, the methane production rate increased by 11.17%, and yields 13.25%.

Having seen that EDDS is effective, we then evaluated the effect of 8 mg/L sulfide on the bioavailability of 10 mg/L iron (Fe2+), with and without EDDS, in the presence of

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sulfide, in both batch assays, and a SAMBR. EDDS was added at 1:1 molar ratio to the Fe2+ (10 mg/L), either simultaneously, or as a Fe-EDDS complex. The addition of 8 mg/L of sulfide limited the bioavailability of Fe2+ by shifting it towards less bioavailable fractions based on BCR, i.e. organic matter/sulfide and residual, resulting in decreasing methane yields and increasing VFAs. A Fe-EDDS complex was found to be more effective in controlling the change in sulfide levels during the SAMBR operation as it helped to reverse the shift in Fe2+ speciation, and increased methane yields by 9.5%.

In order to understand the effects of a TM deficiency on the performance of a SAMBR, we totally excluded them from the feed of a SAMBR. COD removal and methane yield reduced while VFAs in the effluent increased. A reduction of up to 37.5% in the total metal content in the reactor was observed, while the less bioavailable fractions increased up to 13.3%. Pulse addition of trace metals for 7 days at 5-times the daily metal loading was effective in improving the performance of the SAMBR by increasing the amount of TMs in the bioavailable fractions from 2% to 12%, with up to 88% of the added metal retained in the reactor within 24 h. However, a second and third pulse at 5 and 10-times daily metal loading did not result in similar changes in metal speciation and actually inhibited the methanogens.

Finally, based on the work above, we investigated the effect of changes in pH (7, 6.5 and 6), hydraulic retention time (HRT) (6 h, 4 h, and 2 h), solids retention time (SRT) (100 d and 25 d) on the partitioning/speciation of TMs in SAMBRs. The results showed that the metal retention capacity of SAMBRs reduced when the pH, HRT and SRT were reduced i.e. up to 22%, 39%, and 17%, respectively, but it was also found that the speciation of these TMs generally shifted towards highly bioavailable fractions i.e. Soluble and Exchangeable. The degree of shift in speciation depended on the affinity of the TMs for anaerobic sludge, and their sensitivity to the operational changes. TMs with the most and the least significant changes in speciation were Fe and Mn, respectively.

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Effect of Trace Element Limitations on Microbial Communities and Metabolic Pathways in Anaerobic Digesters

Sabine Kleinsteuber*, Babett Wintsche*, Nico Jehmlich**, Denny Popp*

*Dept. Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, Permoserstr. 15, 04318 Leipzig, Germany

**Dept. Molecular Systems Biology, Helmholtz Centre for Environmental Research – UFZ, Permoserstr. 15, 04318 Leipzig, Germany

Keywords: nickel and cobalt deficiency; methanogenic pathways; community dynamics; functional

redundancy; mcrA

Anaerobic digestion (AD) is a widespread and effective way for treating organic waste and biomass residues while producing biogas as renewable energy source. AD is a complex multi-stage process relying on the activity of highly diverse microbial communities including hydrolytic, acidogenic and syntrophic acetogenic bacteria as well as methanogenic archaea. The lower diversity of methanogenic archaea compared to the bacterial groups involved in anaerobic digestion and the corresponding lack of functional redundancy cause a stronger susceptibility of methanogenesis to unfavourable process conditions such as trace element (TE) deprivation, thus controlling the stability of the overall process. However, how methanogens react to TE deprivation specifically and how they adapt their metabolism to such limiting conditions is poorly understood in detail.

We investigated the effects of a slowly increasing TE deficit on the methanogenic community function in a semi-continuous AD process. After parallel operation of two lab-scale reactors that were well supplied with TE, the TE supplementation of one reactor was stopped, resulting in a decline of TE concentrations, especially of cobalt, molybdenum, nickel and tungsten, to insufficient levels. As shown in our recent study (Wintsche et al., 2016), the slowly increasing TE deficit did not significantly affect the reactor efficiency although shifts of the methanogenic community composition and presumably shifts in the methanogenic pathways were observed. The aim of the present study was to understand in more detail how methanogens in biogas reactors cope with TE limitation and sustain their growth and metabolic activity, thus sustaining the overall AD process under sub-optimal TE supply.

Two identical lab-scale continuous stirred tank reactors designated R1 and R2 were operated under mesophilic conditions for 93 weeks as described by Wintsche et al. (2016). The feedstock was dried distillers grains with solubles and the reactors were supplemented with a commercial iron additive and a TE mixture containing cobalt, nickel, molybdenum and tungsten. The reactors were operated at an organic loading rate of 5 g volatile solids per liter and day and a hydraulic retention time of 25 days. Both reactors were operated in parallel for 72 weeks before starting the experimental period in which the TE supply to R2 was altered by omitting the TE solution and reducing the supply of the iron additive from 2.57 g to 0.86 g per day. Batch experiments with 13C-labelled AD metabolites (carbonate, formate, acetate, propionate) to follow the carbon flow in syntrophic interactions, community analyses of the methanogenic populations based on mcrA amplicon sequencing, and mass spectrometry-based proteome analyses were performed on reactor sludge sampled taken at four sampling times. Besides fully labelled acetate, methyl- and carboxyl-group-labelled acetate was applied in separate experiments to quantify the contribution of different methanogenic pathways.

Amplicon sequencing of mcrA genes revealed Methanosarcina (72%) and Methanoculleus (23%) as the predominant methanogens in the undisturbed reactors. With increasing trace element limitation, Methanoculleus increased its relative abundance to 33%. Following the methanogenic pathways by batch tests with methyl- and carboxyl-group labelled acetate, respectively, revealed a predominance of

24

acetoclastic methanogenesis in the fully TE-supplied reactors. In contrast, TE deprivation in R2 caused a shift in the methanogenic pathways towards syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis.

Metaproteome analysis revealed abundance shifts of the enzymes involved in methanogenic pathways. In Methanosarcina, proteins involved in methylotrophic and acetoclastic methanogenesis decreased in abundance while formylmethanofuran dehydrogenase increased, confirming our hypothesis of a shift from acetoclastic to hydrogenotrophic methanogenesis by Methanosarcina. The efforts of both methanogenic genera to stabilize their metabolism and energy balance by increasing the abundance of methyl-H4MPT CoM methyltransferase and methyl CoM reductase were seemingly more successful in Methanoculleus.

The results show that TE supply is a critical factor for methanogenic enzymes. Our previous study on the effects of TE deprivation in anaerobic digestion suggested that limiting concentrations of Co, Mn, Mo, Ni and W cause activity shifts within the methanogenic communities as detected by T-RFLP profiling of mcrA transcripts (Wintsche et al., 2016). While Methanosarcina directly converts acetate and methyl compounds to methane, Methanoculleus utilizes CO2 and H2 or formate and acts as electron sink for syntrophic acetogens and syntrophic acetate-oxidizing bacteria (SAOB). Methanosarcina dominated the well-supplied AD process pointing to acetoclastic methanogenesis as major methanogenic pathway. Although Methanosarcina stayed the predominant methanogen during the whole experiment and was not overgrown by Methanoculleus, it seems that it switched to hydrogenotrophic methanogenesis under TE deprivation. Hydrogenotrophic methanogenesis might be favoured under nickel- and cobalt-deficient conditions as this pathway requires less nickel-dependent enzymes and corrinoid cofactors than the acetoclastic and methylotrophic pathways.

In summary, our study revealed that methanogens react to TE deprivation by adapting their methanogenic metabolism and use different strategies to overcome with this situation. Proteome analysis and tracer experiments revealed that Methanosarcina shifted from trace element expensive pathways (methylotrophic and acetoclastic methanogenesis) to hydrogenotrophic methanogenesis while Methanoculleus increased the hydrogenotrophic activity to sustain energy conservation. Methanosarcina as the versatile and multipotent “heavy duty” methanogen is more fastidious in regard to TE supplementation than Methanoculleus, which is a sufficient substitute not only as partner for SAOB but also as more robust methanogen stabilizing reactor performance under critical conditions.

References Wintsche, B.; Glaser, K.; Sträuber, H.; Centler, F.; Liebetrau, J.; Hauke, H.; Kleinsteuber, S. (2016) Trace

elements induce predominance among methanogenic activity in anaerobic digestion. Front. Microbiol., 7, 2034.

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Biogeochemistry of trace elements in anaerobic digesters: speciation and bioavailability

Eric D. van Hullebusch*,**

*Université Paris-Est, Laboratoire Géomatériaux et Environnement (LGE), EA 4508, UPEM, 77454 Marne-la-Vallée, France

**IHE Delft Institute for Water Education, Department of Environmental Engineering and Water Technology, P.O. Box 3015, 2601 DA Delft, The Netherlands

e.vanhullebusch@un-ihe.org, Eric.vanHullebusch@u-pem.fr

Keywords: anaerobic digestion; trace elements biogeochmistry, speciation, soils, risk assessment

Biogas and digestates are the final products of anaerobic digestion of solid organic waste. While biogas is typically valorised for heat and electricity production, the digestates (liquid and/or solid) are usually recycled back to agricultural fields as fertilizer (Al Saedi et al., 2013; Nkoa, 2014). EU (EU Fertilizer Directive) and national regulations that may restrict the valorisation of digestates consider the total element (e.g. heavy metals and metalloids) concentrations. However, total element concentrations do not always assess the actual environmental risks. For instance, solubility and bioavailability of heavy metals usually decrease during anaerobic digestion, mainly due to precipitation processes with sulfide (S2−), carbonate (CO3

2−) and phosphate (PO43−) (Möller and

Müller, 2012, Fermoso et al., 2015), however it does not mean that long-term heavy metal immobilization in soils is expected. In fact, mobility, transfer in soils, biological up-take by plants and effect of trace elements on microbial diversity and activity is usually strongly connected to the physico-chemical form of the chemical elements considered (Kabata-Pendias and Mukherjee, 2007; Hooda, 2010). Therefore different chemical speciation methods have been developed to assess the speciation of potentially toxic elements with the aim to assess the risks associated with the utilization of digestate as fertilizer.

Therefore, this presentation aims first at reporting who are the toxic elements, their origins (feedstock origin, trace elements dosing for enhanced biogas production (Shakeri Yekta, 2014), and the concentrations usually encountered in digesters (Tampio et al., 2016; Nkao, 2014). A short overview of the biogeochemical processes involved in the partitioning of heavy metals and metalloids in anaerobic digesters (Möller & Müller, 2012) and the analytical methods usually implemented to determine the fate of heavy metals and metalloids in solid substrates will be presented (Valeur, 2011; van Hullebusch et al., 2016; Thanh et al., 2016). Then in the second part of the presentation, the biogeochemical processes regulating the transformation of heavy metals and metalloids speciation and the fate of trace elements in soils will be presented (Hooda, 2010). The current understanding of the fate and effect of trace elements in soils when soils are amended with digestates will be discussed (Insam et al., 2015; García-Sánchez et al., 2015).

References Al Seadi, T., Fuchs, W., ..., & Janssen R. (2013). Biogas digestate quality and utilization. The biogas

handbook: Science, production and applications, 267. Fermoso, F. G., Van Hullebusch, E. D., Guibaud, G., Collins, G., Svensson, B. H., Carliell-Marquet, C., ... &

Frunzo, L. (2015). Fate of trace metals in anaerobic digestion. In Biogas Science and Technology (pp. 171-195). Springer International Publishing.

García-Sánchez, M., Siles, J. A., Cajthaml, T., García-Romera, I., Tlustoš, P., & Száková, J. (2015). Effect of digestate and fly ash applications on soil functional properties and microbial communities. European Journal of Soil Biology, 71, 1-12.

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Hooda, P. (Ed.). (2010). Trace elements in soils. John Wiley & Sons. Insam, H., Gómez-Brandón, M., & Ascher, J. (2015). Manure-based biogas fermentation residues–Friend or

foe of soil fertility?. Soil Biology and Biochemistry, 84, 1-14. Kabata-Pendias, A., & Mukherjee, A. B. (2007). Trace elements from soil to human. Springer Science &

Business Media. Möller, K., & Müller, T. (2012). Effects of anaerobic digestion on digestate nutrient availability and crop

growth: a review. Engineering in Life Sciences, 12(3), 242-257. Nkoa, R. (2014). Agricultural benefits and environmental risks of soil fertilization with anaerobic digestates: a

review. Agronomy for Sustainable Development, 34(2), 473-492. Shakeri Yekta, S. (2014). Chemical speciation of sulfur and metals in biogas reactors: implications for cobalt

and nickel bio-uptake processes (Doctoral dissertation, Linköping University Electronic Press). Tampio, E., Salo, T., & Rintala, J. (2016). Agronomic characteristics of five different urban waste digestates.

Journal of Environmental Management, 169, 293-302. Thanh, P. M., Ketheesan, B., Yan, Z., & Stuckey, D. (2016). Trace metal speciation and bioavailability in

anaerobic digestion: A review. Biotechnology advances, 34(2), 122-136. Valeur, I. (2011). Speciation of heavy metals and nutrient elements in digestate. Diss.:Norwegian University

of life sciences. van Hullebusch, E. D., Guibaud, G., Simon, S., Lenz, M., Yekta, S. S., Fermoso, F. G., ... & Skyllberg, U.

(2016). Methodological approaches for fractionation and speciation to estimate trace element bioavailability in engineered anaerobic digestion ecosystems: An overview. Critical Reviews in Environmental Science and Technology, 46(16), 1324-1366.

27

Platform

28

29

Effect of trace elements supplementation in agricultural biogas plants

Mirco Garuti*, Michela Langone**, Claudio Fabbri*, Sergio Piccinini*

*CRPA – Research Center on Animal Production, Viale Timavo, 43/2 - Reggio Emilia, Italy **University of Trento, Via Mesiano 77 - Trento, Italy

Keywords: biogas; bioenergy; trace elements; full-scale study; process monitoring.

Anaerobic digestion is a promising biotechnological process to produce biogas/biomethane (renewable heat and electricity, biofuel) from agricultural by-products and organic waste and an effluent digestate used as biofertilzer in the concept of circular economy. It is a multi-stage microbiological process (hydrolysis, acidogenesis, acetogenesis and methanogenesis) that involves different groups of microorganisms interacting each other.

At full-scale level, the biogas production is influenced by technological factors (digester size and geometry, solids and hydraulic retention time, stirring intensity), process parameters (temperature, organic loading rate) and biochemical fermentation conditions (pH, H2 content in biogas, trace elements concentration, ammonia level, organic acids accumulation) (Drosg, 2013).

Trace elements (TE) are mainly metals such as cobalt, selenium, nickel, iron, molybdenum, selenium. Substrates used in the anaerobic digestion process show low concentration of TE with respect to other basic macronutrients like carbon, nitrogen, phosphorus and sulphur. Nevertheless many studies have been carried out to provide evidence of TE importance for the effectiveness of the anaerobic degradation process (Pobenheim et al., 2010) and their fundamental interaction with microbial cells (Fermoso et al., 2009).

The present work suggests the applicability of a practical methodological approach for the identification of trace element deficiency and the strategies (i) to optimize the process and (ii) to recover the biogas production (Fig 1). Two full scale case-studies are presented.

30

Figure 1. Methodological approach used in this study for the identification of trace element deficiency and strategies to optimize the process (on the left in yellow) and to recovery the biogas production (on the right in red) during anaerobic digestion process.

In the first case-study the strategy to optimize the process has been undertaken. The agricultural biogas plant had an electrical power of 380 kWel, the feedstock was a mixture of cow slurry, maize silage and triticale silage. An accumulation of volatile fatty acids (VFAs) and a slightly decrease of the TE concentration with respect to historical data were identified by periodical analytical controls of the fermentation process. The optimization of biogas production was achieved by dosing a commercial TE additive with molybdenum, cobalt, selenium and nickel to the digester. The CH4 content in the biogas increased from 52% to 63% and the organic loading rate was 20% lower (keeping constant the biogas production) throughout the days immediately afterwards the TE supplementation.

In the second case-study, the agricultural biogas plant had an electrical power of 999 kWel, the feedstock was a mixture of cow slurry, pig slurry, chicken manure. maize silage, triticale silage, corn meal and glycerine. During the monitoring period a failure in the biogas production occurred and the biological conversion of organic matter in methane was 20-40% underperformed. Inhibition of biological process was confirmed by VFAs accumulation (5550 mg/kg acetic acid, 180 mg/kg propionic acid, 90 mg/kg butyric acid, 110 mg/kg iso-butyric acid, 240 iso-valeric acid). The recovery of biogas production was totally restored in about 5 days after iron, cobalt, molybdenum, selenium supplementation using a commercial TE additive. During the TE supplementation, the CH4 content in the biogas increased from 40% to 59%.

In this study total metals quantification was evaluated by ICP-OES (EN ISO 11885:1997) because it was a cheap, speed and ordinary analysis used by biogas plant operators.

In conclusion this work showed two full-scale case studies about TE supplementation strategy used to optimize the biogas production in agricultural biogas plants and to recover the anaerobic biological process failure, respectively.

The “optimal” TE concentration in anaerobic environment often is not well defined because each biological process could keep its best performing TE concentration on the basis of digester technology, process parameters and metals speciation and bioavailability. The methodological approach used in this work is based on the correlation between the process performances and TE concentration and its comparison with historical data.

References Drosg B. (2013). Process monitoring in biogas plants. IEA Bioenergy, London, U.K. Pobeheim, H.; Munk, B.; Johansson, J.; Guebitz, G.M. (2010). Influence of trace elements on methane

formation from a synthetic model substrate for maize silage. Bioresour. Technol. 101, 836–839. Fermoso, F.G.; Bartacek, J.; Jansen, S.; Lens, P.N.L. (2009). Metal supplementation to UASB bioreactors:

from cell-metal interactions to full-scale application. Sci Total Environ. 407, 3652–3667.

31

Optimization of biogas production from anaerobic digestion of Laminaria sp. through manipulation of trace element availability

J Roussel*, L Austin*, C Carliell-Marquet*

* School of Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom

Keywords: Macro-Algae; Anaerobic Digestion; Trace element; Factorial Design; Biogas.

The production of bioenergy from macro-algae, such as Laminaria sp., has received attention from the early 1970s through a promising high yield of energy conversion (Wise et al., 1979). However, the interest of using macro-algae as anaerobic digestion (AD) feedstock has mainly grown in the last decade in order to meet the UK target of 20% electricity generation from renewable sources by 2020. Overall, macro-algae as feedstock has a greater potential than terrestrial feedstock due to the environmental advantages of farming offshore (no land competition or use of fresh water) and a high biogas yield.

The economic viability of anaerobic digestion as bioenergy producer highly depends on high and fast feedstock degradation. Trace elements (TE) are essential to microbial metabolism and their presence in a sufficiently available form is a necessity to push the AD yields to its maximum. Macro-algae as a sole feed might be at risk of some TE deficiency due to the nature and location of its growth. TE supplementation should prevent the risk of TE deficiency and in addition, it has been shown to increase biogas yield, stabilise digestion and reduce the build-up of volatile fatty acids.

This research looked to establish the influence of four trace elements: Ni, Co, Zn and Fe on biogas production from the seaweed species Laminaria sp. The importance of those four TEs for the metabolism of methanogen was demonstrated by Fermoso et al. (2009), while iron, by its higher concentration, might influence the bioavailability of the other TEs (Roussel and Carliell-Marquet, 2016). Using batch biochemical methane potential tests, the research has adopted a factorial design approach that provides important information on the relative significance of each of the selected trace elements without the need for many experimental runs at different concentrations over long retention times. A range of concentration, from 0 and 1.0 mg.gVS-1, were used to determine the optimal mix of TE and their concentration. The experiment was designed on a factorial framework with each sample containing different combinations of high and low trace element concentrations prepared in triplicate with EDTA used as chelating agent. Biogas production, methane content and volatile solid degradation were used to determine the efficiency of the batch reactors and TEs concentration in the soluble phase or solid phase was measured by ICP-OES to quantify the fate of the supplemented TE. Results were statically analysed using the software minitab.

The main finding from the experiment indicated zinc had a positive effect on the biogas production while cobalt and iron did not exhibit any effect. However, nickel was inhibitory at high concentrations. The results of the factorial analysis indicate that high nickel concentrations dampened the boosting effects of zinc in particular. In contrast to nickel inhibition, zinc appeared to boost biogas production, with a

statistically significant relationship seen over the first 92 hours of the experiment and in the total biogas production. In terms of methane yield, the supplementation of TE in the optimal combination gave a 7% increase in comparison with un-supplemented sample.

The use of EDTA as chelating agent has a strong impact on the fate of those TE and can be linked with their bioavailability (Bartacek et al., 2008). Results showed that

32

nickel remained in the soluble phase at the end of the test while cobalt and zinc were transferred to the solid phase. However, the presence of iron played an important role in the dissolution of Me-EDTA and in the transfer to the solid phase. No statistical relationship was established between the kinetic of dissolution and bioavailaibility of the trace element, further work should be undertaken in order to fully understand the mechanism of the MeEDTA dissolution and to quantify the availability and uptake of the supplemented metals.

The first results showed clearly the importance of the metal supplementation and especially the need to tailor to the feedstock. However, results were not enough to be able to determine the optimal mix and more work is currently being undertaken to optimize the metal supplementation and enhance the methane production. The work is focussing on the cobalt supplementation and the effect of other trace element such as selenium or tungsten.

References Bartacek, J.; Fermoso, F.G.; Baldo-Urruti, A.M.; et al. (2008) Cobalt toxicity in anaerobic granular

sludge:influence of chemical speciation. Journal of Industrial Microbiology & Biotechnology, 35, 1465-1474.

Fermoso, F.G.; Bartacek, J. ; Jansen, S. ; et al. (2009). Metal supplementation to UASB bioreactors: from cell-metal interactions to full-scale application. Science of the Total Environment, 407, 3652-3667.

Roussel, J.; Carliell-Marquet, C. (2016) Significance of Vivianite Precipitation on the Mobility of Iron in Anaerobically Digested Sludge. Front. Environ. Sci. 4:60. doi: 10.3389/fenvs.2016.00060 .

Wise D.L.; et al. (1979). Methane fermentation of aquatic biomass. Resource Recovery Conservation, 4, 217-237

33

Competing demands for trace elements in anaerobic digesters

Y Zhang*, H Song*, N Sriprasert*, CJ Banks*, S Heaven*

*Bioenergy and Organic Resources Research Group, Faculty of Engineering and the Environment, University of Southampton, UK

Keywords: Anaerobic digestion; trace elements; interspecies competition; bioavailability; organic loading

rate

The function of trace elements (TEs) in facilitating the metabolic pathway of anaerobic digestion (AD), and thus enhancing biogas production and organic waste stabilisation, is widely recognised. Challenging research issues remain, however, with regard to how to optimise TE supplementation. This is a complex question involving many areas including chemistry, microbiology and technology optimisation. It is important to minimise TE dosing with respect to both the elements and the concentration used, due to concerns about dispersion into the environment (including to agricultural land), as well as to cost aspects. Attempts have usually been characterised by a trial-and-error approach, however, due to the lack of a clear understanding of the impact of TEs on AD under different process conditions. The aim of this study was therefore to identify some important factors that influence the beneficial effects of TE supplementation. To achieve this, a set of experiments were carried out involving long-term operation of food waste and synthetic organic waste digesters; each focused on a specific effect as listed below.

Effect of interspecies competition between different microbial groups: This experiment used real-world source-segregated domestic food waste at an initial organic loading rate (OLR) of 4 g volatile solids (VS) L-1 day-1 in digesters supplemented with Se, Co, Ni, W. TEs were then allowed to wash out, with Se only maintained at 0.2 mg L-1. After 380 days the OLR was increased to 5 g VS L-1 day-1, and gradual accumulation of volatile fatty acids (VFA) was observed. Co supplementation was restored after VFA accumulation, but could not prevent further VFA accumulation until the OLR was reduced to 2 g VS L-1 day-1. This and follow-up experiments confirmed that TE supplementation has a two-way effect on AD: although its contribution to VFA degradation is well established, it can stimulate VFA production to a great extent, especially when both bacteria and methanogens are lacking TE for their metabolic activities. This observation is supported by another study (Yu et al., 2015). This issue needs particular attention when digesters laden with VFA are operated at moderate or high OLR.

Effect of bioavailability of Fe, Ni and Co: This experiment was to identify the affinity of different digestate fractions for TE. A dilute-out approach was again employed because it is an efficient way of distinguishing the fraction with the highest affinity for TE when TE becomes limiting. Due to the fluctuation of TE concentrations in real-world organic waste, synthetic waste was employed to allow better quantification of the TE concentrations under specific operational conditions. The substrate used comprised whole milk, whole egg powder and rice flour at 20:20:60% (VS basis). Deionised water was added to reduce substrate VS to 10%. TE concentrations in the working substrate were 2.87, 0.03, 0.01, 0.04 and 0.06 mg L-1 for Fe, Ni, Co, Se and Mo, respectively. Four digesters were operated at an OLR of 3 g VS L-1 day-1 with dosing strength of Fe, Co, Ni and Se at 10, 1, 1, 0.1 mg L-1, respectively. From day 175 onwards, Fe, Ni and Co supplementation ceased in digester 1, 2 and 3, respectively, while other elements were maintained in those digesters. Dynamic changes in Fe, Ni and Co distribution profiles in digester 1, 2 and 3 were then monitored over the course of the washout process. The results showed that the intracellular Ni and Co concentrations remained the same during

34

270 days of washout, although the concentrations of extracellular fractions gradually decreased at a similar rate to the total Ni and Co concentrations (Figure 1.1a). This was, however, not true for Fe. Fe concentration in the sulphide-bound fraction remained relatively constant during the course of the experiment, and VFA accumulated when total Fe concentration was still high (Figure 1.1.b).

Figure 1.1. Dynamic changes of TE distribution profiles during their dilute-out phase: (a) Co and (b) Fe.

Effect of metal interaction: the above synthetic waste was used again in this study at the same OLR of 3 g VS L-1 day-1. Various TE and TE combinations were supplemented to eight digesters. The results showed that single element supplementation was unable to satisfy the TE requirement. Binary metal supplemented digesters (Co and Ni) showed VFA accumulation after day 270, although with some delay compared with control (no TE) and single TE dosing digesters. Fe showed antagonistic effects when supplemented with either Co or Ni. When supplemented with Co, Ni and Fe, digesters operated well without VFA accumulation for 400 days, and better than with Co and Ni supplementation. This and follow-up experiments indicated that attention must be paid to the metal ratio in a supplementation matrix. The literature suggests that Ni and Co may have less availability because these can be adsorbed or complexed on FeS (Shakeri Yekta et al., 2014).

Effect of organic loading rate: This experiment was carried out using real-world source-segregated domestic food waste with sufficient TE. The maximum operational loading for this type of digestion, without compromising process stability and food waste conversion efficiency was determined as 8 g VS L-1 day-1. Although higher loadings could be applied, the retention time was reduced to an extent which did not allow complete hydrolysis. Nitrogen mass balance equations were developed to elucidate the nitrogen distribution in digesters. These showed that microbial biomass density increased along with OLR increases, and in turn required an increase in TE addition.

Table 1.1. Effect of organic loading rate on digester operational parameters. Internal normalisation was applied using ratio scale with the data at OLR 5 as baseline.

OLR 5 OLR 7 OLR 8 OLR 9

Organic loading rate 1 1.4 1.6 1.8 Hydraulic retention time 1 0.71 0.63 0.56 Specific methane production 1 1.01 0.98 0.93 Volumetric methane production 1 1.38 1.62 1.67 Residual methane production 1 0.98 - 1.09 Microbial biomass density 1 1.18 1.29 1.26

References Shakeri Yekta, S.; Lindmark, A.; Skyllberg, U.; Danielsson, Å.; Svensson, B.H. (2014) Importance of reduced

sulfur for the equilibrium chemistry and kinetics of Fe(II), Co(II) and Ni(II) supplemented to semi-continuous stirred tank biogas reactors fed with stillage. J. Hazard. Mater., 269, 83-88.

Yu, B. ; Shan, A. ; Zhang, D. ; Lou, Z.; Yuan, H.; Huang, X.; Zhu, N. (2015). Dosing time of ferric chloride to disinhibit the excessive volatile fatty acids in sludge thermophilic anaerobic digestion system. Bioresour. Technol., 189, 154-161.

(b) One-off Fe dosing

due to VFA

accumulation

35

Addition of fly ash from thermal power plant to anaerobic digestion of sewage: Effect of ash particle size.

S. Montalvo*, I. Cahn*, R. Borja**, C. Huiliñir*, A. Barahona***, L. Guerrero***

*Universidad de Santiago de Chile, Av. Lib. Bdo. O`Higgins 3363, Santiago de Chile. Chile,

silvio.montalvo@usach.cl

**Instituto de la Grasa, Campus Universitario Pablo de Olavide - Edificio 46, Ctra. de Utrera, Km. 1, 41013 Sevilla,

España.

***Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso, Chile

Keywords: anaerobic digestion; fly ash; metals; methane production; mixed sludge.

Huiliñir et al (2017) demonstrated that fly ash addition to anaerobic digester operating with sewage mixed sludge (waste activated sludge + primary sludge) enhanced the process, being the best doses 50 mg.L-1 of fly ash; they stated that better behavior of anaerobic digestion was due to the amount of metal present in fly ash and its positive effect on methanogenic archea (Qiang et al., 2013). In this work three experimental runs (ER) were carried out by adding 50 mg·L-1 of fly to lab batch reactors (35 ± 2 ºC) working with sewage mixed sludge in order to evaluate the influence of fly ash particle size from 0.8 to 2.36 mm on methane production and anaerobic biodegradability by comparing the results obtained with those achieved in control reactors (without fly ash addition). In the anaerobic processes that operate with fly ash a higher elimination of total volatiles (VTS), suspended volatile solids (VSS), total chemical oxygen demand (CODT) and soluble chemical oxygen demand (CODS) was achieved than in reactors working without fly ash. An increase of the methane production between 28% and 96% compared to the control reactors was obtained, achieving the highest increases at ash particles size of 1.0-1.4 mm. The metal concentrations in the digestate obtained after anaerobic digestion of sewage sludge are far below those considered as limiting for the use of sludge in soils.

Table 1. Chemical characterization of the fly ash from a thermal power plant

Fly ash

Element Values (mg·kg-1

) Element Values (mg·kg-1

) Element Values (mg·kg-1

)

B 35.03 Ni 6.17 V 13.36

Zn 5.40 Al 1315.42 Ba 44.36

Cr 2.92 Mn 21.05 Fe 3083.58

Cu 2.21 Co <DL Mo <DL

DL: Detection Limit

36

Table 2. Summary of the results achieved in the experimental runs

Parameter Fly ash particle size ER1 ER2 ER3

VTS Removal [%]

Control 33.00 31.20 32.57

< 0.8 mm 36.40 32.59 33.89

1 - 1.4 mm 39.00 34.72 40.11

2 - 2.36 mm 35.84 28.11 31.65

VSS Removal [%]

Control 15.00 27.49 29.00

< 0.8 mm 28.90 30.11 32.73

1 - 1.4 mm 35 34.72 37.00

2 - 2.36 mm 27.27 24.73 31.12

CODT Removal [%]

Control 30.00 34.94 33.56

< 0.8 mm 44.51 46.97 45.38

1 - 1.4 mm 46.27 54.29 57.02

2 - 2.36 mm 40.92 44.44 42.96

CODS Removal [%]

Control 41.72 45.22 43.14

< 0.8 mm 56.15 69.35 64.47

1 - 1.4 mm 61.15 74.19 72.98

2 - 2.36 mm 49.72 68.54 59.47

Methane yield relative to control [%]

Control - - -

< 0.8 mm 59.16 46.52 49.00

1 - 1.4 mm 95.78 85.10 88.58

2 - 2.36 mm 27.82 29.64 34.00

Figure 1. Methane evolution in batch lab reactors

References

Huiliñir, C.; Pinto-Villegas, P.; Castillo, A.; Montalvo, S.; Guerrero, L. (2017). Biochemical methane potential from sewage sludge: Effect of an aerobic pretreatment and fly ash addition as source of trace elements. Waste Management, in press

Qiang, H.; Niu, O.; Chi, Y.; Li, Y. (2013). Trace metals requirements for continuous thermophilic methane fermentation of high-solid food waste. Chemical Engineering Journal, 222, 330-336.

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30 35

mL

CH

4.g

-1 V

SS

Time (days)

< 0.8 [mm] 1 - 1.4 [mm] 2 - 2.36 [mm] Control

37

Metagenomics Reveals The Phylogenetic Distribution Of Selenium-Reducung Microorganisms In Soils And In

Anaerobic Granular Sludge Biofilms

S. Mills*, L. Staicu** and G. Collins*

* Microbial Commnities Laboratory, Microbiology, School of Natural Sciences, National University of Ireland Galway, University Road, Galway, H91 TK33, Ireland

** University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Bucharest, Romania

Keywords: Microbial Selenium Respiration, Metagenomics, Biogenic Selenium, Nanoparticles, 16S

rRNA Sequencing

Selenium (Se) oxyanions are effective electron acceptors, and essential trace

elements, in microbial respiration (Nancharaiah & Lens, 2015). Therefore, Se cycling

in engineered, and natural, environments is highly dependant on microbial activity,

often in complex, mixed-species communities. Microorganisms capable of reducing

soluble Se oxyanions are thought to be phylogenetically diverse (Lenz & Lens,

2009). A deeper, and fuller, understanding of this phylogeny is required to exploit Se

microbiology and develop new environmental biotechnologies.

Anaerobic granular sludge was previously shown to be effective in converting Se

oxyanions in wastewaters to biogenic, less toxic Se (Dessì et al., 2016; Lenz, et al.,

2008). However, the reported conversion rates achieved by various granular sludges

varied greatly (Astratinei., et al 2006); therefore, further investigation of microbial Se-

reducing populations in anaerobic sludge granules is required.

Se has a naturally heterogenous distribution in soils, where it is primarily derived

from bedrock. This leads to ‘hotspots’ of seleniferous soils, which have been

implicated in significant pollution events affecting wildlife and human health (Fan et

al., 1988; Ohlendorf et al., 1990). Therefore, it is also essential to investigate the

microbial ecology of Se-reducers in soils.

The aim of this study was to investigate the phylogeny of microbial populations

underpinning Se reduction in different environments, including seleniferous soils and

waste-conversion biofilms, and to expand our understanding of the microbial ecology

of Se cycling.

Batch incubations of loam soil; Atlantic peatland soil; and both intact and

physically-homogenised, anaerobic sludge granules were used to enrich for selenite-

and selenate-reducing prokaryotic microorganisms. Enrichment cultures, containing

soil, peat or granular biofilms, were set up, and serially sub-cultured to fresh media.

Lactate:acetate (16mM:4mM) or H2:CO2 (80:20v/v) were used as reductants in the

enrichment cultures.

Inductively Coupled Mass-Spectrometry (ICP) was used to measure Se

oxyanions in enrichment cultures. Population dynamics, and community succession,

were monitored by sequencing 16S rRNA genes in temporal sub-cultures, and to

identify key Se-reducing taxa. Shotgun metagenomics were used to comprehensively

characterise selected, highly-enriched, Se-reducing cultures. Both 16S rRNA and

functional mRNA targets were probed, and localised, in 10-um-thick cross-sections of

sludge granules (diameter 0.5-2mm) using fluorescence in situ hybridisations (FISH).

38

Initial isolations of pure cultures of heterotrophic and autotrophic (H2-oxidising)

selenate- and selenite-reducers were achieved at 30°C using a modified Hungate

Roll Tube method (Hungate & Macy, 1973). Selected colonies were purified by

streaking on basal minimal medium containing selenite or selenate along with

appropriate electron donors. To isolate autotrophic microbes using H2 as an electron

donor, Hungate tubes were supplied with Ar:H2 (95:5 v/v). Isolates were

characterised by determining growth rates and optimum growth temperatures.

Scanning Electron Microscopy (SEM) was used to visualise the formation of

elemental Se nanoparticles, which were observed on the surface of spherical sludge

granules and free-floating in culture media, thus indicating selenate- and selenite-

reduction, which was confirmed using ICP analysis.

DNA sequencing of enrichment cultures indicated temporal shaping of diversity in

Se-fed communities, and revealed the breadth of distinct taxa associated with Se

metabolism in each of the soil and sludge enrichment cultures. Heterotrophic

(lactate- or acetate-oxidising), and autotrophic (H2-oxidising), Se-reducing organisms

were purified and isolated from the granular sludge and soil sources, and are

currently undergoing characterisation. FISH experiments revealed highly organised

distributions of Se cyclers across the architecture of sludge granule biofilms.

The results indicate widely distributed potential for Se-reduction across an

expansive phylogeny in previously unexposed samples. Indeed, the presence of both

heterotrophic and autotrophic Se-metabolisers in previously unexposed granular

sludge indicates its potential for the treatment of Se oxyanion-containing

wastewaters. The presence of Se-reducers in each of the soil samples tested

suggests the ubiquitous nature of these phylogenetically diverse microorganisms. It

is likely that these organisms play a role in maintaining low levels of toxic Se

oxyanions in seleniferous soils.

References

Astratinei, V., van Hullebusch, E., & Lens, P. (2006). Bioconversion of selenate in methanogenic anaerobic granular sludge. Journal of Environmental Quality, 35(5), 1873–1883. https://doi.org/10.2134/jeq2005.0443

Dessì, P., Jain, R., Singh, S., Seder-Colomina, M., van Hullebusch, E. D., Rene, E. R., … Lens, P. N. L. (2016). Effect of temperature on selenium removal from wastewater by UASB reactors. Water Research, 94, 146–154. https://doi.org/10.1016/j.watres.2016.02.007

Fan, A. M., Book, S. A., Neutra, R. R., & Epstein, D. M. (1988). Selenium and human health implications in California’s San Joaquin Valley. J Toxicol Environ Health, 23(4), 539–559. https://doi.org/10.1080/15287398809531135

Hungate, R. E., & Macy, J. (1973). The Roll-Tube Method for Cultivation of Strict Anaerobes. Bulletins from the Ecological Research Committee, (17), 123–126. Retrieved from http://www.jstor.org/stable/20111550

Lenz, M., Hullebusch, E. D. Van, Hommes, G., Corvini, P. F. X., & Lens, P. N. L. (2008). Selenate removal in methanogenic and sulfate-reducing upflow anaerobic sludge bed reactors. Water Research, 42(8), 2184–2194. https://doi.org/10.1016/j.watres.2007.11.031

Lenz, M., & Lens, P. N. L. (2009). The essential toxin: The changing perception of selenium in environmental sciences. Science of the Total Environment, 407(12), 3620–3633. https://doi.org/10.1016/j.scitotenv.2008.07.056

Nancharaiah, Y. V, & Lens, P. N. L. (2015). Ecology and Biotechnology of Selenium-Respiring Bacteria, 79(1), 61–80. https://doi.org/10.1128/MMBR.00037-14

Ohlendorf, H. M., Hothem, R. L., Bunck, C. M., & Marois, K. C. (1990). Bioaccumulation of selenium in birds at Kesterson Reservoir, California. Archives of Environmental Contamination and Toxicology, 19(4), 495–507. https://doi.org/10.1007/BF01059067

39

High-throughput characterisation of whole-

ecosystem microbial communities: anaerobic

granules in ‘micro-sequencing batch

reactors’ Sarah O’Sullivan, Estefanía Porca Belío and Gavin Collins

Microbiology, School of Natural Sciences, National University of Ireland,

Galway, University Road, Galway, Ireland

(e-mail: gavin.collins@nuigalway.ie)

Anaerobic digestion (AD) comprises the degradation of complex organic materials in the

absence of oxygen to produce biogas, a renewable fuel. In some configurations of

engineered AD systems, the process is underpinned by anaerobic granules, which are

self-immobilised, spherical biofilms. Granules comprise of complex consortia of

microorganisms from each of the trophic groups involved in anaerobic digestion including

bacteria and archaea. They are naturally occurring within anaerobic bioreactors and are

intricate to the treatment of many types of wastewaters. They facilitate and allow the

interspecies transfer of various substrates to ensure complete degradation of organic

constituents from the wastewater present in the bioreactor (Grotenhuis, 1991,

O’Flaherty, 1997, Stams and Plugge, 2009).

In recent, previous work from our laboratory, specific methanogenic activity assays using

large (1.4-2.0mm), medium (800μm-1.399mm) and small (400-799μm) granules against

key substrates found that large granules were most active – although this is not in

agreement with some similar investigations we have also done. The aims of this study

were threefold; to determine (1) differences in methanogenic activity across granule size

fractions and from different wastewater sources; (2) the extent to which anaerobic

granules can be considered replicates with respect to physical and ecological

characteristics; and (3) whether there are distinct and replicated shifts in the profiles of

the active communities of individual anaerobic granules in response to environmental

cues.

Volatile solids (VS) concentrations of 100 individual large granules, as well as cDNA

sequencing targeting 16S rRNA from sixteen (16) large granules, indicated that granules

can be considered as distinct, closely replicated, whole ecosystems. On this basis, and

by using novel, ‘micro-sequencing batch reactors’ (micro-SBRs), a series of high-

throughput experiments was set up to investigate the single-whole-ecosystem

microbiology of granules. Individual granules in micro-SBRs were fed every 48 hours

over 42 days. Granules were subjected to various, distinct conditions of pH (4, 7, 10),

carbon source (acetate, cellulose, glucose), trace elements availability (e.g. cobalt-

deprivation) and temperature (37°C, 23°C). For each condition, assays were set up with

and without the methanogenic inhibitor, 2-bromoethanesulfonate (BES), to also

investigate non-methanogenic pathways. DNA and RNA was co-extracted from

individual granules at the conclusion of micro-SBR trials using an adapted protocol

(Griffiths et al., 2000). 16S rRNA gene sequences were amplified using the universal

bacterial and archaeal forward primer 515f and reverse primer 806r, and ampliconswere

sequenced on an Illumina MiSeq platform. Volatile fatty acids analysis of BES-treated

granules indicated homoacetogenic activity. Community structure succession shifted in

40

response to environmental conditions and activity differed according to granule size.

Community structure analyses indicated significant differences between sludge types but

not between sludge sizes. Individual single granules were considered replicates with

respect to VS content and the active community profile (Fig. 1). The uSBR study

indicated distinct and replicated shift in active community structure in response to

specific environmental condition conditions (Fig. 2), suggesting an opportunity for high-

throughput, whole-ecosystem studies on the biofilms, which could support mathematical

models of microbial community behaviour.

Figure 1. Barplots indicating relative structure of active communities in individual granules.

Figure 2. Canonical correspondence analysis of granules fed with and without cobalt.

41

Influence of Trace Elements on the Methanogenic Pathway at

High Ammonium Concentrations

Rahim Molaey, Alper Bayrakdar, Recep Önder Sürmeli, Bariş Çalli

Environmental Engineering Department, Marmara University, 34722 Kadikoy, Istanbul, Turkey

Keywords: Ammonia; Anaerobic mono-digestion; Methanoculleus; Selenium; Syntrophic acetate oxidation

The long-run influence of trace element (TE) deficiency and supplementation on anaerobic mono-digestion of chicken manure (CM) at elevated total ammonia nitrogen (TAN) concentration (>6000 mg/l) was investigated in this study. Three identical daily-fed anaerobic reactors were used in the experiments. The 1.3 L continuously stirred bio-reactors were operated with 0.8 L working volume at 36±1 oC. They were fed manually once per day with raw chicken manure diluted with tap water according to the applied organic loading rate (OLR). Throughout the study, the OLR and hydraulic retention time (HRT) were kept constant at 3.65 kg/m3.d and 30 days, respectively.

Unlike what is considered, in our former study we found that chicken manure does not contain sufficient amounts of W, Se and Co, which are essential TEs for AD (Molaey et al., Submitted). Therefore, throughout this study while reactor 2 (R2) was supplemented with Se (0.2 mg/l) and reactor 3 (R3) with a TE mix including Co (1 mg/l), Mo (0.2 mg/l), Ni (1 mg/l), Se (0.2 mg/l) and W (0.2 mg/l), reactor 1 (R1) was operated as control without TE supplementation.

Before starting this study, all the reactors had been operated for about 9 months and the acetate concentration in R1 reached to 20000 mg/l. After starting this study with a new chicken manure the acetate concentration decreased rapidly below 500 mg/l within 40 days. The decrease in VFAs concentration enhanced the methane production and the methane yield rose from 0.22 to 0.28 l/gVS in average. The improvement in the performance of R1 which was not supplemented with TEs was attributed to the relatively higher TE concentrations in the new chicken manure used in this period. However, in the long run the acetate concentration increased again gradually and reached to 15000 mg/l at Day 113. As a consequence, the CH4 yield of R1 decreased and was 0.21±0.05 l/gVS between day 50 and 113. In the meanwhile, the acetate concentration in R2 and R3 was quite low and never exceeded 3000 mg/l. In this period, 0.27±0.03 and 0.26±0.05 l/gVS of CH4 yields were achieved in R2 and R3, respectively. After Day 113, the acetate concentration in R1 increased sharply and exceeded 25000 mg/l at Day 140. Accordingly, the CH4 yield steadily decreased and dropped below 0.10 l/gVS. In the same period, the acetate concentration increased slightly to 2900 mg/l and the CH4 yield did not change in R2. Unexpectedly, after day 132 the acetate and propionate concentrations increased sharply and in 12 days reached to 13000 mg/l and 3000 mg/l, respectively. As a result, the CH4 yield dropped to 0.12 l/gVS. The TE supplementation was stopped at day 110 in R3 to evaluate how long the enhancement effect of TE supplementation will last. The CH4 production performance was not adversely affected from the termination of TE supplementation and even slightly increased to 0.31±0.03 l/gVS. Meanwhile, the acetic acid concentration in R3 was 1156±440mg/l. These results showed that the TEs added formerly to the reactors continued to stimulate the methane production after the termination of supplementation. The long-lasting effect of TEs was presumably related to the accumulation of TEs in the reactor because of precipitation and/or adsorption and the subsequent release of insolubilized TEs after the termination of dosing.

The influence of TE supplementation was also evaluated by investigating the methanogenic population dynamics. The metagenomic analyses of the digestate taken

42

from R2 and R3 showed that TEs supplementation was critically important on the diversity methanogens. The results of metagenomic analyses revealed that the dominant methanogens in R1 and R3 were related to the hydrogenotrophic methanogens of the order of Methanomicrobiales and Methanobacteriales and an uncultured archaeon clone ARCM3 and M4_D05. On the species level, the dominant methanogen was identified as Methanoculleus bourgensis. It has been reported that Methanoculleus bourgensis easily adapts to elevated ammonium concentrations in such as anaerobic manure digesters (Maus et al., 2015).

Figure 1.1. The methane yield (a) and acetate concentration (b) graphs

The findings of this study indicated that at elevated TAN concentration (>6000 mg/l) Se supplementation is critical for enhanced methane production. However, in the long-run addition of a TE mix containing Co, Mo, Ni, Se and W is more beneficial than the addition of Se alone. It was revealed that the addition of TEs stimulated the growth of the hydrogenotrophic methanogen M. bourgensis in the anaerobic CM mono-digester. It is known that, M. bourgensis utilizes the CO2 and H2 as substrate for CH4 production (Sowers et al., 2009) and collaborate with syntrophic acetate oxidizing bacteria for the consumption of acetate (Fotidis et al., 2013). In the light of these results, it is concluded that in anaerobic mono-digestion of CM at TAN concentrations above 6000 mg/l, the TE supplementation is crucial for stable methane production which is carried out via syntrophic acetate oxidation followed by hydrogenotrophic methanogenesis.

References

Fotidis, I.A., Karakashev, D., Angelidaki, I., 2013. Bioaugmentation with an acetate-oxidising consortium as a tool to tackle ammonia inhibition of anaerobic digestion. Bioresour. Technol. 146, 57–62. doi:10.1016/j.biortech.2013.07.041

Maus, I., Wibberg, D., Stantscheff, R., Stolze, Y., Blom, J., Eikmeyer, F.G., Fracowiak, J., König, H., Pühler, A., Schlüter, A., 2015. Insights into the annotated genome sequence of Methanoculleus bourgensis MS2T, related to dominant methanogens in biogas-producing plants. J. Biotechnol. 201, 43–53. doi:10.1016/j.jbiotec.2014.11.020

Sowers, A.D., Gaworecki, K.M., Mills, M.A., Roberts, A.P., Klaine, S.J., 2009. Developmental effects of a municipal wastewater effluent on two generations of the fathead minnow, Pimephales promelas. Aquat. Toxicol. 95, 173–181. doi:10.1016/j.aquatox.2009.08.012

43

Trace elements as pH controlling agents support microbial chain elongation

H. Sträuber*, M. Dittrich-Zechendorf**, F. Bühligen*, S. Kleinsteuber*

*UFZ – Helmholtz Centre for Environmental Research, Department of Environmental Microbiology, Permoserstr. 15, 04318 Leipzig, Germany

**Deutsches Biomasseforschungszentrum gemeinnützige GmbH (DBFZ), Department Biochemical Conversion, Torgauer Str. 116, 04347 Leipzig, Germany

Keywords: acidogenic reactor; VFA; manganese; iron; Megasphaera

For the production of carboxylic acids by anaerobic fermentation, pH control is required. However, adding buffer solutions is ineffective in leach-bed reactors due to their spatially uneven distribution. We investigated the effect of solid alkaline iron and manganese additives on maize silage fermentation and microbial communities. Without additives, the pH dropped to 3.9 and lactic acid bacteria were favoured. Total product yields of 207 ± 5.4 g organic acids (C2-C6) and alcohols per kg volatile solids were reached. The addition of trace elements increased the pH value and the product spectrum and yields changed. With a commercial iron additive, the product yields were higher (293 ± 15.2 g kg-1 volatile solids) and clostridia used lactic acid for microbial chain elongation of acetic acid producing n-butyric acid. With the addition of pure Fe(OH)3 or Mn(OH)2, the total product yields were lower than in the other reactors. However, increased production of medium-chain fatty acids and the occurrence of distinct clostridial taxa (Lachnospiraceae, Ruminococcaceae and Megasphaera) related to this metabolic function were observed. Thus, the application of alkaline trace metal additives as pH stabilizing agents can mitigate spatial metabolic heterogeneities when trace metal deficient substrates like specific crops or residues thereof are applied.

44

ADM1-based mechanistic model for trace elements

bioavailability in anaerobic digestion processes

L. Frunzo*, F.G. Fermoso**, V. Luongo*, M.R. Mattei*, and G. Esposito***

* Department of Mathematics and Applications "Renato Caccioppoli", University of Naples Federico II,

Complesso Monte Sant’Angelo, 80124 Naples, Italy

** Instituto de la Grasa (C.S.I.C.), Campus Universidad Pablo de Olavide. Edificio 46.

Ctra. de Utrera km. 1, 41013-Sevilla, Spain

*** Department of Civil and Mechanical Engineering, University of Cassino and Southern Lazio, via Di Biasio

43, 03043 Cassino, Italy

Keywords: Trace elements, Anaerobic digestion, Mathematical modelling, Ordinary differential equations

Predicting the ultimate fate of trace elements (TE) in anaerobic processes has received

increased attention across the research community (Van Hullebusch et al. 2016,

Fermoso et al. 2015). TE represent essential cofactors for the growth of anaerobic

microorganisms and are subjected to complex interactions, with the biomass and the

liquid phase (e.g., precipitation, organic complexation, bio-sorption, adsorption, inorganic

complexation), which affect the trace metal bio-availability and in turns process stability.

Although there have been attempts to model physicochemical processes in anaerobic

digestion (AD) in special cases, to the best of our knowledge, an overall model able to

take into account the several processes affecting TE bioavailability is missing. This might

be attributed to the wide range of components to be taken into account and to the

complexity and heterogeneity of the processes that should be defined in order to model

such phenomena.

In this study a new mechanistic model based on anaerobic digestion model no.1 (ADM1)

approach (Batstone et al. 2002) has been proposed in order to simulate the TE fate and

their effects on anaerobic digestion in terms of total methane production.

Figure 1. Schematic representation of the proposed model.

45

Although the ADM1 model predicts the dynamic concentration of biochemical

components fairly, its predictions are limited when the TE effect is relevant due to

insufficient consideration of physicochemical processes (e.g. ADM1 ignores the liquid-

solid precipitation reactions, the TE interactions with particulate and soluble ligands, the

TE microbial uptake). The major reason of sidelining physicochemical processes can be

attributed to wide range of components to be taken into account and to the complexity of

the processes that should be defined in order to model such processes.

The main processes and mechanisms affecting TE speciation and bio-availability and the

related variables needed to model such phenomena have been defined. In particular,

three main process categories have been taken into account: i) TE bio-uptake,

production and release; ii) TE reaction with liquid compounds; iii) TE reaction with solid

compounds. Such processes have been incorporated in the ADM1 in the form of

different submodules and have been combined with the pre-existing ADM1 biochemical

compartment. The latter includes the modeling of the main biodegradation steps of AD

(e.g. hydrolysis, acidogenesis, acetogenesis, methanogenesis), the acid-base

equilibrium and gas transfer kinetics. The defined relationships between the processes

are represented in Figure 1. More precisely, the developed model takes into account: the

release of TE during hydrolysis of complex organic matter; the fate of sulfur in the AD

process; the microbial uptake of the TE; the release of TE during microbial decay.

Furthermore, the bio-chemical module of the ADM1 has been modified in order to take

into account: reaction of TE and organic acids (acetic, propionic, butyric, valeric, and

LCFA); precipitation of TE with carbonate and sulfide; sorption of TE on microbial

biomass; sorption of TE on solid inert compounds.

The differential equations governing TE, substrates, products and bacterial

groups dynamics involved in the AD processes have been synthesized based on mass

balance considerations. For the n1 liquid (including dissolved substrates, microbial

groups and solid precipitates) and n2 gas components, denoted as liqS and gasS

respectively, they take the following form:

,,...,1

),(

1

,_,,1,,1,,1,,

, 321

ni

vvvVSqSqdt

SdVitransgjijprec

m

jjijsorp

m

jjijbio

m

jliqiliqoutiinin

iliqliq

,,...,1, 2,_

,,ni

V

V

V

qS

dt

dS

gas

liq

itransg

gas

gasigasigas

where liqV and gasV denote the liquid and gas phase volume respectively; inq and outq

are the inlet and outlet flow rate; iinS

, represents the ith inlet liquid component

concentration; 321 ,, mmm denote the number of biochemical, sorption and precipitation

processes which affect the dynamics of the ith liquid component.

The terms itransgjijprecjijsorpjijbio vvv ,_,,,,,, ;;; represent respectively: the biochemical

reaction rate, the sorption rate both on soluble and particulate ligands, the precipitation

rate and the gas/transfer rate for the ith model component and for the jth process. The

model has been tested through numerical simulations related to the case of a single

anaerobic digestion reactor fed with the organic fraction of municipal solid waste.

46

References Van Hullebusch, Eric D. et al., (2016). Methodological approaches for fractionation and speciation to

estimatetrace element bioavailability in engineered anaerobic ecosystems: an overview, Critical reviews in Environmental science and Technology. 46, 1324-1366.

Fermoso, F. G., Van Hullebusch, E. D., Guibaud, G., Collins, G., Svensson, B. H., Carliell-Marquet, C., ... & Frunzo, L. (2015). Fate of trace metals in anaerobic digestion. In “Biogas Science and Technology”(pp. 171-195). Springer International Publishing.

Batstone, DJ. et al. (2002). The IWA anaerobic digestion model no 1 (ADM1), Water Science and Technology, 45, 65-73.

47

Stability of ZnS formed during anaerobic digestion

Maureen Le Bars*, Clément Levard**, Samuel Legros***, Jean-Paul Ambrosi**, Jérôme

Rose**, Daniel Borschneck**, Emmanuel Doelsch*

* UPR Recyclage et risque, CIRAD, France

** CEREGE UM34, Aix-Marseille Université, CNRS, IRD, France

*** UPR Recyclage et risque, CIRAD, Sénégal

Keywords: organic waste recycling; metal contamination; zinc sulfides; nanoparticles; speciation

Recycling of digestates is a strategic aspect for anaerobic digestion (AD) plants

suitability. Many studies have shown the fertilizing properties of digestate on croplands

(Nkoa, 2014). However, application of digestate on lands is not riskless, particularly

regarding metal contamination. Indeed, organic wastes used as input in AD can be

highly concentrated in zinc partly due to the addition of high amount of zinc in pig and

cattle feeding (Alburquerque et al., 2012). Repeated land application of digestate causes

long-term accumulation of zinc in soil. To prevent risk of contamination, some countries

implemented regulation on digestate application. Unfortunately, those regulations only

focus on total metal concentration while zinc speciation is the crucial parameter to

consider when evaluating its bioavailability and potential toxicity (Harmsen, 2007).

It has been shown that AD changes zinc speciation: zinc is mostly found as sulfides in

digestates. ZnS formed during AD transform very quickly under oxic conditions (total

transformation within 2 months (Lombi et al., 2012)) compared with what is observed

with natural ZnS (i.e. sphalerite) (<2% of sphalerite transformed in one year (Robson et

al., 2014)) or synthetic ZnS with a crystallite size of 20-40 nm (7 to 76% of ZnS

transformed in 2 years depending on the soil type (Voegelin et al., 2011)). This

difference remains unexplained and is the main focus of our study in order to assess the

fate of ZnS after spreading on crops.

We assumed that ZnS formed in organic waste have nanometric sizes due to the

presence of high amount of organic matter that inhibit ZnS growth. First, we synthetized

nano-ZnS of different sizes to assess the influence of size and crystallinity on ZnS

stability in controlled systems and in soil samples. We obtained new accurate data on

ZnS-NPs transformation kinetics. These nano-ZnS have been characterized by:

- Transmission Electronic Microscopy (TEM) to determine particle morphology (figure 1);

- Wide Angle X-ray Scattering with Pair Distribution Function Analysis to obtain crystallite

sizes and lattice constrains;

- X-ray Absorption Spectroscopy (XAS) at Zn k-edge for local structure.

Those well characterized nano-ZnS were also used as references for XAS Zn k-edge in

situ measurements to assess the properties (size and crystallinity) of ZnS presents in

digestates sampled in various industrial plants. This study evidences the formation of

nano-ZnS during anaerobic digestion, the latter being spread in large quantities on

cultivated lands during digestate recycling. We gained a new understanding on how AD

conditions control the precipitation of ZnS-NPs and on ZnS-NPs fate in soil after

digestate application on cultivated lands.

48

Figure 1. TEM images of three of the ZnS-NPs synthetized, estimated sizes: 5.9 nm (left), 4.5 nm (middle)

and 2.5 nm (right, dark field imaging)

References

Alburquerque, J. A., de la Fuente, C., Ferrer-Costa, A., Carrasco, L., Cegarra, J., Abad, M., & Bernal, M. P.

(2012). Assessment of the fertiliser potential of digestates from farm and agroindustrial residues.

Biomass and Bioenergy, 40, 181-189.

Harmsen, J. (2007). Measuring bioavailability: from a scientific approach to standard methods. Journal of

environmental quality, 36(5), 1420-1428.

Lombi, E., Donner, E., Tavakkoli, E., Turney, T. W., Naidu, R., Miller, B. W., & Scheckel, K. G. (2012). Fate

of Zinc Oxide Nanoparticles during Anaerobic Digestion of Wastewater and Post-Treatment

Processing of Sewage Sludge. Environmental Science & Technology, 46(16), 9089-9096.

Nkoa, R. (2014). Agricultural benefits and environmental risks of soil fertilization with anaerobic digestates: a

review. Agronomy for Sustainable Development, 34(2), 473-492.

Robson, T. C., Braungardt, C. B., Rieuwerts, J., & Worsfold, P. (2014). Cadmium contamination of

agricultural soils and crops resulting from sphalerite weathering. Environmental Pollution, 184, 283-

289.

Voegelin, A., Jacquat, O., Pfister, S., Barmettler, K., Scheinost, A. C., & Kretzschmar, R. (2011). Time-

Dependent Changes of Zinc Speciation in Four Soils Contaminated with Zincite or Sphalerite.

Environmental Science & Technology, 45(1), 255-261.

49

A simultaneous study of organic matter and trace elements accessibility in substrate and digestate from an anaerobic

digestion plant

A. Laera*, S. Shakeri Yekta**, R. Buzier ***, Mattias Hedenström****, Mårten Dario**, G.

Guibaud***,G. Esposito*****, E.D. van Hullebusch*,******

* Laboratoire Géomatériaux et Environnement, Université Paris-Est, 77454 Marne-la-Vallée, France **The Department of Thematic Studies - Environmental Change, Linköping Universitet, 58183 Linköping,

Sweden *** GRESE, Université de Limoges, 87060 Limoges, France

**** Department of chemistry, Umeå Universitet, 90187 Umeå, Sweden ***** Dipartimento di Ingegneria Civile e Meccanica, Università degli Studi di Cassino e del Lazio

Meridionale, 03043 Cassino, Italy ****** Department of Environmental Engineering and Water Technology, IHE Delft Institute for Water

Education, 2601 Delft, The Netherlands

Keywords: Substrate;

Digestate;

Organic matter fractionation method;

Trace elements;

Organic matter bio-accessibility.

Digestate is a residual product of the anaerobic digestion (AD) of organic wastes

(substrate). The chemical characteristics of the digestate mostly depends on the

chemical composition of the substrate (Fuchs & Drosg, 2013; Nkoa, 2014) and the

operational conditions of the AD process (Möller & Müller, 2012). Generally, the

digestate contains trace elements such as cobalt (Co), copper (Cu), iron (Fe),

manganese (Mn), molybdenum (Mo), nickel (Ni) and zinc (Zn) (Fuchs & Drosg, 2013;

Rintala et al. 2010). Concentration, chemical speciation and bioavailability of trace

elements in the digesters determine their nutritious or toxic effects on microbial

degradation pathways as well as the digestate quality, when applied as biofertilizer.

Trace elements speciation is regulated by the presence of organic and inorganic

metal-binding ligands (Fermoso et al., 2015; Gustavsson et al., 2013; van Hullebusch et

al., 2016). Organic matter compounds, such as the extracellular polymeric substances

(EPS), have been found to play an important role in trace elements immobilization

(Sheng et al., 2010).

The present work aims to assess the interrelationship between bioaccessibility of

different organic matter fractions, including EPS, and trace elements by performing an

organic matter fractionation procedure known as the EPS method (Jimenez et al., 2014)

and simultaneous analyses of trace elements associated with each organic fraction. The

method is applied on samples of substrate and digestate collected from an AD plant,

treating organic fraction of household waste in Linköping, Sweden.

The EPS method allow the sequential extraction of five operationally defined

organic matter fractions. The extracted fractions have different degree of solubility and

reactivity to the extracting reagents, thus different degree of bioaccessibility. The first

extracted fraction is dissolved organic matter (DOM), then soluble extracellular polymeric

substances (S-EPS), readily extractable extracellular polymeric substances (RE-EPS),

humic like substances (HLS) (Jimenez et al., 2014) and finally poorly extractable organic

matter (PEOM) (Jimenez et al., 2015). A new faction, defined as organic matter bound to

50

minerals (i.e. carbonates, sulphides and hydroxides) (CSH), was included in the EPS

method before HLS fraction extraction. In addition to the original EPS method, a modified

EPS procedure is tested by moving the CSH fraction at the beginning of the extraction

procedure. The purpose of this modification is to extract trace elements associated with

inorganic ligands at the beginning of the extraction procedure to assess the potential

contribution of inorganic sources to trace elements released during the sequential

extraction of organic matter fractions. In this work the original EPS method is named

Procedure 1, while the modified method is called Procedure 2.

Organic carbon and trace element concentrations were measured after each step

of the sequential extraction using total carbon analyser and inductively coupled plasma

mass spectrometer, respectively.

The preliminary results show different distributions of organic carbon among the

organic matter fractions in substrate and digestate. Figure 1 shows that, in both

procedures, a large proportion of extracted organic carbon is contained in DOM, S-EPS

(only in Procedure 1), HLS and PEOM fractions in substrate. In digestate most of the

organic carbon is extracted in HLS (only in Procedure 2) and PEOM fractions during

Procedure 1 and 2. A substantial decrease in concentration of organic carbon associated

with DOM and S-EPS fractions suggests that these fractions contain the most

bioaccessible organic substances in the substrate which have been degraded by

microorganisms during the AD process. In contrast, the concentration of organic carbon

extracted in PEOM (only in Procedure 1) fraction in digestate indicates an enrichment of

these organic matter fraction during the AD process.

Figure 1. Dissolved organic carbon (DOC) concentration in the organic matter fractions extracted in substrate and digestate by performing Procedure 1 and 2. Except of DOM fraction extracted by Procedure 1, results are mean of triplicates. Note that the value with * expresses methodological LOQ.

The analysed trace elements are Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb and Zn. However, the concentrations of As, Cd, Cu, Mo and Pb, simultaneously released after each extraction step, were below the limit of quantification in almost all extracted liquid fractions, for this reason those elements are not shown here. Moreover, the elements’ concentration extracted in PEOM fraction are not discussed since there is a high uncertainty on the measured values due to the dilution factor required before quantification by ICP-MS.

The concentrations of trace elements are shown in Figure 2, which displays the distribution of trace elements in organic fractions in substrate and digestate. Similar trend is found when both procedures are performed. DOM fraction is extracted by centrifuging the fresh samples and the trace elements quantified in this fraction represent the chemical species in ionic forms and bound to aqueous inorganic and organic ligands (e.g. soluble EPS). Trace elements extracted in S-EPS fraction, mainly in Procedure 1,

0

50

100

150

DOM S-EPS RE-EPS CSH HLS PEOM

mg

DO

C/g

TS

in

Substarte Digestate

[CE

LL

RE

F]

0

50

100

150

DOM CSH S-EPS RE-EPS HLS PEOM

mg

DO

C/g

TS

in

Substrate Digestate

Procedure 1 Procedure 2

51

by using CaCl2 solution are likely extracted by ion exchange reaction. Thus it is not possible to deduce whether S-EPS fraction is involved in immobilization of those elements, rather representing the exchangeable fraction of trace elements in solid phase of the samples. The elements extracted in HLS fraction by using 0.1 M NaOH solution are likely associated with humic like substances in samples. This assertion is supported by the fact that the concentrations of some elements such as Al, Cr and Zn in substrate is almost unvaried after the initial extraction of CSH fraction, representing the trace elements bound to the inorganic fraction in the solid phase. Instead, in digestate the amount of Cr extracted in HLS by the two procedures is unvaried, while more Al, Co, Ni and Fe are extracted by Procedure 1 compared to Procedure 2.

To validate the trace elements extraction performance of the two procedures, the total element concentration extracted by acid digestion in the two samples is compared with the sum of elements extracted at each step of the two procedures. Indeed, by performing Procedure 1, the recovery ranges from a minimum of 29% (Cr) to a maximum of 93% (Zn) in substrate and from 18% (Cr) to 88% (Mn) in digestate, whereas in Procedure 2 the recovery ranges from 34% (Cr) to 111% (Co) in substrate and from 19% (Cr) to 114% (Ni) in digestate. The results suggest that the two extraction procedures do not have the same extraction efficiency for all trace elements. Moreover, the present results reveal that for some extraction step the used reagents strongly interact with trace elements determining their leaching or precipitation.

Figure 2. Trace elements distribution in the extracted fractions by Procedure1 and 2 in substrate and digestate. Results are expressed in percentage of total element extracted by the respective procedures.

This study highlights limitations for applying an organic matter fractionation method (Jimenez et al., 2014) to extract trace elements, most probably due to the strong interactions of the reagents used to extract organic matter with the trace elements chemistry.

References Fermoso, F. G.;van Hullebusch, E. D.;Guibaud, G.;Collins, G.;Svensson, B. H.;Carliell-Marquet, C.;…

Frunzo, L. (2015). Fate of Trace Metals in Anaerobic Digestion. In Biogas Science and Technology (pp.

0%

20%

40%

60%

80%

100%

120%

Al Fe Cr Mn Co Ni Zn

% o

f to

tal e

lem

en

t

DOM S-EPS RE-EPS CSH HLS

0%

20%

40%

60%

80%

100%

120%

Al Fe Cr Mn Co Ni Zn

DOM S-EPS RE-EPS CSH HLS

0%

20%

40%

60%

80%

100%

120%

Al Fe Cr Mn Co Ni Zn

% o

f to

tal e

lem

en

t

DOM CSH S-EPS RE-EPS HLS

0%

20%

40%

60%

80%

100%

120%

Al Fe Cr Mn Co Ni Zn

DOM CSH S-EPS RE-EPS HLS

Substrate_Procedure 1 Digestate_Procedure 1

Substrate_Procedure 2

Digestate_Procedure 2

52

171–195); Springer International Publishing, Switzerland. Fuchs, W.;& Drosg, B. (2013). Assessment of the state of the art of technologies for the processing of

digestate residue from anaerobic digesters. Water Science and Technology, 67(9), 171-195. Gustavsson, J.;Yekta, S. S.;Karlsson, A.;Skyllberg, U.;& Svensson, B. H. (2013). Potential bioavailability and

chemical forms of Co and Ni in the biogas process-An evaluation based on sequential and acid volatile sulfide extractions. Engineering in Life Sciences, 13(6), 572-579.

Jimenez, J.;Aemig, Q.;Doussiet, N.;Steyer, J. P.;Houot, S.;& Patureau, D. (2015). A new organic matter fractionation methodology for organic wastes: Bioaccessibility and complexity characterization for treatment optimization. Bioresource Technology, 194, 344-353.

Jimenez, J.;Gonidec, E.;Cacho Rivero, J. A.;Latrille, E.;Vedrenne, F.;& Steyer, J. P. (2014). Prediction of anaerobic biodegradability and bioaccessibility of municipal sludge by coupling sequential extractions with fluorescence spectroscopy: Towards ADM1 variables characterization. Water Research, 50(2004), 359-372.

Möller, K.;& Müller, T. (2012). Effects of anaerobic digestion on digestate nutrient availability and crop growth: A review. Engineering in Life Sciences, 12(3) 242-257.

Nkoa, R. (2014). Agricultural benefits and environmental risks of soil fertilization with anaerobic digestates: A review. Agronomy for Sustainable Development, 34(2), 473-492.

Rintala, J.;Tampio, E.;& Salo, T. (2010). Valorisation of food waste to biogas. Sheng, G.;Yu, H.;& Li, X. (2010). Extracellular polymeric substances (EPS) of microbial aggregates in

biological wastewater treatment systems: A review. Biotechnology Advances, 28(6), 882-894. van Hullebusch, E. D.;Guibaud, G.;Simon, S.;Lenz, M.;Yekta, S. S.;Fermoso, F. G.;… Collins, G. (2016).

Methodological approaches for fractionation and speciation to estimate trace element bioavailability in engineered anaerobic digestion ecosystems: An overview. Critical Reviews in Environmental Science & Technology, 46(16), 1324-1366.

53

Ability of anaerobic granules for metal-mediated direct

interspecies electron transfer

C.-D. Dubé*, S.R. Guiot*

* National Research Council Canada, 6100 Royalmount Avenue, Montreal, H4P 2R2 Canada

Keywords: Anaerobic granular sludge; interspecies electron transfer; bioactivity; conductive particles

The long-established efficacy of granular sludge for high-rate anaerobic wastewater treatment is namely due to the granule compactness and cells proximity that facilitate metabolites exchange and interspecies electron transfer (IET). It has long been known that granulation is aided by metal cations and precipitates (Guiot et al. 1988), namely as cations help binding negatively charged cells together to form microbial nuclei and cation precipitates serve as surface for adhesion of bacteria (Yu et al. 2000, Show 2006). It has also been shown more recently that granulation may benefit from the addition of conductive materials (granular activated carbon (GAC), charcoal, metallic minerals), through mineral-mediated direct interspecies electron transfer (mDIET) (Shrestha and Rotaru 2014, Zhao et al. 2015, Dubé and Guiot 2015). DIET excluding hydrogen and formate, could happen between obligate H2-producing acetogenic (OHPA) bacteria and methanogenic archaea in some environments (Stams et al. 2006), including brewery wastewater anaerobic granules where Geobacter sp. play a key role (up to 30% of all bacteria) (Morita et al. 2011).

To further investigate the question whether DIET could occur in Geobacter-deprived granules, specific activities of disintegrated granules, where Geobacter represented only 0.2% of total bacteria, with and without electrically conductive and non-conductive microparticles were compared to each other, and to those of the whole granules. Three materials were tested: GAC (conductive), stainless steel (ProPak) (highly conductive) and porcelain (non-conductive). GAC is known to help methanogenesis during digester start-up or recovery (Liu et al. 2012, Lü et al. 2016). GAC provides a large specific surface area for biofilm development, just like porcelain, while ProPak offers the greatest conductivity but a lower specific surface area for colonisation.

The layered architecture of granules, which promotes the physical proximity between syntrophic cells (Guiot et al. 1992), could also promote DIET, as previously seen in granules formed with Geobacter species (Summers et al. 2010). Consequently, any disruption of such a structure should result in reduced methane-producing specific activities (MPA), and in most cases, reduced substrate-consuming activities (SCA) also. As shown in Table 1, the disintegration of the granules had a negative effect, resulting in a decrease of the ethanol SCA and MPA at 41% and 38%, respectively, of those of the integral granules. When incubated with ProPak and GAC, the cells from disintegrated granules had a higher MPA, reaching 190±10% and 175±13% of the MPA obtained with disintegrated granules without microparticles, respectively. When porcelain (non-conductive) microparticles were added, the MPA was reduced at 65±4% of the control without microparticles. Thus, the conductivity of the microparticles added to cells from disintegrated granules seems instrumental and suggests the reality of mDIET.

Scanning electron microscopy (SEM) clearly showed that GAC and porcelain microparticles were colonized to a greater extent than the stainless steel microparticles by the end of assay. By contrast, SEM showed that ProPak was ineffective for cell colonisation, where most cells observed were still separated (Figure 1). This was expected since the former ones presented a higher porosity and ruggedness than the stainless steel. The porosity and ruggedness of non-conductive microparticles (e.g. porcelain) facilitated cells from disintegrated anaerobic granules to reform a biofilm

54

without the methanogenic activity to be recovered as compared to that of whole original granules. On the contrary, cells in the presence of non-porous but highly conductive microparticles (e.g. ProPak) did recover a significant level of the whole granule activity without reformation of biofilm. This suggests that anaerobic granule architecture facilitates IET not only because of the reduced distance of diffusion for the electron-transfer molecules, but likely also because they provide an enabling matrix for DIET.

Table 1. Specific activities of both ethanol consumption (SCA) and methane production (MPA) obtained with

disintegrated granules without and with conductive and non-conductive microparticles

Microbial materials Ethanol SCA MPA

mmol/gVSS·d % mmol/gVSS·d %

Whole granules 5.4 ± 0.3 245 ± 18 5.2 ± 0.3 260 ± 15

Disintegrated granules 2.2 ± 0.1 100 2.0 ± 0.1 100

Disintegr. gran. + GAC 5.4 ± 0.4 245 ± 9 3.5 ± 0.4 175 ± 13

Disintegr. gran. + stainless steel 2.1 ± 0.3 95 ± 15 3.8 ± 0.3 190 ± 10

Disintegr. gran. + porcelain 2.1 ± 0.4 95 ± 20 1.3 ± 0.0 65 ± 4

Figure 1. Scanning electron microscope images of colonized GAC (A), porcelain (B), and ProPak (C) microparticles at the end of the assays. Bar = 5 µm.

References Dubé, C.-D.; Guiot, S.R. (2015) Direct interspecies electron transfer investigation in anaerobic digestion: a

review. Adv. Biochem. Eng., 151, 101-115. Guiot, S.R.; Gorur, S.S.; Kennedy, K.J. (1988) Nutritional and environmental factors contributing to microbial

aggregation during upflow anaerobic sludge bed-filter (UBF) reactor start-up. Hall, E.R. and Hobson, P.N. (eds), pp. 47-53, Pergamon Press, Oxford, UK.

Guiot, S.R.; Pauss, A.; Costerton, J.W. (1992) A structured model of the anaerobic granule consortium. Wat. Sci. Technol., 25, 1-10.

Liu, F.; Rotaru, A.E.; Shrestha, P.M.; Malvankar, N.S.; Nevin, K.P.; Lovley, D.R. (2012) Promoting direct interspecies electron transfer with activated carbon. Energy Envir Sci, 5, 8982-8989.

Lü, F.; Luo, C.; Shao, L.; He, P.-J. (2016) Biochar alleviates combined stress of ammonium and acids by firstly enriching Methanosaeta and then Methanosarcina. Water Res., 90, 34–43.

Morita, M.; Malvankar, N.S.; Franks, A.E.; Summers, Z.M.; Giloteaux, L.; Rotaru, A.E.; Rotaru, C.; Lovley, D.R. (2011) Potential for direct interspecies electron transfer in methanogenic wastewater digester aggregates. mBio, 2, e00159-00111.

Show, K.-Y. (2006) Biogranulation technologies for wastewater treatment. Tay, J.-H., Tay, S.T.-L., Liu, Y., Show, K.-Y. and Ivanov, V. (eds), pp. 35-56, Elsevier, Amsterdam.

Shrestha, P.M.; Rotaru, A.-E. (2014) Plugging in or going wireless: strategies for interspecies electron transfer. Front. Microbiol., 5, 1-8.

Stams, A.J.M.; De Bok, F.A.M.; Plugge, C.M.; Van Eekert, M.H.A.; Dolfing, J.; Schraa, G. (2006) Exocellular electron transfer in anaerobic microbial communities. Envir. Microbiol., 8, 371-382.

Summers, Z.M.; Fogarty, H.E.; Leang, C.; Franks, A.E.; Malvankar, N.S.; Lovley, D.R. (2010) Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria. Science, 330, 1413-1415.

Yu, H.Q.; Fang, H.H.P.; Tay, J.H. (2000) Effects of Fe2+ on sludge granulation in upflow anaerobic sludge blanket reactors. Water Sci. Technol., 41, 199-205.

Zhao, Z.; Zhang, Y.; Woodard, T.L.; Nevin, K.P.; Lovley, D.R. (2015) Enhancing syntrophic metabolism in up-flow anaerobic sludge blanket reactors with conductive carbon materials. Bioresource Tech., 191, 140–145.

55

Strategies in Ni and Co recovery with biogenic sulphide

Y.Liu*, M. Yao*, I. Yoong*, M. Peces**, J. Voughan**, G. Southam***, D.Villa-Gomez*

*School of Civil Engineering, The University of Queensland, 4072 QLD, Australia

**School of Chemical Engineering, The University of Queensland, 4072 QLD, Australia

***School of Earth and Environmental Sciences, The University of Queensland, 4072 QLD, Australia

Keywords: metal recovery; sulphide; precipitation; Nickel; Cobalt

Recovery of metals from mine tailings not only avoids the release of harmful chemicals

that causes severe environmental damage, but is also an economic resource as tailings

contain significant amount of unrecovered metals with market potential.

Australia is the third largest nickel producer generating huge quantities of tailings mostly

containing high levels of unrecovered nickel (Ni) and cobalt (Co). Cobalt is frequently to

co-occur in Ni ores (Crundwell, Moats et al. 2011), which leads to the co-existence of

both metals in leaching solutions, hindering the selective metal recovery.

Among the options for metal recovery, metal precipitation with biogenic sulphide

produced in sulphate reducing bioreactors is gaining attention as it can be locally

produced and avoid the transportation of toxic compounds as compared with traditional

chemical sulphide (Hatami, Bissonnette et al. 2010). Also, sulphate and substrate that

are needed by the production of biogenic sulphide exist in many wastes, providing an

opportunity to achieve both environmental and economic benefits. However, unlike

chemical sulphide, sub-products accompanying biogenic sulphide are inherent to

changes in microbial pathways due to the type of substrate used, which in turn may

affect the metal sulphide characteristics. Since the separated recovery of Ni and Co

sulphide is already a challenge due to their similarities in chemical behaviour, it is

essential to determine if the metals precipitated with biogenic sulphide have key physical

differences such as settling rates that can lead to selective recovery through gravity

separation(Crittenden, Trussell et al. 2012).

Therefore, the objective of this study was to characterize Ni and Co precipitation with

biogenic sulphide produced in sulphate reducing bioreactors fed with three different

substrates- volatile fatty acids (VFA), ethanol and leachate from the fermentation of

municipal solid waste, to evaluate their physical differences that can facilitate selective

metal recovery. Firstly, batch precipitation experiments were carried out to determine

differences in metal precipitates characteristics, where Ni and Co were added

individually and as mixture at different concentrations into chemically produced sulphide

(sodium sulphide) and VFA-fed biogenic sulphide (Fig 1.1). Secondly, settling rate

experiments were conducted to evaluate the viability of selective recovery by gravimetric

techniques. A 1.5L column with three sampling ports at 3, 10 and 17 cm of height was

used to determine Ni and Co solids removal rate as a function of height and overflow

rate. Metal concentration and particle size distribution (PSD) were measured by atomic

absorption spectrophotometer (AAS) and AccuSizer 780 AD, respectively.

Batch experiments showed that Ni-enriched precipitates were larger when biogenic

sulphide was used, whereas Co-enriched precipitates did not exhibit any change in PSD

when using different sulphide sources. Acetate and phosphate were mainly responsible

56

for the presence of bigger Ni precipitates (7.80±0.52 μm and 12.28±0.75 μm of mean

size) over Co precipitates (3.70±0.27 μm and 6.18±0.79 μm of mean size), which could

prevent the formation of Ni fines and enhance its growth and settlement. On the other

hand, the presence of sulphate did not improve Ni removal efficiency by enlarging its

particle size (Table 1.1). Ni removal efficiency was always lower than that of Co when

single metal reacted with biogenic sulphide (Table 1.1), possibly because the

stoichiometric excess of sulphide could induce the formation of soluble Ni polysulphide

complexes (Lewis and van Hille 2006). However, when the initial Ni concentration

increased from 100 ppm to 500 ppm, Ni removal efficiency increased from 77.5±2.2% to

98.3±0.3%, indicating that higher Ni ion concentration could destabilise the Ni

polysulphide complexes, thus resulting in the formation of Ni precipitates. In addition, the

existence of Co-sulphide precipitates could also improve Ni removal efficiency due to co-

precipitation, as observed by D.Fortin et al. (1994) on co-precipitation of Ni-sulphide with

iron (Fe)-sulphide in sulphate reducing medium. Co, on the other hand, presented a

removal efficiency of >99% regardless of sulphide sources or initial concentration (Table

1.1).

Results of settling rate experiments indicated that Ni and Co solids can be more easily

separated with lower overflow rate and higher retention time, and Co presented a

relatively higher settling rate than Ni (80.9±0.3% for Ni and 88.3±0.5% for Co after 23h of

settlement in VFA-fed biogenic sulphide). Metal settling rate displayed differences

depending on the substrate used for the production of the biogenic sulphide. For

example, in leachate-fed biogenic sulphide, averaged metal solids settling rate was only

around two thirds of that in VFA-fed sulphide during a 23-hour period. Such differences

were mainly attributed to the residual phosphate contained in the biogenic sulphide that

affected the settlement. Overall results confirm that the main driver of the differences in

Co and Ni settling rate is the biogenic sulphide source, which in turn can be used for the

separate recovery of both metals. Further SEM-EDX analysis will help to identify the

chemical structure of the precipitates and thus, the other critical compounds that have an

influence on settling rate.

Figure 1.1. Schematic presentation of the experimental set-up for batch and column experiments

57

Table 1.1. Metal ions removal efficiency and mean particle size of metal precipitates in batch experiments,

with initial metal concentration of 500 ppm

Metal ions removal efficiency (%)* Mean size of metal precipitates (μm)*

Ni Co Ni Co

Ni(Co)+Na2S 93.01±5.10 99.98±0.001 2.50±0.24 4.95±0.08

Ni(Co)+Na2S+sulphate 90.55±1.27 99.90±0.07 9.90±1.22 3.09±0.35

Ni(Co)+Na2S+acetate 98.35±0.61 99.96±0.004 7.80±0.52 3.71±0.27

Ni(Co)+Na2S+phosphate 98.93±0.38 99.95±0.01 12.28±0.75 6.18±0.79

Ni(Co)+biogenic sulphide 98.29±0.34 99.85±0.02 22.29±0.08 8.37±0.37

*Average values of triplicate samples with their corresponding standard deviations.

References

Crittenden, J. C., R. R. Trussell, D. W. Hand, K. J. Howe and G. Tchobanoglous (2012). Gravity Separation.

MWH's Water Treatment: Principles and Design, Third Edition, John Wiley & Sons, Inc.: 641-725.

Crundwell, F., M. Moats, V. Ramachandran, T. Robinson and W. G. Davenport (2011). Extractive Metallurgy

of Nickel, Cobalt and Platinum Group Metals. Burlington, Burlington : Elsevier Science.

D.Fortin, G. S. a. T. J. B. (1994). "Nickel sulfide, iron-nickel sulfide and iron sulfide precipitation by a newly

isolated Desulfotomaculum species and its relation to nickel resistance."

FEMS_Microbiology_Ecology 14: 121-132.

Hatami, H., B. Bissonnette, Y. Choi, H. Dijkman and N. Verbaan (2010). Sulfide precipitation of copper and

zinc from dilute acidic solution using biologically produced diluted H2S gas. Conference of

metallurgics. Vancouver, Canada.

Lewis, A. and R. van Hille (2006). "An exploration into the sulphide precipitation method and its effect on

metal sulphide removal." Hydrometallurgy 81(3-4): 197-204.

58

Barium role in the pathway of the anaerobic digestion process

V.Wymana,b

, A.Serranoc, R. Borja

c, A. Jiménez

a, A. Carvajal

b, F. G. Fermoso*

c

a Universidad Pablo de Olavide, Carretera de Utrera, 1, 41013 Seville, Spain

b Universidad Técnica Federico Santa María, Avenida Vicuña Mackenna 3939 San Joaquín, Santiago, Chile.

c Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Avenida Padre García

Tejero, 4. 41012 Seville, Spain

*e-mail address: fgfermoso@ig.csic.es

Keywords: Anaerobic pathway; Barium; Hydrolysis; Methane production; Trace elements

Anaerobic digestion (AD) constitutes a technology for organic waste treatment and

valorisation trough energy generation in form of biogas. This process is carried out by

microorganisms with an anoxic metabolism which degrade organic matter releasing

methane (CH4), carbon dioxide (CO2) and other gases. In this process four stages are

distinguished: hydrolysis, acidogenesis, acetogenesis and methanogenesis (Appels et

al., 2008). Anaerobic digestion is a complex process which can be affected by several

factors. Among these factors, the lack of trace elements (TE) causes instability and even

the total failure of the process. The supplementation with iron (Fe), nickel (Ni), cobalt

(Co), molybdenum (Mo) in the digestion system had successfully manifested a promotion

in the anaerobic digestion performance with different substrates (Choong et al., 2016).

The presence of this elements was related with a decrease in volatile fatty acids (VFA)

concentrations and therefore with an increase in biogas production and biogas yield

(Nordell et al., 2016).

Barium (Ba) is an element belonging to IIA group and is considerate a TE. It is the least

studied of the heavier alkali and alkaline earth metals in terms of functionally replacing

elements in biological systems (Wackett et al., 2004). It’s effects on the enzyme that

catalyzes anaerobic digestion have not been reported in depth to date.According to this

background, it is proposed to study the influence of different concentrations of barium

dosage in the stages of the AD process using synthetic and a real complex substrates.

To achieve this objective, triplicates BMP assays were performed with different barium

supplementation dosages on substrates of cellulose, glucose, mix of short chain acids

(propionic, butyric and valeric) and sodium acetate, for the evaluation of hydrolysis,

acidogenesis, acetogenesis and methanogenesis stages, respectively (Angelidaki et al.,

2009). The influence of Ba+2 in a real complex substrate was evaluated over dried green

folder (DGF), which presented a particle size smaller than 355 µm. Barium was dosed as

barium chloride dihydrate (BaCl2·2H2O) in concentrations of 0, 2, 20, 200 and 2000

mgBa+2/L. Methane production was quantified by soda volume displacement.

The effects of Ba+2 dosages in accumulated methane production are shown in Figure 1.

For hydrolysis stage (Figure 1.a), the assays with the highest concentration of Ba+2

showed a strong inhibition respect to experiments with lower Ba+2 dosages.

Acidogenesis and acetogenesis stages (Figure 1.b and 1.c) showed final methane

productions similar for all the cases studied, varying in a short range from 292.41 to

342.25 mL CH4/gCOD. Finally, in the acetoclastic methanogenesis stage (Figure 1.d) it

is observed that the presence of Ba+2 decreased the maximum methane production at

Ba+2 dosages of 200 and 2000 mg Ba+2/L.

59

(a) (b)

(c) (d)

Figure 1. BMP assays curves obtained for synthetic substrates: (a) cellulose, (b) glucose, (c) mix of short

chain acids (d) sodium acetate.

Responses of DGF (Figure 2) were different in presence of Ba+2. Concentrations of 2

and 20 mgBa+2/L showed an improvement in the methane production respect BMP

without Ba+2 supplementation. Higher concentrations of Ba+2 resulted in a decrease of

the methane production. These differences could be attributed to an inhibition of the

activity of hydrolytic bacteria, which was affected at the higher Ba2+ dosages according to

Figure 1.a.

Figure 2. BMP assays curves obtained for dried green folder.

References

Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J.L., Guwy, A.J., Kalyuzhnyi, S., Jenicek, P., Van Lier, J.B., 2009. Defining the biomethane potential (BMP) of solid organic wastes and energy crops: A proposed protocol for batch assays. Water Sci. Technol. 59, 927–934.

Appels, L., Baeyens, J., Degrève, J., Dewil, R., 2008. Principles and potential of the anaerobic digestion of waste-activated sludge. Prog. Energy Combust. Sci. 34, 755–781.

Choong, Y.Y., Norli, I., Abdullah, A.Z., Yhaya, M.F., 2016. Impacts of trace element supplementation on the performance of anaerobic digestion process: A critical review. Bioresour. Technol. 209, 369–379.

Nordell, E., Nilsson, B., Nilsson P??ledal, S., Karisalmi, K., Moestedt, J., 2016. Co-digestion of manure and

60

industrial waste - The effects of trace element addition. Waste Manag. 47, 21–27. Romero-güiza, M.S., Vila, J., Mata-alvarez, J., Chimenos, J.M., Astals, S., 2016. The role of additives on

anaerobic digestion : A review 58, 1486–1499. Wackett, L.P., Dodge, A.G., Lynda, B.M., Ellis, L.B.M., 2004. Microbial Genomics and the Periodic Table

MINIREVIEW Microbial Genomics and the Periodic Table. Appl. Environ. Microbiol. 70, 647–655.

61

Fate of heavy metals over the anammox process treating the

liquid fraction digestate of OFMSW effluents: A case study

Pichel, A.*, Val del Río, A.*, Méndez, R.*, Mosquera-Corral, A.*

* Department of Chemical Engineering, Institute of Technology, Universidade de Santiago de

Compostela, E-15705 Santiago de Compostela, Spain

Abstract

The content of heavy metals (HMs) was evaluated within the partial nitritation-anammox (PN-An) process for

the operation of two lab-scale reactors (R1 and R2). The feeding consisted in the liquid fraction of digestate

(LFD) from an anaerobic digestion (AD) treatment of OFMSW, with (R2) and without (R1) ammonia stripping

prior to the AD step. The performance of the PN-An process indicated no inhibition due to HMs for both

reactors, with Al and Fe as the most significant HMs (in the range of 5-7 mg/L) and average dissolved

fractions of HMs of 36% and 72% for R1 and R2, respectively.

Keywords: heavy metals, OFMSW, anammox, inhibition, ammonia stripping

Introduction

The combination of the anaerobic digestion (AD) and partial nitritationanammox (PN-An)

processes is a sustainable and cost-effective alternative to remove organic matter and

nitrogen from high strength wastewater sources like landfill leachate or the organic

fraction of municipal solid wastes (OFMSW) (Ahmed, 2012). These effluents are

characterized by large concentrations of nitrogen, which can be reduced by further

ammonia stripping pre-treatment prior to the AD step (Ortega-Martínez, 2016), as well as

heavy metals (HMs) which exert inhibitory effects over the biomass activity and

constitute one of the major bottlenecks when treating such effluents (Renou, 2008; Bi,

2014). Previous research works addressed the anammox ability to withstand HMs

inhibition, e.g. via IC50 estimation by batch assays (Li, 2014). Nevertheless, few reports

studied HMs throughout the operation of biological reactors fed with industrial

wastewater (Chen, 2014; Zhang, 2015). This work addresses the fate and influence of

HMs in two lab-scale PN-An reactors fed with the liquid fraction of digestate (LFD) from

an AD treating of OFMSW.

Materials and Methods

Two sequencing batch reactors (R1 and R2) with a working volume of 1.4 L were

operated at 30-33 ºC. R1 was fed with a LFD1 from a previous AD step, while R2 was

fed with a LFD2 from previous ammonia stripping and subsequent AD steps. Raw and

filtered (0.45 μm) samples of the influent and effluent were collected at day 167 from R1

and R2, as well as biomass samples and an initially stored fraction of the inoculum. The

total and dissolved concentration of HMs (listed in Table 1) were determined by triplicate

through ICP-MS spectroscopy (Agilent 7700x). At this point, the total nitrogen removal

was 60% and 80% for R1 and R2, with nitrogen concentrations of 1.3 and 1.4 g N-NH4+/L

and biomass concentration of 1.5 and 2.8 g VSS/L, respectively.

Results and discussion

62

Table 1. Total concentration of HMs (μg/L) in LFD1 and LFD2 fed to R1 and R2 (respectively) at day 167 of

operation.

LFD1 LFD2 LFD1 LFD2 LFD1 LFD2 LFD1 LFD2

Fe 4946 ± 4 6137 ± 2 Ni 98.0 ± 4.2 286 ± 2 Co 20.7 ± 4.2 42.4 ± 1.1 Hg 2.5 ± 2.5 1.2 ± 4.1 Al 4124 ± 4 6427 ± 3 Cr 40.7 ± 4.6 115 ± 1 V 20.0 ± 3.9 50.8 ± 1.6 Cd 1.9 ± 9.8 1.7 ± 9.8 Zn 451 ± 4 371 ± 2 As 56.3 ± 6.0 95.8 ± 1.0 Mo 11.2 ± 11.8 14.2 ± 2.8 Be 0.5 ± 26.4 0.3 ± 24.3 Cu 372 ± 4 225 ± 1 Mn 161 ± 5 137 ± 1 Se 6.4 ± 16.8 27.3 ± 7.8 Ti 235 ± 5 413 ± 3 Pb 41.3 ± 1.9 49.0 ± 1.0 Ag 7.3 ± 3.8 5.1 ± 6.8

The concentrations of the HMs in the studied LFD effluent were in the observed range

for OFMSW-like effluents (Renou, 2008), except for Al and Fe which were above the

reported range. Several HMs frequently studied along with the anammox process like

Cu, Zn, Cr and Ni were below the reported IC50 values (Li, 2014).

Figure 1. Concentration of relevant HMs at day 167 of operation: (a), (b) in the influent (inf) and effluent

(eff) of R1 and R2; (c), (d) in the biomass of R1, R2 and in the inoculum.

The dissolved fraction of HMs is known to provoke inhibition over the anammox bacteria

(Li, 2014). In this case, the dissolved fraction of the HMs between the influent and

effluent was very similar in both reactors, with the exception of Cu and Ni in the influent

of R1 which increased by 89% and 47%, respectively (Figure 1.a). The average fraction

of dissolved HMs for LFD2 (72%) was higher than for LFD1 (36%) (e.g. the case for Al in

Figure 1.b), although no related inhibitory effects were observed in R2, probably due to

the low concentration of HMs (Table 1).

Regarding the accumulation of HMs in the biomass of both reactors, the HMs at low

concentration levels (μg/g) had the highest increase compared to the inoculum (like Pb,

Cr and Ni, Figure 1.c). However, the HMs present in higher concentrations (mg/g) are

expected to have a more significant effect despite the lower differences with the

inoculum (like Al and Zn, Figure 1.d). Nevertheless, no detrimental effects due to HMs in

higher concentrations were observed over the PN-An performance.

In conclusion, the PN-An process treating the LFD of an OFMSW effluent was capable of

withstanding the presence of HMs at the measured concentrations, including the case

where an ammonia stripping step is implemented prior to the AD. This indicates its

applicability for treating this kind of effluents regarding HMs content.

63

Acknowledgements

LIFE METHAmorphosis (http://www.life-methamorphosis.eu) consortium would like to

thank the European Commission for her support through LIFE financial instrument

(LIFE14/CCM/ES/000865).

References

Ahmed, F.N.; Lan, C.Q. (2012) Treatment of landfill leachate using membrane bioreactors: A review. Desalination, 287, 41-54.

Bi, Z.; Qiao, S.; Zhou, J.; Tang, X.; Cheng, Y. (2014). Inhibition and recovery of Anammox biomass subjected to short-term exposure of Cd, Ag, Hg and Pb. Chem. Eng. J., 244, 89-96.

Chen, H.; Yu, J.J.; Jia, X.Y.; Jin, R.C. (2014). Enhancement of anammox performance by Cu(II), Ni(II) and Fe(III) supplementation. Chemosphere, 117, 610-616.

Li, G.; Puyol, D.; Carvajal-Arroyo, J.M.; Sierra-Álvarez, R.; Field, J.A. (2014). Inhibition of anaerobic ammonium oxidation by heavy metals. J. Chem. Technol. Biotechnol., 90(5), 830-837.

Ortega-Martínez, E.; Sapkaite, I.; Fernández-Polanco, F.; Donoso-Bravo, A. (2016) From pre-treatment toward inter-treatment. Getting some clues from sewage sludge biomethanation. Bioresour. Technol., 212, 227-235.

Renou, S.; Givaudan, J.G.; Poulain, S.; Dirassouyan, F.; Moulin, P. (2008). Landfill leachate treatment: Review and opportunity. J. Hazard. Mater., 150, 468-493.

Zhang, Z.Z.; Zhang, Q.Q.; Xu, J.J.; Shi, Z.J.; Guo, Q.; Jiang, X.Y.; Wang, H.Z.; Chen, G.H.; Jin, R.C. (2016). Long-term effects of heavy metals and antibiotics on granule-based anammox process: granule property and performance evolution. Environ. Biotechnol., 100(5), 2417-2427.

64

Copper plant uptake from liquid digestate – influence of the

presence of enrofloxacin

S. Sayen*, E.Vulliet**, E. Guillon*, C. M. R. Almeida***

* Institut de Chimie Moléculaire de Reims (ICMR), UMR CNRS 7312, Université de Reims Champagne-Ardenne, BP 1039, Reims Cedex 2, France.

** Université de Lyon, Institut des Sciences Analytiques, UMR 5280 CNRS, Université Lyon 1, ENS-Lyon, 5 rue de la Doua, Villeurbanne, France.

*** Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, Matosinhos, Portugal.

Keywords: plant uptake; metal; digestate; copper; antibiotic

Sludges originate during biological and chemical processes in wastewater treatment

plants and may contain a wide spectrum of organic and inorganic substances such as

metal trace elements and pharmaceuticals1. Large amounts of sludges are produced

worldwide and necessitate disposal every year. As they are rich in nutrients and organic

matter, their application to agricultural fields is commonly used as a soil amendment.

This practice may bring additional benefit to soil and crop systems and reduce the need

for fertilizer application, however, it may also pose environmental risks by introducing

various pollutants -including metal trace elements and pharmaceuticals- to amended

soils and the underlying groundwater.

In most cases sludges must be first stabilized to decrease their risk to introduce

pollutants, and anaerobic digestion is one of the most widely used stabilization

technologies. Moreover, the high cost of sludge management and the growing interest in

alternative energy sources have prompted proposals for different strategies to optimize

biogas production during anaerobic sludge treatment2. However, there is a lack of

information about these treated sludges by anaerobic digestion (digestates) and their

potential environmental impacts or benefits connected to agronomic reuse or disposal.

Pharmaceuticals and metal trace elements can be both present in digestates and they

are likely to interact by coordination complex formation which may modify their reactivity,

behaviour in soils, bioavailability, and plant uptake3,4.

Thus, it seems very important to consider the impact of pharmaceuticals presence on

metal uptake by plants from digestates. This is the purpose of this presentation which

focuses on the uptake by plants of a metal (copper) from a liquid digestate in the

absence and in the presence of a pharmaceutical (enrofloxacin). These two pollutants

are selected since they can be both present in digested sludges and are known to

interact together via complex formation5. The main objectives are to investigate (i)

whether the presence of digestate modify the uptake of copper and enrofloxacin by

plants and (ii) if there is a change in the metal and enrofloxacin plant uptake by the

presence of each other.

Phragmites australis plants were collected in a river estuary in Portugal and an

agricultural digestate was obtained from a French farm. For plant uptake experiments,

three concentrations of digestate were considered, and for each digestate concentration,

three systems were investigated: (i) plants in presence of copper, (ii) plants in presence

of copper and enrofloxacin, and (iii) plants in presence of enrofloxacin, in order to study

the influence of the antibiotic presence in the digestate on copper uptake by plants.

65

The determination of copper amounts remaining in the digestate and taken up by plants

was performed using atomic absorption spectroscopy after acidic high pressure

microwave digestion. In a same way, the amounts of enrofloxacin in the digestate and in

plant tissues were determined using liquid chromatography after a pre-concentration and

clean up step.

During experiments, vessels were wrapped in aluminum foil to prevent the exposure of

roots and digestate solutions to light and to avoid enrofloxacin photodegradation.

However, the contents of ciprofloxacin (metabolite of enrofloxacin) were also measured

in solution and in plant tissues in order to follow the eventual enrofloxacin degradation

during plant uptake.

References

1 P. Verlicchi, E. Zambello, Pharmaceuticals and personal care products in untreated and treated sewage

sludge: Occurrence and environmental risk in the case of application on soil - A critical review,

2015, Sci. Total Environ., 538, 750-767. 2 P. Neumann, S. Pesante, M. Venegas, G. Vidal, Developments in pre-treatment methods to improve

anaerobic digestion of sewage sludge, 2016, Rev. Environ. Sci. Biotechnol., 15, 173-211. 3 D. Jia, D. Zhou, Y. Wang, H. Zhu, J. Chen, Adsorption and cosorption of Cu(II) and tetracycline on two soils

with different characteristics, 2008, Geoderma, 146, 224-230. 4 M. Graouer-Bacart, S. Sayen, E. Guillon, Macroscopic and molecular approaches of enrofloxacin retention

in soils in presence of Cu(II), 2013, J. Colloid Interface Sci., 408, 191-199. 5 H. Ftouni, S. Sayen, S. Boudesocque, I. Dechamps-Olivier, E. Guillon, Structural study of the copper(II)-

enrofloxacin metallo-antibiotic, 2012, Inorg. Chim. Acta, 382, 186-190.

66

Bioaugmentation with autochthonous microbial consortia to potentiate phytoremediation of cadmium contaminated

sediments

Ana P. Mucha *, Catarina Teixeira*, C. Marisa R. Almeida *

*Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal

Keywords: autochthonous bioaugmentation; microbial consortia; cadmium contamination; phytoremediation;

salt-marsh sediments

Recovering of estuarine environments is in need and microbial assisted

phytoremediation is a promising, though yet poorly explored, new remediation technique.

This technology takes advantage of naturally present plant-microorganisms association

and could be a valid option in order to reduce pollution while preserving natural

biodiversity.

The aim of this study was to develop and characterize autochthonous microbial consortia

resistant to cadmium that could enhance phytoremediation of salt-marsh sediments

contaminated with this metal. The microbial consortia were selectively enriched (Teixeira

et al. 2014) from rhizosediments colonized by Juncus maritimus and Phragmites

australis. In parallel, a microbial consortium was also enriched from non-vegetated

sediment in order to investigate the effect of bioaugmentation on metal mobility,

independently of the presence of plants. The bacterial community present in the initial

sediments and in the different microbial consortia was characterized by automated rRNA

intergenic spacer analysis (ARISA) and by next generation sequencing using the

pyrosequencing platform 454. The effect of the bioaugmentation with the developed

consortia on cadmium speciation and uptake by plants was assessed in microcosm

experiment carried out in greenhouses with an automatic irrigation system that simulated

estuarine tidal cycles.

Bacterial community of the initial rhizosediments was more similar to each other than to

that of non-vegetated sediment. Nevertheless, the consortium obtained from the P.

australis rhizosediment was more similar to the consortium from the non-vegetated

sediments than from the one obtained from the J. maritimus rhizosediment (Figure 1).

Despite the differences in the community structure, the different consortia presented

similar microbial abundance and were all dominated by the genus Sporolactobacillus

(53-69%). Results from the microcosm experiments showed that bioaugmentation with

the respective microbial consortia changed metal speciation, decreasing Cd

bioavailability in P. australis rhizosediment and in non-vegetated sediment, but not in J.

maritimus rhizosediment (Nunes da Silva et al. 2014). Moreover, the addition of the

microbial consortium increased cadmium accumulation in J. maritimus roots and

rhizomes, enhancing its phytostabilization potential, and promoted cadmium

translocation to P. australis stems, increasing its phytoextraction potential.

This study provides new evidences that the development of autochthonous microbial

consortia for enhanced phytoremediation of metals might be a simple, efficient, and

67

environmental friendly remediation procedure for the recovery of moderately impacted

sediments.

Figure 1.1. Cluster analysis based on Bray - Curtis similarities of ARISA fingerprints of microbial communities and the relative abundance of the different bacterial groups (relative abundance > 2%) in the initial sediments (initial) and in the microbial consortia obtained for bioaugmentation (bioaug) (J - J. maritimus rhizosediment, P - P. australis rhizosediment, S – non-vegetated sediment),.

References

Teixeira C, , Nunes da Silva M., Rocha A.C., Gomes C.R., Almeida C.M., Mucha A.P. (2014). Development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium. Science of the Total Environment, 493, 757–765.

Nunes da Silva M., Mucha A.P., Rocha A.C., Teixeira, C., Gomes C.R., Almeida C.M.R. (2014). A strategy to potentiate Cd phytoremediation by saltmarsh plants - autochthonous bioaugmentation. Journal of Environmental Management, 134, 136-144.

68

69

Short communications

70

71

Use of trace elements addition for anaerobic digestion of brewer’s spent grains

C. Bougrier*, D. Dognin*, C. Laroche* and J.A. Cacho Rivero*

*Veolia Recherche & Innovation, zone portuaire de Limay, 291 avenue Dreyfous Ducas, Limay F-78520, France

(E-mail: claire.bougrier@veolia.com)

Keywords: Anaerobic digestion; Brewer’s spent grains; Supplementation; Performance

The brewery industry generates large amount of by-products and notably Brewer’s Spent Grains (BSG). Anaerobic digestion seems to be a promising option to convert BSG into energy although a previous study by Sežun et al. (2011) has shown that anaerobic digestion of BSG as mono substrate cannot be successful. Other lignocellulosic agricultural feedstock, like maize silage, are anaerobically digested only when nutrients and trace elements are added. Addition of trace elements ensures stable performance (Bougrier et al., 2013). The aim of the study was to evaluate the effect of trace elements addition on the anaerobic degradation of 4 different BSG samples.

Mesophilic anaerobic digestion was conducted in 4L lab-scale reactors with a hydraulic retention time of 40 days. The 4 BSG samples used had similar characteristics and a 2.2 to 2.5-fold dilution with tap water was realised in order to comply with process recommendations (feeding with VS content around 90 g.L-1 and N content below 5 g N.L-

1). Feeding was manually done 3 times a week dividing proportionally the weekly load. Process performance was evaluated by measuring biogas production and digestate quality. Macro and trace elements content was measured in the soluble phase. As initial trace elements concentrations in all 4 BSG samples were similar, the same additive solutions were used in all cases. Three additives solutions were defined according to a literature review: without macro and trace elements (Control), based on the lowest concentration and considering a bioavailability of 50% of trace elements already present in the BSG (LC: Low Concentration) and based on the highest concentration and considering 0% bioavailability of trace elements present in the BSG (HC: High Concentration).

Table 1.1 presents the main performance observed with the four different BSG samples. For all BSG samples, except M, Control reactors failed before 3 months of operation. pH decreased while VFA content increased and methane production stopped. For the M sample, although the Control reactor did not fail during the 4 months of operation, it showed lower performance compared to the others, especially in terms of methane yield. Therefore, additive solution is necessary to maintain stable biodegradation of BSG.

In terms of performance, no significant differences were observed between LC and HC reactors for all four BSG samples. Considering global performance, COD removal was around 60-65%, soluble COD content was 10% except for G sample (VFA concentration was high due to feeding troubles during holidays) and methane yield represented more than 80% of the measured BMP (Biological Methane Potential), except for H sample (only 70%).

72

Table 1.1. Impact of trace elements solution addition on the average performances for the BSG samples

Reactor COD rem

(%)

sCOD/tCOD

(%)

[VFA]

(g.L-1

)

YCH4

(NLCH4.kg-1

CODin)

Y’CH4

(NLCH4.kg-1

VSin)

G-CTL Not stable after 3 months

G-LC 62 ± 3% 22 ± 6% 1 110 ± 667 227 ± 12 282 ± 14

G-HC 58 ± 5% 18 ± 5% 1 026 ± 578 229 ± 20 285 ± 25

H-CTL Not stable after 2 months

H-HC 64 ± 2% 10 ± 2% 78 ± 49 188 ± 17 235 ± 22

M-CTL 56 ± 4% 16 ± 1% 2 131 ± 229 190 ± 38 240 ± 48

M-LC 60 ± 2% 11 ± 2% 246 ± 234 246 ± 16 311 ± 20

M-HC 59 ± 6% 9 ± 2% 91 ± 58 248 ± 16 314 ± 20

S-CTL Not stable after 3 months

S-LC 66 ± 4% 12 ± 2% 183 ± 272 266 ± 12 374 ± 17

S-HC 67 ± 3% 12 ± 2% 171 ± 254 247 ± 13 348 ± 18

Based on these results, LC solution could be considered efficient enough to maintain stable anaerobic digestion of BSG with good performance. However, considering each element separately the behaviour was different. Table 1.2 presents the concentrations of each added element in the soluble phase compared to the recommendations found in the literature. Grey box represents concentrations below the recommendations. Based on these results the definition of a new solution was possible.

Table 1.2. Fate of trace elements during digestion: digestate soluble fraction concentrations

Reactor

Ca Mg K Na Fe Cu Mn Co Ni

(mg.L-

1)

(mg.L-

1)

(mg.L-

1)

(mg.L-

1)

(mg.L-

1)

(mg.L-1

) (mg.L-1

) (mg.L

-

1)

(mg.L-

1)

Conc.

range

in

literature

75-200 75-300 100-

600 50-200 1-10

0.05-

0.30

0.50-

1.50

0.05-

0.50

0.05-

0.50

G40-LC 320 45 83 63 12 1.15 1.13 0.04 0.15

G40-HC 327 44 97 77 17 1.05 1.27 0.20 0.33

HKN40-HC 267 37 99 71 18 1.12 0.49 0.24 0.14

MHU40-LC 80 22 45 34 6 0.08 0.33 0.02 0.02

MHU40-HC 96 31 59 43 8 0.12 0.56 0.08 0.05

SMG40-LC 73 14 76 33 3 0.14 0.20 0.02 0.01

SMG40-HC 80 14 102 43 3 0.17 0.30 0.10 0.05

Magnesium and nickel should be added at HC concentration. Medium concentrations were suggested for potassium and cobalt. As sodium is not one of the major macro elements, LC concentration was enough even if Na content in digestate was lower than recommended. For calcium, iron, copper and manganese, low addition was efficient. Calcium was added in the tap water used for dilution (no extra Ca in LC solution). Therefore, on an industrial plant, addition might be needed depending on the dilution strategy applied.

References Bougrier C., Dognin, D., Laroche, C. and Cacho-Riveiro, J.A. (2013) Impact of nutrients addition on

biological stability of mono-substrate anaerobic digestion of maize silage. AD14, Viña del Mar (Chili). Sežun, M., Grilc, V., Zupančič, G.D. and Marinšek Logar, R. (2011) Anaerobic digestion of brewery spent

grains in a semi-continuous bioreactor: inhibition by phenolic degradation products. Acta Chimica Slovenica, 58, 158-166.

73

Is there trace metal deficiency in anaerobic membrane bioreactor

(AnMBR) for municipal wastewater treatment?

A.Ilic*, P.Dolejs*, M.Polaskova**, J.Bartacek*,

*Department of Water Technology and Environmental Engineering, Technicka 5, 166 28 Prague 6, Czech

Republic

**Research and development division of ASIO, spol. s r.o, Kšírova 552/45, 619 00 Brno, Czech Republic

Keywords: Trace metals; AnMBR; Municipal wastewater; BMP

Introduction

The application of anaerobic digestion for municipal wastewater treatment has gained

more attention in the last decade, since it can recover chemical energy from the

wastewater in the form of biogas, which can be further utilized for heating or the

production of electricity. The other main advantages of anaerobic over aerobic

wastewater treatment are its capability to treat higher organic loads and smaller amount

of sludge produced. As an emerging technology for wastewater treatment, an anaerobic

membrane bioreactor (AnMBR) can provide effluent with high quality and the possibility

for nutrient recovery as a post treatment.

The research on the influence of the main trace metals (TM) (Fe, Co, Ni, Zn, Mn and Cu)

on anaerobic digestion systems such as Upflow Anaerobic Sludge Blanket (UASB) and

Expended Granular Sludge Bed (EGSB) reactors has been reported (Fermoso et al.

2009, Zandvoort et al. 2006). However, a system such as AnMBR, treating municipal

wastewater, has not been thoroughly studied in terms of trace metal content and

bioavailability. This research focuses on the bioavailability of trace metals in a pilot

AnMBR treating municipal wastewater from Pilsen (the Czech Republic) since February

2017. We assess the limitation of the methanogenic potential of anaerobic sludge by the

lack of trace metals and the speciation of trace metals in the solid and liquid phase of the

reactor.

Materials and methods

The AnMBR pilot plant description

The AnMBR pilot plant is located in the area of WWTP in Pilsen, with the inflow water

being pumped after the preliminary treatment. The reactor consists of one tank (reactor

volume 2886 L) with two submerged micro- und ultrafiltration membranes, each of them

having separate outflow tank (Figure 1.1). The mixing in the tank is achieved by the

recirculation of digester content. The hydraulic retention time is at the moment 3 d, with

planned decrease up to 12 hours. The reactor is operated under psychrophilic conditions

i.e., at the temperature of the inflow wastewater, (5°C to 20°C).

Biomethane Potential (BMP) assay

In order to determine if there is a deficiency of trace metals in the system, BMP assay

test will be performed (Angelidaki et al. 2009). The BMP of the wastewater in a sample

containing a mixture of trace metals (Fe, Co, Ni, Zn, Mn and Cu) will be compared to a

sample without the mixture. Each sample will contain raw wastewater as a substrate and

sludge from bioreactor as an inoculum.

74

The BMP test will be done in triplicate, under mesophilic (37°C) and psychrophilic (20°C)

conditions. The BMP will be assessed by volumetric measurement of the produced

biogas.

If the BMP tests show increased production of methane as a result of trace metal

addition, further batch tests, with single trace metals supplementation will be done to

gain deeper understanding of trace metal requirement for the given system. Moreover,

the bioavailability of trace metals in the raw wastewater will be determined, since trace

metal supplementation experiments will depend on that factor also. Trace metal

speciation will be assessed with sequential extraction (solid phase) and with the DGT

technique (liquid phase).

Figure 1.1. Schematic of the pilot AnMBR unit treating municipal wastewater

Expected outcome

The main outcome of the research is to determine whether the supplementation of the

TM to the AnMBR for municipal wastewater treatment will enhance methane production.

The influence of temperature in the system on the methane production in terms of TM

addition will also be assessed.

Acknowledgments

This work was accomplished with the financial support of the European Commission

through the Joint Doctorate SuPER-W programme (Sustainable Resource, Product and

Energy Recovery from Wastewater).

References

Fermoso, F. G., Bartacek, J., Jansen, S., & Lens, P. N. L. (2009). Metal supplementation to UASB bioreactors: from cell-metal interactions to full-scale application. Science of The Total Environment, 407(12), 3652–3667.

Zandvoort, M. H., van Hullebusch, E. D., Gieteling, J., & Lens, P. N. L. (2006). Granular sludge in full-scale anaerobic bioreactors: Trace element content and deficiencies. Enzyme and Microbial Technology, 39(2), 337–346.

Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J. L., Guwy, A. J.Pavel, Lier, J. B. van. (2009). Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays. Water Science and Technology, 59(5), 927–934.

75

Improvement of anaerobic digestion systems co-digesting food waste and pig manure with addition of trace metals

Yan Jiang*, Conor Dennehy*, Peadar G. Lawlor**, Gillian E. Gardiner***, Xinmin Zhan*

*Civil Engineering, College of Engineering & Informatics, National University of Ireland, Galway, Ireland **Teagasc, Pig Development Department, Animal and Grassland Research and Innovation Centre,

Moorepark, Fermoy, Co. Cork, Ireland ***Department of Science, Waterford Institute of Technology, Waterford, Ireland

* Corresponding author’s e-mail: xinmin.zhan@nuigalway.ie

Keywords: Selenium; molybdenum; co-digestion; methane production rate

INTRODUCTION Trace metals (TM) are essential cofactors in enzymes, which play important roles

in anaerobic digestion systems. A deficiency of TM may affect the synthesis of enzymes and limit the growth of methanogens, so as to limit the microbial activity and even result in process failure (Thanh et al., 2016). In this study, selenium (Se) and molybdenum (Mo) were selected to assess the effect of TM on co-digestion of food waste (FW) and pig manure (PM).

MATERIALS AND METHODS

FW was collected from 5 local residents in Galway, Ireland. Prior to use, FW was ground to less than 2 mm and diluted 5 times (w/w) using tap water. PM was taken from the storage tank of a local farm and was stored at 11oC before use. Sludge was obtained from an anaerobic digester in a local wastewater treatment plant (WWTP). The inoculum and substrates (mixture of FW and PM) ratio was 3:1 based on volatile solids (VS), and the FW and PM ratio was 1:1 on VS base.

Four 10 L stainless steel reactors were operated with the working volume of 8 L. The reactors were fed and discharged every weekday. Agitation was conducted for half an hour every two hours at the speed of 100 rpm. The hydraulic retention time (HRT) was 25 days and the temperature was controlled at 35 oC. The experiment was conducted in duplicate, R1 and R2 worked as control without TM addition. The initial concentrations of both Se and Mo were adjusted to 0.2 mg/L in R3 and R4, and feedstock containing 0.2 mg/L Se and Mo was fed every weekday. Biogas was collected every day by sampling bags to measure CH4 (%), CO2 (%) and H2S (ppm). Digestate was taken twice a week for analysis of total solids (TS), VS, soluble chemical oxygen demand (SCOD), volatile fatty acid (VFA), total ammonia nitrogen (TAN), alkalinity and phosphate.

RESULTS AND DISCUSSION

The reactors have been operated for 30 days, and the methane production is shown in Figure 1.1. The methane production rate fluctuated, but the values in TM systems were significantly higher than those in control systems (p=0.007). The systems haven’t become stable yet.

76

Figure 1.1. Variations of methane production rate in control and TM systems

The statistical analyses of the parameters in control and TM systems are shown in Table 1.1.

Table 1.1 Statistical analyses of the parameters in control and TM systems

Control TM P-value

CH4 production rate (mL/h) 62±49 97±38 0.007

H2S (ppm) 1463±268 1389±349 0.601

pH 7.51±0.11 7.44±0.08 0.168

SCOD (mg/L) 4042±2360 3545±2421 0.704

VS removal rate (%) 46.7±4.4 42.7±3.1 0.138

Alkalinity (mg/L) 7984±803 8096±834 0.789

TAN (mg/L) 1798±303 1798±312 0.999

Significant difference was only observed in methane production rate, which was 56.5% higher in TM system than that in control system. There were no significant differences in all the other parameters between the two systems, including H2S concentration, pH, SCOD, VS removal rate, alkalinity and TAN. It possibly indicated that TM mainly affected methanogenesis during co-digestion of FW and PM. Se and Mo were reported to be found in enzymes such as formate dehydrogenase (FDH), which can relieve the accumulation of propionate in digesters (Banks et al., 2012). However, since the reactors are not stable yet, it is too early to make strong conclusions. The detailed mechanism will be studied in future experiment.

CONCLUSION

Addition of Se and Mo in FW and PM co-digestion systems significantly increased the methane production rate by 56.5%.

References Banks, C.J., Zhang, Y., Jiang, Y., Heaven, S. 2012. Trace element requirements for stable food waste

digestion at elevated ammonia concentrations. Bioresour Technol, 104, 127-35. Thanh, P.M., Ketheesan, B., Yan, Z., Stuckey, D. 2016. Trace metal speciation and bioavailability in

anaerobic digestion: A review. Biotechnol Adv, 34(2), 122-36.

77

Balance of Selected Trace Elements at Cup-plant Cultivation for

Biogas Production Compared to Reference Maize

S. Ustak, J. Munoz

Crop Research Institute, Drnovska 507/73, 16106 Prague 6 – Ruzyne, Czech Republic

Keywords: biogas production; cup-plant; maize; nutrient balance; digestate fertilizing.

Material and methods

As a non-traditional crop suitable for biogas production was selected cup-plant (Silphium

perfoliatum L.) and as a reference crop maize. Unlike maize, cup-plant forms a perennial

vegetation. In our trials was proven cup-plant vegetation long life, namely 15 - 20 years

or more. At both crops were obtained at least 3-years data about yields within parcel field

trials in 3 different variants of nitrogen fertilization (50, 100 and 150 kg N per 1 ha) and at

single fertilization of P and K (50 kg/ha of P2O5 and K2O). The plant yield, chemical

analysis and fermentation testing were carried out based on individual crops and

fertilization variants. All analyses and experiments were performed in 3 repetitions. When

dry matter (d.m.) of above-ground phytomass at harvest was less than 25 %, such crops

were ensilaged in fresh state and after withering 24 hours. The ensilaging fermentation

tests were performed by laboratory mini-silos with capacity of 10 L. The ensilaging time

was set at 90 days.

The assessment of biogasification followed the silage experiments. Laboratory tests

were conducted on the assembly of 48 pieces of 3-liter glass anaerobic fermenters at

temperature of 37 ± 1 °C and stirred for 15 minutes every 2 hours. Biogas production

(methane) testing used methodology VDI 4630. The input ratio of organic matter to the

inoculum was 3:10. The inoculum was adjusted fermentate from operating BGS, which

processes animal excrements, maize and grass silage in ratio 40:40:20. The total period

of biogas digestion was set at 49 days. The chemical analyses were performed based on

common procedures.

Results and Discussion

The cultivation of agricultural crops for green biomass to biogas production, or for green

forage, is associated with higher macro- and micro-nutrients uptake than harvesting for

dry matter to combustion. The annual nutrient uptake of phytomass and soil nutrient

content are the basis to determine compensatory fertilization doses. The comparison of

nutrients application related to the limit of average dose in the Czech Republic for

digestate from agricultural BGS (10 t of d.m. in total at 3-years), and the need of

compensatory fertilization (nutrient uptake) in cut plant yield average of 15 tons of d.m.

from 1 ha, shows that digestate substitutes a big part of compensatory fertilization at

essential nutrients. For example, fertilization by digestate with d.m. average of 8 % at

dose of 42 t/ha per year, corresponding to 10 t/ha of d.m. for 3 years, covers more than

90 % of phosphorus nutritional requirements at cut plant, and more than 80 % of nitrogen

and potassium. The results showed a low coverage needs of sulphur, calcium and

magnesium (ranging 30 – 45 %).

78

Table 1. Nutrient supply ratio from digestate (10 t of d.m. per 3-years) and uptake by the above-ground

biomass of cup-plant (for average yield 15 t/ha of d.m.).

Parameter Amount of essential nutrients (kg/ ha) N P2O5 K2O CaO MgO S

Uptake with a yield of cup-plant green biomass of 15 t/ha of d.m. 176 92,8 368 409 103 12 Digestate fertilizing supply (annual dose of 42 t/ha, 8 % of d.m.) 151 87 303 153 46 4 Uptake difference and nutrient supply (kg/ha) -25 -6 -65 -256 -57 -8 Digestate share of total compensatory nutrient requirements (% ) 85,8 93,7 82,2 37,4 44,6 32,5

In table 2 is presented selected trace elements (micro-nutrients) average content in cut

plant above-ground biomass for comparison to maize. We see that cup-plant has higher

requirements on micro-nutrients, except on Cu and Zn. The differences are in B uptake

(9 times higher), followed by Co (5 times), Fe and Mn (2 times). Therefore, we expect a

positive yield using these micro-nutrients.

Table 2. Average content of selected trace elements in crops.

Crop Average content (mg/kg of dry phytomass)

B Fe Mn Co Cu Mo Ni Zn Maize (K) 3,7 72 25 0,07 11,5 0,36 0,64 22,5 Cup-plant (M) 31,7 136,1 41,0 0,32 7,22 0,52 0,85 15,8 M : K ratio 8,6 1,9 1,6 4,6 0,6 1,4 1,3 0,7

As forage crop, cup-plant stands out at growth stages with high feed quality, although

lower in comparison to maize (tab. 3). Thanks to above-ground biomass high yield and

chemical composition, is a promising high-forage production crop and suitable for biogas

production. The specific methane production is high, only about 6 – 8 % lower compared

to maize. The methane yield per 1 ha ranges from 3.6 to 4.6 thousands of Nm3, often

comparable to maize with methane yields from 3.6 to 5.6 thousands of Nm3/ha.

Table 3. The basic yield parameters, biomass quality and methane production of the cup-plant compared

with maize (trials of CRI, long-time average).

Parameter Cup-plant Maize Green biomass yield (t/ha) 62,8 49,8 d.m. content (%) 24,6 30,2 d.m. yield (t/ha) 15,4 15,0 Raw ash (% of d.m.) 8,04 4,2 Yield of organic d.m. (t/ha) 14,3 14,4 Specific methane production (Nm

3/t of d.m.) 256 282

Methane yield (Nm3/h) 3955 4241

Raw protein (% of d.m.) 7,5 11,6 BNVL – extracted nitrogen-free substances (% of d.m.) 38,5 46,9 – of that reduced sugars (% of d.m.) 6,25 15,3 Lipids (% of d.m.) 2,63 4,04 Raw fibre (% of d.m.) 26,4 21,4

The results highlight that cup-plant biomass addition to maize at biogasification improve

the balance of micro-nutrients for methane yield (B, Co, Fe, and Mo). Therefore, this

crop is recommended as suitable supplement into the raw materials composition of

agricultural BGS for the partial substitution of maize.

79

The favorable ecological effect of cup-plant cultivation is related to its perennial

character, ensuring erosion control and soil conservation effect. Based on long-time

results, cup-plant is recommended as suitable replacement of maize at biogas

production.

Acknowledgment

The article was developed within the project of Czech Ministry of Education No.LD15164

and the Czech Ministry of Agriculture No.RO0416.

80

Full-scale agricultural biogas plant metal content and process parameters in relation to bacterial and archaeal microbial

communities over 2.5 year span

Sabina Kolbl1, Domen Zavec2, Katarina Vogel Mikuš3, Fernando Fermoso4, Blaž

Stres2,5

1 University of Ljubljana, Faculty of Civil and Geodetic Engineering, Hajdrihova 28, SI-1000

Ljubljana, Slovenia

2 University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Group for

Microbiology and Microbial Biotechnology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia

3 University of Ljubljana, Biotechnical Faculty, Department of Biology, Chair of Botany and Plant

Physiology

4 Instituto de la Grasa (C.S.I.C.). Avda. Padre García Tejero, 4. 41012-Sevilla, Spain

5University of Ljubljana, Faculty of Medicine, Vrazov trg 2, 1000 Ljubljana, Slovenia

A start-up of 4 MW agricultural biogas plant in Vučja vas, Slovenia, was monitored from

2011 to 2014. The start-up was carried out in 3 weeks with the intake of biomass from

three operating full-scale 1-2 MW donor agricultural biogas plants. The samples were

taken (i) from donor digesters at the start-up, (ii) at different time points (days 0-70; days

521-547; days 928-958) (n=13) from two serial digesters (F1-F2) and (iii) at one time

point form all 6 serial digesters (F1-F6).

Biogas plant process was monitored through measurements of produced biogas,

electrical energy, operating temperature, pH, total soluble organic carbon (tSOC), UV-

VIS absorption spectra of aromatic compounds, analyses of total short chain fatty acid

(SCFA) content and their respective profiles. The content of trace metals was

determined using XRF profiling. In addition, a fast molecular profiling technique T-RFLP

was used to assess the changes in bacterial and archaeal microbial community profiles

over time (Kolbl et al., 2017).

The changes in microbial community were more rapid during the phase of the start-up

than at later phases. Microbial communities where functionally stable and produced

biogas throughout the whole observed time frame, whereas SCFA were the most

variable parameter. Microbial community diverged from the composition of donor

digesters due to different environmental conditions over time, however, the

physiochemical parameters and microbial community at the same time did not differ

between serial digesters F1 and F2. Concentration of acetate was highly associated with

microbial community changes over time, followed by tSOC. In contrast, archaeal

communities showed little connection with these parameters although the archaeal and

bacterial community dynamics was correlated. Surprisingly, the digesters F1 to F6

showed little differences in the structure of microbial community and other

physiochemical parameters at the same time, except SCFA.

Variation partitioning was conducted in order to identify the most important parameters

associated with changes in microbial communities. The results show that a large fraction

of variability in bacterial and archaeal microbial communities (>55%) remained

unexplained although the biogas production process was not affected. This shows that

81

additional parameters would need to be measured, possibly at different scales, in order

to fully elucidate the variables responsible for reorganization of microbial communities

beyond random noise. The results also show that reorganization of microbial

communities does is not necessarily directly associated with impact on performance in

full scale biogas reactors.

References

Kaulich B, Gianoncelli A, Beran A, et al. Low-energy X-ray fluorescence microscopy opening new

opportunities for bio-related research. Journal of the Royal Society Interface. 2009;6(Suppl 5):S641-

S647. doi:10.1098/rsif.2009.0157.focus.

Kolbl, S., Forte-Tavčer, P., Stres, B., 2017. Potential for valorization of dehydrated paper pulp sludge for

biogas production: Addition of selected hydrolytic enzymes in semi-continuous anaerobic digestion

assays. Energy 126, 326–334. doi:10.1016/j.energy.2017.03.050

Kolbl, S., Paloczi, A., Panjan, J., Stres, B., 2014. Addressing case specific biogas plant tasks: Industry

oriented methane yields derived from 5L Automatic Methane Potential Test Systems in batch or semi-

continuous tests using realistic inocula, substrate particle sizes and organic loading. Bioresour.

Technol. doi:10.1016/j.biortech.2013.12.010

Kolbl, S., Panjan, J., Stres, B., 2016. Mixture of primary and secondary municipal wastewater sludge as a

short-term substrate in 2 MW agricultural biogas plant: site-specific sustainability of enzymatic and

ultrasound pretreatments. J. Chem. Technol. Biotechnol. 91, 2769–2778. doi:10.1002/jctb.4883

Murovec, B., Kolbl, S., Stres, B., 2015. Methane Yield Database: Online infrastructure and bioresource for

methane yield data and related metadata. Bioresour. Technol. 189, 217–223.

doi:10.1016/j.biortech.2015.04.021

82

Influence of Trace Element Supplementation on Anaerobic Mono-Digestion of Chicken Manure

Alper Bayrakdar*,**, Rahim Molaey*, Recep Önder Sürmeli*,***, Bariş Çalli*

* Environmental Engineering Department, Marmara University, 34722 Kadikoy, Istanbul, Turkey

**Environmental Engineering Department, Necmettin Erbakan University, 42140, Meram, Konya, Turkey *** Environmental Engineering Department, Bartın University, 74100, Merkez, Bartın, Turkey

Keywords: Ammonia; Chicken manure; Trace elements; BMP

Chicken manure contains high amount of biodegradable organic matter. Anaerobic digestion (AD) is a favourable option to treat and stabilize the organic matter in chicken manure with biogas production. The high amount of organic nitrogen in chicken manure, which exists in the form of undigested protein and uric acid is hydrolysed to ammonia under anaerobic conditions (Bolan et al., 2010). Therefore, in practice ammonia inhibition is experienced as the major problem in AD of chicken manure (Bujoczek et al., 2000). In order to alleviate the ammonia inhibition in AD, stripping, zeolite adsorption, membrane separation, struvite precipitation, dilution and co-digestion have been applied in numbers of studies (Huang et al., 2015). Although these processes mitigate the ammonia inhibition, in practice they will make the operation of AD process difficult. Therefore, the main challenge is to develop a reliable method for using nitrogen rich organic wastes as mono-substrate.

At high ammonia concentrations, acetate using methanogens are more vulnerable than hydrogenotrophic species and methane production takes place via syntrophic acetate oxidation (SAO) (Schnürer et al., 1999; Westerholm et al., 2011). It is reported that the redox-enzyme, formate dehydrogenase (FDH), plays an important role in SAO and depends on the availability of trace elements (TEs) such as Se, Co, Mo and W (Banks et al., 2012; Plugge et al., 2009). Nickel and iron are also essential trace elements in the syntrophic acetate oxidation (Thauer et al., 2008). On the other hand, excess TEs supplementation may be toxic for anaerobic consortium (Plugge et al., 2009) and high concentrations of TEs can limit the use of digestate in agriculture and cause environmental pollution.

In the past, researchers assumed that manures contain relatively high amounts of TEs and therefore did not consider any TE deficiency problem in AD of manures. However, recent findings are in contrast to this assumption (Schattauer et al., 2011). There number of studies investigating the effect of TEs supplementation on AD of food waste (Banks et al., 2012; Feng et al., 2010; Zhang et al., 2015), to our knowledge there is no study about TE requirements for AD of chicken manure at elevated TAN levels, although chicken manure is deficient in some essential TEs for anaerobic microorganisms. In this study, the effects of TE supplementation were studied for the first time on AD of chicken manure with the bio-methane potential (BMP) tests by increasing total ammonia concentrations till to 6000 mg/l.

Two BMP tests were performed. In BMP test-1, 10 different TEs concentration were

examined by increasing the concentrations in TE mix from 1- to 50 fold at moderate total

ammonia concentration (3000 mg/l). In BMP test-2, the effect of TEs supplementation on

the biogas production was investigated at 420, 3000, 4000 and 6000 mg/l of TAN

concentrations. In BMP tests, 2 gTS/l of raw chicken manure was used as substrate and

TAN concentrations were adjusted by adding NH4Cl externally. The inoculum/substrate

(I/S) ratio and total TS content were kept at 2 and 0.6% respectively. Higher TS contents

83

were not tested because of difficulties in mixing. The BMP tests were conducted in 250

ml glass bottles with 100 ml active volume in triplicate. After BMP tests, Gompertz model

was applied to find the maximum methane production rate (Rm, ml CH4/gVS/day) (Lay et

al., 1997).

In BMP test-1, the optimum TE concentrations were determined according to CH4

production rate. TEs concentration up to 10-fold did not significantly affect the results.

CH4 production rate was predicted as 21.3 ± 3.08 ml CH4/gVS.d between 1- and 10-fold

TE concentrations. When the TE concentrations increased more than 10-fold, both the

cumulative CH4 production and the production rate decreased considerably. The results

have indicated that 1-fold TE mix is adequate.

For BMP test-2, the highest CH4 production rate of 41.0±1.65 ml/gVS.d was achieved at

420 mg/l of TAN. However, CH4 production rates decreased to 25.5±0.75, 11.5±0.22 and

5.1±0.08 ml/gVS.d as TAN concentration increased to 3000, 4000 and 6000 mg/l,

respectively. Although, the TE supplementation at 420 mg/l of TAN has increased the

cumulative methane production slightly by 3%, there is no clear difference between the

CH4 production rates of sets with and without TE supplementation. At 3000 and 4000

mg/l of TAN, the TE supplementation improved the CH4 production rate by about 5-6%.

On the other hand, at 6000 mg/l of TAN, the CH4 production rate of TE supplemented set

was visibly higher (28.3%) than that of non-supplemented one. A similar tendency was

observed in cumulative methane production. With TE supplementation, the cumulative

methane production increased by 7-8% at TAN of 3000 and 4000 mg/l. The improvement

with TE supplementation exceeded 20% at 6000 mg/l. These results clearly show that

the TE mix consisting of Co, Se, W, Mo, Ni and Fe is more effective on methane

production at elevated TAN concentrations.

References Banks,C.J.; Zhang,Y.; Jiang,Y.; & Heaven,S. (2012). Trace element requirements for stable food waste

digestion at elevated ammonia concentrations. Bioresour. Technol. 104, 127–135.

Bolan,N.S.; Szogi,A.A.; ChuasavathIt,T.; Seshadri,B.; JR.,M.J.R.; & PanneerselvaMm,P. (2010). Uses and

management of poultry litter. Worlds. Poult. Sci. J. 66, 673–698.

Bujoczek,G.; Oleszkiewicz,J.; Sparling,R.; & Cenkowski,S. (2000). High Solid Anaerobic Digestion of

Chicken Manure. J. Agric. Eng. Res. 76, 51–60.

Feng,X.M.; Karlsson,A.; Svensson,B.H.; & Bertilsson,S. (2010). Impact of trace element addition on biogas

production from food industrial waste - linking process to microbial communities. FEMS Microbiol.

Ecol. 74, 226–240.

Huang,H.; He,L.; Lei,Z.; & Zhang,Z. (2015). Contribution of precipitates formed in fermentation liquor to the

enhanced biogasification of ammonia-rich swine manure by wheat-rice-stone addition. Bioresour.

Technol. 175, 486–493.

Lay,J.-J.; Li,Y.-Y.; & Tatsuya,N. (1997). Influences of pH and moisture content on the methane production in

high-solids sludge digestion. Water Res. 31, 1518–1524.

Plugge,C.M.; Jiang,B.; De Bok,F.A.M.; Tsai,C.; & Stams,A.J.M. (2009). Effect of tungsten and molybdenum

on growth of a syntrophic coculture of Syntrophobacter fumaroxidans and Methanospirillum hungatei.

Arch. Microbiol. 191, 55–61.

Schattauer,A.; Abdoun,E.; Weiland,P.; Plöchl,M.; & Heiermann,M. (2011). Abundance of trace elements in

demonstration biogas plants. Biosyst. Eng. 108, 57–65.

Schnürer,A.; Zellner,G.; & Svensson,B.H. (1999). Mesophilic syntrophic acetate oxidation during methane

formation in biogas reactors. FEMS Microbiol. Ecol. 29, 249–261.

Thauer,R.K.; Kaster,A.-K.; Seedorf,H.; Buckel,W.; & Hedderich,R. (2008). Methanogenic archaea:

ecologically relevant differences in energy conservation. Nat. Rev. Microbiol. 6, 579–591.

84

Westerholm,M.; Dolfing,J.; Sherry,A.; Gray,N.D.; Head,I.M.; & Schnürer,A. (2011). Quantification of

syntrophic acetate-oxidizing microbial communities in biogas processes. Environ. Microbiol. Rep. 3,

500–505.

Zhang,W.; Wu,S.; Guo,J.; Zhou,J.; & Dong,R. (2015). Performance and kinetic evaluation of semi-

continuously fed anaerobic digesters treating food waste: Role of trace elements. Bioresour. Technol.

178, 297–305.

85

Modelling the Effect of Trace Elements in Anaerobic Digestors

A. Hatzikioseyian, P. Kousi, E. Remoundaki, M. Tsezos

Laboratory of Environmental Science and Engineering, School of Mining and Metallurgical Engineering, National

Technical University of Athens (NTUA)

Heroon Polytechniou 9, 15780, Athens, Greece e-mail: artin@metal.ntua.gr

Keywords: Modelling; Trace Elements (TE); Anaerobic Digestion; Sulphate-Reducing Bacteria (SRB);

Sulphide.

Trace elements (TEs) have an important biological function in anaerobic digestion (AD)

processes. Elements such as Fe, Zn, Ni, Co, Mo, Cu, Mn, W and Se in trace amounts

noticeably increase biogas production (van Hullebusch et al. 2016). Most of these

elements are part of essential enzymes involved in methane production. In addition,

some of these, e.g. Fe or Ni, remove sulphide toxicity acting as scavenger by forming

insoluble metal sulphides. On the other hand, supplements such as EDTA, NTA and

other chelating agents, also increase biogas production by increasing the bioavailability

of these elements. Although numerous studies appear in the literature, the reported

optimum concentrations for TEs differ significantly due to diverse experimental

conditions. Often the stimulatory effect of TEs is ignored and only the inhibitory action is

considered. Thus the need to further elucidate the physicochemical and biological

interactions of TEs in AD is emerging. Modelling of anaerobic processes for biogas

production has also gained increased attention. Anaerobic Digestion Model 1 (ADM1) is

still considered today as a state-of-the-art model in this area (Batstone et al. 2002). The

model includes a significant number of chemical and biological processes. However, it

could be further extended by considering the action of TEs in the reactors. Fedorovich et

al. were the first to include the production of biogenic hydrogen sulphide by SRB as a

parallel and competing biological action in ADM1 (Fedorovich et al. 2003). This

extension is not only useful for sulphate-containing wastes but also affects the

implementation of trace metals (TMs) in the model, which interact with the biogenic

sulphide produced by SRB. This affects the dosing and the bioavailability of TMs;

Significantly higher amounts of metals are required due to the precipitation of metal

cations and less TMs are bioavailable under sulphate reducing conditions. Therefore, it

is well recognised that extended modelling modules are needed to overcome the

limitations and omissions of ADM1 for widening the application field of the model.

Implementing the action of TEs in anaerobic digestion models is a challenging task. A

number of issues should be considered. Some TEs are usually present in feedstock.

However, the chemical form and bioavailability may not render them directly utilizable by

the microbial consortia in the reactor. Often the reactor may be deficient in some of

them, so external dosing is necessary. The amount and chemical form of the

supplemented TEs increase the operating cost and raise issues concerning digestate

management. On the other hand, the benefit from increased biogas production is

significant.

The present work discusses some aspects of physicochemical and biological interactions

of TEs in AD models. Indicative physicochemical processes are: (a) TE speciation for

cations and oxyanions as a function of pH and redox potential, (b) TE precipitation

86

chemistry as insoluble sulphides, phosphates, carbonates, hydroxides, (c) complexation

of TE with natural (DOM) and synthetic chelating agents, (d) adverse or synergetic

effects of common ions e.g. Na+, Mg2+, Ca2+, alkalinity, ionic strength, (e) surface

chemistry e.g. adsorption processes on the surface of particulate material.

Toxicity/speciation models such as the Free Ion Activity Model (FIAM), Biotic Ligand

Model (BLM), Windermere Humic Aqueous Model (WHAM) and the Non-Ideal

Competitive Adsorption (NICA) Model, incorporate biotic and abiotic interactions of

cations. The principles from these models could be incorporated in any TE

implementation in AD systems.

The biological action of TEs could be implemented as a dose response relationship. This

factor could appear as a modifier in the Monod kinetic equation for the class of

microorganisms that TEs are more likely to act as growth limiting factor.

max 1 2 nf(S )f(S )...f(S )...f(pH)f(T)...f(TE) (0.1)

Assuming that an element can act either as stimulating or inhibiting agent and depending

on the concentration range, both actions should be foreseen. A simple dose response

formula, analogue to the Haldane equation, is presented in Figure 1.1. There is

extensive evidence that neither total nor dissolved aqueous metal concentrations are

good predictors of metal bioavailability and toxicity. Thus, CTE should stand for the

concentration of trace elements in bioavailable form affecting the microbial growth and a,

b0, b1 and b2 are parameters of the equation. There is significant uncertainty of which TE

species are considered bioavailable. Dissolved free ionic metal species are far more

bioavailable than most complexed metal species. However metal complexes can act as

a pool for free ion species.

Figure 1.1. A tentative multiplication factor in Monod equation to simulate the effect of TE on biomass

growth.

Acknowledgments: This work has been initiated in the frame of COST Action ES1302 European Network on

Ecological Functions of Trace Metals in Anaerobic Biotechnologies.

References Batstone, D. J.; J. Keller; I. Angelidaki; S. V. Kalyuzhnyi; S. G. Pavlostathis; A. Rozzi; W. T. M. Sanders; H.

Siegrist; V. A. Vavilin (2002). Anaerobic Digestion Model No.1 (ADM1), IWA Publishing.

Fedorovich, V.; P. Lens; S. Kalyuzhnyi (2003). Extension of Anaerobic Digestion Model No. 1 with processes of sulfate reduction. Applied biochemistry and biotechnology, 109 (1-3), 33-45.

van Hullebusch, E. D.; G. Guibaud; S. Simon; M. Lenz; S. S. Yekta; F. G. Fermoso; R. Jain; L. Duester; J. Roussel; E. Guillon; U. Skyllberg; C. M. R. Almeida; Y. Pechaud; M. Garuti; L. Frunzo; G. Esposito; C. Carliell-Marquet; M. Ortner; G. Collins (2016). Methodological approaches for fractionation and speciation to estimate trace element bioavailability in engineered anaerobic digestion ecosystems: An overview. Critical Reviews in Environmental Science and Technology, 46 (16), 1324-1366.

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88

Biogenic selenium particles: linking water treatment with resource recovery

L.C. Staicu

University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Bucharest, Romania (lucian.staicu@upb.ro)

Keywords: Selenium; Bioremediation; Particles; Resource; Recovery.

Selenium (Se) has attracted considerable attention because of its dual nature: toxicant and micro-nutrient. Following the advent of the Industrial Revolution, the anthropogenic release of Se has contributed to the disruption of its biogeochemical cycle. Industrial activities such as energy production based on fossil fuel combustion, crude oil processing and ore smelting contributed significant amounts of Se to the environment. Selenium has an increased tendency to bioaccumulate and concentrate within aquatic trophic networks, leading to ecological disasters where entire fish and bird populations were virtually eliminated (e.g., Lake Belews and Kesterson Reservoir, USA) (Lemly, 2004). Importantly, in industrial effluents, Se is mainly present as high-valence, water-soluble and bioavailable oxyanions: selenate, SeO42-, and selenite, SeO32-. Among the treatment technologies, bioremediation, founded on the microbial reduction of Se oxyanions to elemental Se, Se0 (Figure), has emerged as a “greener” alternative to the energy-intensive and cost-prohibitive physical-chemical systems.

As a solution to industrial Se release, biological treatment technologies have been tested for the clean-up of Se-bearing wastewaters. Phylogenetically-diverse bacteria can use Se for cellular respiration under anaerobic conditions, generating energy to sustain growth (Stolz and Oremland, 1999). A number of bioreactor settings using Se-respiring bacteria have been tested and some are commercially available. These include (i) granular sludge bioreactors (e.g., upflow anaerobic sludge blanket, UASB), (ii) fluidized-bed bioreactors (FBBR), (iii) packed-bed bioreactors, (iv) hydrogen-based hollow fiber membrane biofilm reactor (MBfR), and (v) electro-biochemical reactor (EBR).

When Se-bearing effluents are treated biologically, water-insoluble Se0 nanoparticles are often the main end product. Because biogenic Se0 has colloidal properties that make it stable and prone to long-distance transport in aquatic ecosystems, the deployment of a post-treatment (polishing) stage (e.g., media filtration, chemical coagulation and electrocoagulation) is envisaged to recover it before exiting the bioreactor set-up. From an economic standpoint, the recovery of biomaterials with industrial applications could help offset the treatment costs. Elemental Se possesses unique photo-optical and semiconducting properties that are exploited by electronics and energy industries. Consequently, linking the treatment with the recovery of Se in an integrated platform sustains the fundamental goal of the circular economy framework.

89

Figure. UASB reactor treating selenium-laden wastewater (adapted from Staicu et al., 2017).

References

Lemly, A.D. (2004). Aquatic selenium pollution is a global environmental safety issue. Ecotox. Environ. Safe., 59, 44-56. Staicu, L.C.; van Hullebusch, E.D.; Rittmann, B.E.; Lens, P.N.L. (2017). Industrial selenium pollution. Sources and biological treatment technologies. In: “Bioremediation of selenium contaminated wastewaters”, van Hullebusch, E.D., Springer (in press). Stolz, J.F.; Oremland, R.S. (1999). Bacterial respiration of arsenic and selenium. FEMS Microbiol. Rev., 23,

615-627.

90

The impact of crude glycerol from biodiesel production on

biomethane production and trace metal content in batch

experiment

Bojana Danilović1, Leon Deutsch

2, Urška Magerl

3, Dragiša Savić

1, Sabina Kolbl

3, Blaž

Stres2,4

1 University of Niš, Facaulty of Techology, Leskovac, Serbia

2 University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia

3 University of Ljubljana, Faculty of Civil Engineering, Ljubljana, Slovenia

4 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia

Keywords: glycerol; crude; biodiesel; biomethane; trace metal; batch

The crude glycerol remaining after biodiesel production can be used as carbon source in

biogas production (Kolesárová et al., 2011) and can contain up to 58% of carbon

depending on the feedstock used for biodiesel production (Thompson and He, 2006).

On the other hand, crude glycerol generally contains minerals and salts that can have

negative impact on the biogas production and its methane content. Inorganic salts at

concentration higher than 15 g/l can cause a decrease in the production of biogas (Bodík

et al., 2008). In this paper, the trace metal content analysis of the substrate components

used for biogas production was performed (waste water treatment plant sludge, cow

manure, whey, crude glycerol). In order to examine the influence of crude glycerol and

the underlying trace element content on the efficiency of biomethane production a batch

experiment using Automatic Methane Potential Test System (AMPTS) was performed.

The 500 ml glass flask containing 325-330 ml of the digestive mixture were placed in

water bath (39°C) and mixed for 1 min every 5 minutes. Crude and pure glycerol were

used as substrate and control at 2% and 3% vol/vol.

The content of twenty-three trace elements the substrates was determined using ICP-

OES analysis with argon as a carrier gas (ARCOS FHE12, SPECTRO, Germany). The

samples were prepared by wet digestion (1 ml of a sample with 9 ml of concentrated

nitric acid) followed by filtration through syringe filter with a 0.45 µm pore size.

Compared to the other substrates used for digestion the crude glycerol from biodiesel

production contained significantly higher concentration of Si (fivefold) and P (two to six

fold higher), while approximately the same or lower concentration of all other examined

trace elements were detected. No Ag, Cd, Co, Li and Tl, were detected in the substrates,

while B was present only in cow`s manure (1.4 mg/L)

Additionally, the contribution of each component to the total content of trace elements in

the mixture was calculated based on the mixture composition (Table 1.1.). The crude

glycerol significantly contributed only to the content of K (14.4%), Si (17.3%) and P

(11.6%), while heavy metals content was not significantly influenced (Table 1.1). This

was confirmed in laboratory experiments simulating anaerobic digestion of prepared

mixture with approximately the same quantity of pure and crude glycerol (2 and 3%).

Namely, after six days of monitoring, slightly lower quantity of methane was produced in

experiments with crude glycerol compared to the experiments containing same quantity

91

of pure glycerol (Figure 1.1). As can be expected, the prolonged lag phases were only

noticed in the experiments conducted with crude glycerol.

The results confirmed that crude glycerol remaining after biodiesel production can be

used as an additional carbons source for biogas production.

Table 1.1. The trace elements quantity (mg/L) added by each substrate, and participation of crude glycerol in total quantity of trace elements in the digestive mixture

Figure 1.1. The methane formation during anaerobic digestion (39oC, AMPTS) of mixtures supplemented

with additional 2% (∆) and 3 % (o) of either pure glycerol (open symbols) or crude glycerol from biodiesel production (filled symbols) (vol/vol). Control mixtures contained cow manure, whey and waste water

treatment plant sludge only.

Acknowledgments

This work was supported by STSM to BD within the COST Action ES1302

References Kolesárová, N.; Hutňan, M.; Bodík, I.; Špalková,V. (2011) Utilization of biodiesel by-products for biogas

production. J.Biomed. Biotechnol., 2011, 1-15 Thompson, C., He, B. (2006) Characterization of crude glycerol from biodiesel production from multiple

feedstocks. App. Eng. Agricult,. 22, 261–265 Bodík, I.; Hutňan, M.; Petheöová, T.; Kalina, A. Anaerobic treatment of biodiesel production wastes,

Proceedings of the Book of Lectures on 5th International Symposiumon Anaerobic Digestion of Solid Wastes and Energy Crops, Hammamet, Tunisia, 2008.

92

The impact of nano ZnO on anaerobic digestion of the

organic fraction of municipal solid waste

I. Temizel, B. Demirel*, N.K. Copty and T.T. Onay

Institute of Environmental Sciences, Boğaziçi University, Bebek, 34342, Istanbul, Turkey

(*E-mail: burak.demirel@boun.edu.tr)

Keywords: Anaerobic digestion; biogas; municipal solid waste; nanomaterials; methane

Abstract

Engineered nanomaterials (ENMs) are commonly used in commercial products at

greater amounts. However, despite widespread use of ENMs in commercial products

and their eventual disposal in sanitary landfills, the fate and behaviour of ENMs in solid

waste environments are still not well understood yet. Even though the impacts of metallic

and metal oxide ENMs (such as titanium dioxide-TiO2, silver-Ag, zinc oxide-ZnO, cerium

oxide-CeO2 and copper oxide –CuO) on conventional activated sludge (AS) wastewater

treatment and anaerobic digestion (AD) of sewage sludge systems have been discussed

in literature, there exists relatively little information about the impacts of these inorganic,

metal oxide ENMs during anaerobic waste stabilization phase in landfills. In this phase,

the organic fraction of the municipal solid waste (MSW) is converted into biogas, mainly

methane (CH4) and carbon dioxide (CO2), by a mixed group of microorganisms (Bacteria

and Archaea). The adverse impacts of metal oxide ZnO, CuO and CeO2 nanomaterials

on biogas and methane production from landfills particularly warrant further research,

since some recent studies show that these inorganic ENMs can pose adverse impacts

on biological activity in biological waste treatment systems. Therefore, this study was

designed to investigate the impacts of three selected metal oxide ENMs, namely, ZnO,

CuO and CeO2, on biological activity and biogas production stage during anaerobic

conversion of the organic matter available in municipal solid waste (MSW) into biogas,

using batch tests. Preliminary results from this ongoing research work will be presented

at the conference.

93

Adsorption mechanism of nanoparticles ZnO and CuO on anaerobic granular sludge EPS: Contributions of EPS fractional

polarity and nanoparticle diameters

Liangliang Wei*, Ming Xin*, Sheng Wang*, Junqiu Jiang*, Qingliang Zhao*,

* State Key Laboratory of Urban Water Resources and Environment (SKLUWRE); School of Environment, Harbin Institute of Technology, Harbin 150090, China.

∗ Corresponding author: weill333@163.com (L.L. Wei); zhql1962@163.com (Q.L. Zhao)

Keywords: Extracellular polymeric substances; Nanoparticles; Adsorption; Mechanisms

Abstract: Worldwide application of nanotechnology led to an increasingly release of

nanoparticles in wastewater treatment system, and thus into sewage sludge, which

potentially impairs the digestion efficiency of sewage sludge. In this study, the binding

quality, adsorption mechanism, as well as the chemical fractional contribution of the

anaerobic granular sludge EPS to the adsorption of nano- ZnO and CuO was

investigated. In brief, nano CuO could be more easily adsorbed by sludge EPS than that

of nano ZnO (1.31g/g VS vs 0.53g/g VS), and a smaller nanoparticles diameter benefited

to the adsorption processes. Hydrophobic EPS (HPO-A and HPO-N) within the

anaerobic granular sludge was more efficient in adsorbing nano CuO and nano ZnO than

that of the hydrophilic EPS. For example, EPS fractions of HPO-A and HPO-N extracted

from the sludge sample exhibited a relatively higher adsorption ability of 2.09g/g VS and

2.27 g/g VS for nano CuO, respectively, much higher than that of HPI (0.76 g/g VS).

Since HPO-A fraction was the predominant fraction within the granular sludge EPS, thus

it played a major role in nanoparticles removal. The adsorption of the nano CuO onto the

unfractionated EPS could be better described by the Freundlich isotherm, while

Langmuir models fitted to the adsorption of nano ZnO. In addition, structures changes of

the EPS pre- and after nanoparticles were evaluated via the analysis of infrared

spectroscopy and scanning electron microscopy, and results demonstrated that

functional structures of hydroxyl, carboxyl, amino, amide groups and C-O-C groups

played a major role in nanoparticles removal.

Objectives

EPS of anaerobic granular sludge, obtained from lab-scale expanded granular sludge

bed (EGSB) reactor, was extracted using NH4OH and subsequently fractionated using

XAD-8/XAD-4 resins. The principal objective of this studies was to evaluate which EPS

fractions were responsible for nano CuO and ZnO adsorption. Adsorption kinetic

characteristics of the different EPS fractions were comparably studied. In addition,

possible changes of the chemical structures in EPS during nanoparticles adsorption

were examined.

Results and Findings

Chemical composition of extracted EPS from anaerobic granular sludge

For each gram of volatile suspended solids (VS) in sludge, as much as 248.4 mg DOC of

EPS was extracted from the anaerobic granular sludge. Specifically, HPO-A was the

predominant fraction of the extracted sludge EPS, accounting for 40.6% of the bulk

DOC. HPI fraction was another major component, and constituted 29.7% of the bulk

94

DOC, and other three fraction were quite low and decreased in the trend of TPI-A

(10.4%) > TPI-N (10.2%) > HPO-N (9.1%). In unit of mg/g VS, about 106.9 of protein

could be extracted from the anaerobic granular sludge. Similar to the distribution of

sludge DOC, the majority of the sludge EPS related proteins were in the form of HPO-A

and HPI. Specifically, the percentage distribution of protein within the anaerobic granular

sludge exhibited a decreased trend of HPI (46.1%) > HPO-A (35.9%) > TPI-N (10.2%) >

HPO-N (5.5%) > TPI-A (2.2%). For comparison, the carbohydrate content was lower in

the NH4OH extractable EPS in comparison with that of the protein, and as much as 49.5

mg/g VS of carbohydrates was extracted from anaerobic granular sludge. In brief, the

carbohydrates were mainly existed as HPO-A (29.2%), HPI (25.1%) and HPO-N (21.2%)

within the anaerobic granular sludge, while that of the TPI-A and TPI-N was quite lower.

Adsorption characteristics of nanoparticles by EPS and its fractions

Generally, as much as 1.31 g/g VS of nano CuO could be efficiently adsorbed by the

EPS extracted from the anaerobic granular sludge, was much higher than that of the

nano ZnO (0.53g/g VS). In addition, fractional adsorption capacities of the sludge EPS

varied widely depending on both the characteristics of the nanoparticles and the types of

EPS fractions employed (Fig. 1). For the anaerobic granular sludge, HPO-N exhibited

the highest nano-CuO adsorption rate (2.27 g/g VS), followed by HPO-A (2.09 g/g VS),

while that of the other three fractions were quite low and declined in the trend of HPI

(0.76 g/g VS) > TPI-N (0.63 g/g VS) > TPI-A (0.20 g/g VS). The adsorption trend of

nano-ZnO by different EPS fractions were similar to that of nano-CuO, and distributed in

the trend of HPO-A (0.77) > HPO-N (0.62) > TPI-N (0.51) > HPI (0.44) > TPI-A (0.39). In

overall, hydrophobic EPS (HPO-A and HPO-N) within the anaerobic granular sludge was

more efficient in adsorbing than that of the hydrophilic EPS regardless of the chemical

characteristics of the nanoparticles. Since HPO-A fraction was the predominant fraction

within the granular sludge EPS, thus it played a major role in nanoparticles removal.

Table 1.1. (a) Adsorption of nano- CuO and ZnO by EPS and its fractions extracted from anaerobic granular sludge; (b) FT-IR spectrum variation pre- and after nanoparticles adsorption

To evaluate the effect of nanoparticle diameters on the adsorption processes, the

nanoparticles of ZnO and CuO with averaged diameters of 30 nm and 100 nm were

selected, and the adsorption characteristics was comparably analyzed with the

controlling test (with a diameter of 50 nm). Experimental results demonstrated that the

increasing of the nanoparticles diameters declined the maximum adsorption capacities of

the unfractionated/fractionated EPS. For example, the corresponding adsorption

capacities of the 30 nm nano-ZnO onto the unfractionated EPS was 0.59 g/g VS, slightly

higher than that of the 50 nm and 100 nm nanoparticles (0.53 g/g VS and 0.49 g/g VS,

respectively).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Bulk EPS HPI HPO-A TPI-A HPO-N TPI-N

Ad

so

rpti

on

cap

acit

y (g

/g V

S)

Nano-CuO Nano-ZnO

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Binding mechanisms of nano- ZnO and CuO on anaerobic granular sludge

The adsorption of the nano CuO onto the unfractionated EPS could be better described

by the Freundlich isotherm (R2=0.998) than that of Langmuir equation (R2=0.975), while

Langmuir models fitted to the adsorption of nano ZnO (R2=0.975). Moreover, functional

structures of hydroxyl, carboxyl, amino, amide groups and C-O-C groups played a major

role in nanoparticles removal.

References Lombi, E.; Donner, E.; Tavakkoli, E.; Turney, T.W.; Naidu, R.; Miller, B.W.; Scheckel, K.G. (2012). Fate of

zinc oxide nanoparticles during anaerobic digestion of wastewater and post-treatment processing of sewage sludge. Environ. Sci. Technol., 46, 9089−9096.

Mu, H.; Chen, Y.G.; Xiao, N.D. (2011). Effects of metal oxide nanoparticles (TiO2, Al2O3, SiO2 and ZnO) on waste activated sludge anaerobic digestion. Bioresource Technology 102, 10305–10311.

Ganesh, R., Smeraldi, J., Hosseini, T., Khatib, L., Olson, B.H., Rosso, D., 2010. Evaluation of nanocopper removal and toxicity in municipal wastewaters. Environ. Sci. Technol., 44, 7808–7813.

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Metals Removal from Incineration Ashes of Anaerobically Digested Sewage Sludge

F. Arroyo Torralvo*, A. Serrano**, M. Rodríguez-Galán*, B. Alonso-Fariñas*, F. Vidal*

* University of Seville, Department of Chemical and Environmental Engineering, Higher Technical School of Engineering, Camino de los Descubrimientos, s/n, Seville, Spain

**Instituto de la Grasa, Spanish National Research Council (CSIC), Campus Universitario Pablo de Olavide-Ed. 46, Ctra. de Utrera, km. 1, Seville, Spain

Keywords: anaerobic digestion; sewage sludge; incineration; ashes; metals recovery

Sewage sludge is generated from municipal waste water treatment. Anaerobic digestion

of sewage sludge produces a high calorific value biogas, which can be utilized as fuel to

reduce the energy consumption of the wastewater treatment plants (Cao and

Pawłowskia, 2012). Anaerobically digested sludge can be employed as organic

amendment because of its high content of phosphorus and nitrogen. But, in addition to

various organic substances, heavy metals may end up in the sludge. Then, as heavy

metals can pollute the environment, their presence can limit the use of the digested

sludge as organic amendment and other waste management alternative should be

considered (Risberg, K. 2015).

Sludge incineration has become more common in recent years. Although the digested

sludge can be energy profitably, containing considerable organic matter, it have not been

the main driver for sludge incineration (Cao and Pawłowskia, 2012). In the EU, the

Waste Framework Directive (Directive 2008/98/EC) encourage the material recycling of

sludge and limit the disposal of organic matter to landfills. On other hand, the heavy

metals contain in digestate sludge are limited for disposal in agriculture land by Directive

86/278/CEE. These requirements and the fact than the amount of sludge generated is

very large compared to the land area available for the disposal or treatments as

composting have promoted the use of incineration for sludge management (Pure, 2012).

The main advantages of incineration are: large reduction of waste volume, thermal

destruction of toxic organic compounds and minimization of odour generation. As a

disadvantage, part of the solids remains as ashes, containing most heavy metals

originally contained in the sludge (Fytili and Zabaniotou, 2008). Incineration ashes of

anaerobically digested sewage sludge are usually sent to landfill and, in some cases,

they have to be previously stabilised to reduce the risk of heavy metals leaching.

Acid extraction is a widely developed methodology for metal removal from solid wastes

(Oliver and Carey, 1977). But, the extractive potential of this technic highly depends of

the physicochemical characteristics of the waste (Bunge, 2015). On other hand, high

cost of chemicals (i.e. H2SO4) is a handicap respect to other separation process based in

mechanical operations. Deep Eutectic Solvents (DES) have showed high metal removal

potential from solid wastes in previous works (Bakkar and Neubert, 2016). In this sense,

DES could be a low-cost alternative to acids for metal recovery from ashes of

anaerobically digested sludge. In addition, DES are eco-friendly solvents with high

biodegradability.

97

This work aims to contribute to the further applicability of technologies that might be used

to remove heavy metals from Incineration ashes of anaerobically digested sewage

sludge. Two runs of experiments using a H2SO4 solution and DES as metal extraction

agent respectively are being carried out. Anaerobically digested sewage sludge samples

from a real wastewater treatment plant were calcined at 550 ºC at laboratory to obtain

the ashes employed in the experiments. Main results will be reported in IMAB17.

References Bunge, R. (2015). Recovery of metals from waste incinerator bottom ash. Institut für Umwelt und

Verfahrenstechnik UMTEC, Switzerland. Available on www.umtec.ch.

Bakkar, A.; Neubert, V. (2016). Recycling of steelmaking dusts through dissolution and electrowinning in

deep eutectic solvents. 2nd World Congress and Expo on Recycling. Berlin, Germany.

Risberg, K. (2015). Quality and function of anaerobic digestion resources. PhD. thesis, Department of

Microbiology, University of Agricultural Sciences, Uppsala, Sweden. ISSN 1652-6880.

Fytili, D.; Zabaniotou, A. (2008). Utilization of sewage sludge in EU application of old and new methods—A

review. Renewable and Sustainable Energy Reviews, 12, 116–140.

Cao, Y.; Pawłowskia, A. (2012). Sewage sludge-to-energy approaches based on anaerobic digestion and

pyrolysis: Brief overview and energy efficiency assessment. Renewable and Sustainable Energy

Reviews, 16, 1657-1665.

Pure (Project on Urban Reduction of Eutrophication). (2012). ISBN 978-952-5725-91-9. Available on

www.purebalticsea.eu.

Oliver, B. G.; Carey, J.H. (1977). Acid solubilization of sewage sludge and ash constituents for possible

recovery. Water Research 10, 1077-1081.

98

Improving Gas Counter for Measurement of BioMethane Potential and Methanogenic Activity

O K BEKMEZCİ*,**

, E Piro*, D Ucar

***

* Department of Environmental Engineering, Bitlis Eren University, Bitlis, Turkey ** Department of Environmental Engineering, Marmara University, Istanbul, Turkey

*** GAP Renewable Energy and Energy Efficiency Center, Harran University, Sanliurfa, Turkey

Keywords: Biomethane Potential; Methanogenic Activity; Gas Counter

Two of the most common tools for biogas researchers are biomethane potential and methanogenic activity tests. Both of those tests require quantification of methane production for a given sludge-substrate couple under controlled conditions. Some researchers employ manual or semi-manual measurement methods. Since trivial tasks in those methods require so much time of the research personnel, they limit the number of the reactors to be used in the test, which in return limits the number of parameters to be tested. Other researchers utilize gas counters. Unfortunately, there is a limited number of suitable gas counters in the market. Currently, available gas counters are based on liquid replacement method. Although their initial cost limits the number of reactors, they can require minimal labour from the researcher. Likewise, they generally work with acceptable errors. Those errors can occur due to inherent and practical calibration uncertainties, water depth (when applicable), and most inevitably ambient pressure changes.

Researchers need comparable measurements among their own data and between reported data in the literature. The inherent errors in measurement methods obstruct the comparability. For example, while ambient pressure change due to weather causes daily or hourly fluctuations in measurement error, altitude causes high variations between different laboratories. “Normal volume” is used to sustain comparability but currently available systems do not include means for such normalization.

The advances in sensor and microcontroller technologies allow more precise and affordable gas counters. In this study, a new gas counter system is designed, prototyped, and tested. The prototype:

- measures absolute pressure and temperature of each released gas pocket of fixed volume.

- calculates normal volume of each released gas pocket. - measures and records the gas production in real-time (for per released gas

pocket). - can measure 42 gas lines simultaneously. - is scalable. - requires no operator attendance. - includes a data acquisition software (OzanGet).

Figure 1.1 illustrates the principal parts of a single gas counter. The gas to be measured enters to the gas counter from a normally open solenoid valve (NOSV). The opening of the gas counter is closed by a normally close solenoid valve (NCSV). As the gas to be measured accumulates, the pressure increases in the gas collection chamber. The chamber pressure is constantly monitored by a microprocessor using a pressure sensor. When the pressure reaches to a predetermined level, the micro controller initiates an exhaust sequence of 8 steps: close NOSV, measure pressure and temperature, open NCSV, wait for chamber pressure to become equal to ambient pressure, close NCSV, measure pressure and temperature, send data to a computer, opening NOSV. Then, OzanGet acquires the data and stores it together with the gas release time in an Excel

99

file. Since the data includes the volume and temperature values in the gas collection chamber before and after the release and the volume of the chamber is known, the amount of released gas can be calculated by ideal gas law and can be represented in terms of normal volume.

Figure 1.1. Principal parts of the gas counter.

Acknowledgement: This study is supported by HUBAK, Harran University (project no: 16121).

100

Thermal treatment for olive oil wastes utilization: bioactive

compounds and substrate for soil contaminated remediation.

Guillermo Rodríguez Gutiérrez, Juan Fernández-Bolaños Guzmán, Antonio Lama-Muñoz. Fátima Rubio-Senent, África Fernández-Prior, Elisa María Rodríguez Juan, Aranzazu García

Borrego, Alejandra Bermúdez Oria and Blanca Vioque-Cubero.

Instituto de la Grasa, (CSIC), ctra de Utrera Km 1, Campus Pablo de Olavide, Edif. 46. CP 41013, Seville, Spain.

Keywords: olive oil wastes; phenols; bioactive compounds; functional oil; soil remediation.

New techniques for total utilization of olive oil wastes are required to solve the

environmental problems of this industry. Olive oil wastes, like alperujo, are rich sources

in bioactive compounds and they are appropriate substrates for conversion into useful

products. The nature of alperujo makes necessary to apply specific treatments to

process it successfully. It is the case of a novel thermal pre-treatment (hydrothermal

reactor) that hydrolyzes bioactive compounds to liquid phase getting a final solid rich in

cellulose, oil and proteins in which the calorific value has been increased. The new

technology is based in the steam explosion system in which the sample is treated under

higher pressures (up to 36 Kg/cm2) and temperatures (up to 240 ºC) followed by rapid

depressurization. The new conditions and the novel reactor have been designed to

operate under lower pressure and temperatures (up to 10 Kg/cm2 and 190ºC) without

depressurization, obtaining similar results. The application at the first time and

combination of this technology with energy valorization methods to alperujo could solve

olive oil waste problems. The alperujo is partially autohydrolysed in the hydrothermal

reactor and phases are easily separated by decantation, filtration or centrifugation. After

separation the liquid is rich in phenols that are currently extracted in the industry, and

sugars. The solid fraction is concentrated in oil richer in minor components that could be

extracted as functional oil. Other components like stones and cellulose are also

concentrated in the final solid. The higher fragments of stones are partially removed from

the alperujo into the industry, diminishing its percentage from 45 to 15%, keeping the

remaining fragments the lower sizes. The potential of these fragments of stones for

metal absorption has been studied, increasing the effect for a lower particle size of

stones. The thermal treatment increases the concentration of stones close to 30-35%

referred to dry matter, obtaining a final solid free of phytotoxic agents like phenols and

rich in natural heavy metal absorbent. Thus, three fractions could be obtained after

treatment, a liquid phase rich in phenols and new compounds, a new pomace olive oil

rich in minor components and a final solid more suitable for agroindustrial uses in

contaminated soils.

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Posters

102

103

Understanding the impact of Fe, Ni and Co on the hydrolytic and

acidogenic stage of anaerobic digestion

DK Villa-Gomez*, A Lomate*, N Islam*, M Peces*

* School of Civil Engineering, The University of Queensland, 4072 QLD, Australia.

Keywords: trace metals, anaerobic digestion, hydrolysis, acidogenesis.

Agro-industrial residues an abundant source of biomass produced in millions of tons in

Australia (Victoria. Department of Primary Industries. Farm Services 2010). These large

quantities represent a potential biomass resource that can be used for bioenergy

production via anaerobic digestion (AD) otherwise diverted to landfills. However, such

biomass is one of the least tapped since its exploitation is limited by its slow hydrolysis

(Ye et al. 2013). The latter is related to its lignocellulosic structure, wherein cellulose

and hemicelluloses (holocellulose) are trapped in the lignocellulosic structure (Ye et al.

2013). Iron (Fe), Nickel (Ni) and Cobalt (Co) are essential trace metals (TM) present in

the enzymes involved in the metabolic pathways of AD and their effect has been widely

reported in the acetogenic and methanogenic stage but not in the hydrolytic stage

(Fermoso et al. 2015). Nevertheless, there is a strong evidence that micronutrients

deficiency also occurs in agro-industrial residues where the limiting step is hydrolysis

(Pobeheim et al. 2011; Vintiloiu et al. 2012; Gustavsson et al. 2013). Understanding the

effect of these TM in the hydrolytic step is important to improve the bioenergy production

from agriculture residues. Moreover, the effect of TM in the other key AD steps (i.e.

acidogenesis) is also relevant for the production of intermediates such as volatile fatty

acids, key pre‐cursors to produce higher-value end-products (Kleerebezem et al. 2015).

The objective of this study is to assess the impact of Fe, Co and Ni on the anaerobic

digestion of lignocellulosic compounds with special focus on the hydrolytic and

acidogenic step.

Batch AD experiments were carried out in triplicate in 160 mL serum bottles placed in a

temperature‐controlled room at 35 °C. Sludge (5 g volatile solids-VS/L) from a

wastewater treatment plant was used as inoculum. Lignin, cellulose or glucose were

added as substrates at 5 g/L (±0.7) of chemical oxygen demand (COD). These

compounds were selected as these are main components of agro-industrial residues and

their degradation require passing the hydrolytic and acidogenic step. Fe (20-50 ppm), Co

(0.5-1 ppm), or Ni (2-5 ppm) were supplemented from concentrated stock-solutions

prepared with chloride salts (CoCl2, NiCl2 and FeCl2). Control experiments were carried

out without metal supplementation. The bottles headspace was flushed with nitrogen (N2)

to maintain anaerobic conditions, and sealed bottles with a rubber septum and

aluminium crimp. The experiments were carried out at pH 5.5-6 to avoid metal

precipitation using HCl. Specific Biogas production (LN biogas/kg VSsubstrate·day) and

composition (% of CH4 and CO2) were monitored over time. To monitor hydrolysis and

acidogenesis, 2 mL of liquid samples of the experimental bottles were taken during the

exponential phase of biogas production for volatile fatty acids (VFA) analysis. COD

analyses were carried out at the beginning and end of the experiments.

The experiments carried out with glucose represent the acidogenic step, as glucose is a

model acidogenic substrate and VFA are the direct product of this step. Overall results in

104

the experiments with glucose exhibited consistent VFA production with variations in the

concentrations of these upon metals addition. Butyrate was the main VFA accumulated,

followed by hexanoic acid and acetate. Co at 0.5 ppm displayed a lower butyrate

accumulation (Fig 1.2) suggesting a better use of this compound as reflected also by the

biogas yield results (Fig 1.1 A) and as in agreement with other authors that showed that

butyrate is better used in the presence of Co (Karlsson et al. 2012). Accordingly, the

results of biogas yield with glucose indicated that Fe (20 ppm) greatly increased the

biogas yield, suggesting a stimulatory effect over methanogenesis, while Ni (2ppm) and

Co (1 ppm) also influenced biogas production but in a lesser extent. Ni at 5 ppm resulted

in less biogas yield suggesting an inhibitory effect on methanogenesis (Fig 1.1 A), but

not acidogeneis, as VFA were produced.

Using cellulose as a model substrate for hydrolysis, the VFA results on the experiments

carried out with cellulose showed no variations in respect to the controls and were below

the detectable range (Fig 1.2). Only propionate known as a leftover from the hydrolysis

and acidogenesis reactions (Choong et al. 2016) was detected, suggesting that

hydrolysis is limiting the biogas production, thus no accumulation of intermediates are

observed. Ni and Co at the two concentrations studied displayed a stimulatory effect on

biogas production in the experiments carried out with cellulose (Fig 1.1 B). The addition

of Fe at 50 ppm did not increase the biogas production and at 20 ppm it even decreased

the biogas production (Fig 1.1 B). The addition of Co at 1 ppm also boosted the methane

(CH4) composition in the biogas (46%) as compared to the control (32%) suggesting a

shift in the microbial pathway due to the presence of this metal (Pobeheim et al. 2011).

Finally, the evaluation of lignin in AD, showed the lowest biogas production among the

three substrates studied and additionally, none of the metals and concentrations studied

in the experiments carried out with lignin increased the biogas yield (Fig 1.1 C). This

could be due to the structure of the lignin molecule compromising the added trace

elements by limiting speciation and thus bioavailability (Fermoso et al. 2015). Adsorption

of TM to particulate matter such as lignin has been reported (Gustavsson et al. 2013).

Nevertheless, Ni and Co at both concentrations and Fe at 50 ppm demonstrated to be

inhibitory for biogas production. The soluble chemical oxygen demand increased with Co

at 1 ppm from (61.3 (±13.3) to 98.6 (±15.7) and Ni from 69.6 (±14.18) to 152.7 (±7.09),

demonstrating that these TM enhanced solubility and that the inhibition was only at the

methanogenic stage and not for the hydrolytic and acidogenic stage. This was also in

agreement with the resulting acetate and the rest of VFA in minor concentrations, which

demonstrates a conversion of linginin to VFA aided by the addition of Co and Ni.

105

Figure 1.1 Cumulative biogas yield in the experiments carried out with glucose (G), cellulose (C) and lignin (L) with Fe,

Ni and Co and without TM supplementation (CT).

Figure 1.2. Volatile fatty acids values measured during the maximum biogas production rate on the experiments.

References Choong Y. Y., Norli I., Abdullah A. Z. and Yhaya M. F. (2016). Impacts of trace element supplementation on

the performance of anaerobic digestion process: A critical review. Bioresource Technology 209,

369-79.

Fermoso F. G., van Hullebusch E. D., Guibaud G., Collins G., Svensson B. H., Carliell-Marquet C., Vink J. P.

M., Esposito G. and Frunzo L. (2015). Fate of Trace Metals in Anaerobic Digestion. In: Biogas

Science and Technology Guebitz GM, Bauer A, Bochmann G, Gronauer A and Weiss S (eds),

Springer International Publishing, pp. 171-95.

Gustavsson J., Shakeri Yekta S., Sundberg C., Karlsson A., Ejlertsson J., Skyllberg U. and Svensson B. H.

(2013). Bioavailability of cobalt and nickel during anaerobic digestion of sulfur-rich stillage for

biogas formation. Applied Energy 112, 473-7.

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Karlsson A., Einarsson P., Schnürer A., Sundberg C., Ejlertsson J. and Svensson B. H. (2012). Impact of

trace element addition on degradation efficiency of volatile fatty acids, oleic acid and phenyl acetate

and on microbial populations in a biogas digester. Journal of Bioscience and Bioengineering 114(4),

446-52.

Kleerebezem R., Joosse B., Rozendal R. and Van Loosdrecht M. C. M. (2015). Anaerobic digestion without

biogas? Reviews in Environmental Science and Bio/Technology 14(4), 787-801.

Pobeheim H., Munk B., Lindorfer H. and Guebitz G. M. (2011). Impact of nickel and cobalt on biogas

production and process stability during semi-continuous anaerobic fermentation of a model

substrate for maize silage. Water Research 45(2), 781-7.

Victoria. Department of Primary Industries. Farm Services V. (2010). Bioenergy from agriculture in Victoria :

the value chain / [compiled by Paul Turnbull, Brian Kearns and Kathryn Robertson]. DPI,

[Melbourne].

Vintiloiu A., Lemmer A., Oechsner H. and Jungbluth T. (2012). Mineral substances and macronutrients in the

anaerobic conversion of biomass: An impact evaluation. Engineering in Life Sciences 12(3), 287-

94.

Ye J., Li D., Sun Y., Wang G., Yuan Z., Zhen F. and Wang Y. (2013). Improved biogas production from rice

straw by co-digestion with kitchen waste and pig manure. Waste Management 33(12), 2653-8.

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Lessons learnt and areas of interest for research in biological sulphate reduction for metal recovery

D. K. Villa-Gomez*

* School of Civil Engineering, University of Queensland, 4067 QLD, Australia.

Keywords: sulphide; metals; bioreactor

The metal mining industry faces the increasing need to process low-grade ores as

accessible higher grade ores become depleted (Pokhrel & Dubey 2013), thus increasing

the volume of fine-grained tailings produced (Mudd 2007). The produced tailings often

lead to environmental problems, including the release of water bearing elevated metal

concentrations to downstream river and groundwater systems. Treatment has not only

an environmental benefit, but also an economic one as it might become commercially

viable given appropriate techniques. The challenge is then to be able to recover the

remaining metals in a way that waste value competes with the direct extraction of the

metals from the source.

Biological sulphate reduction (BSR) is an attractive process for the production of

sulphide to precipitate metals from wastewaters coming from mining activities. To date,

this technology has been assessed at full scale for the recovery of valuable metals such

as Cu, Ni and Zn. Despite this, research gaps are still encountered in this technology for

improving and expanding its scope to: unexplored cost-effective electron donors and

bioreactor configurations that allows metal recovery. One research gap is on the factors

affecting metal sulphide precipitation in bioreactors. Metal sulphide precipitates have low

solubility thus forming small particles that are difficult to recover (Lewis 2010). In

addition, biomass and the substances present in bioreactors hinder metal sulphide

recovery. This review discusses important findings on sulphate reduction and metal

recovery on the principles and factors affecting metal sulphide-precipitation in BSR

bioreactors, comparing them with state-of-the-art literature and suggesting areas of

interest for research.

Current contribution

Figure 1 shows the main pathways affecting metal sulphide recovery in a single stage

bioreactor identified in this study: A) metal complexation with dissolved organic matter,

B) metal precipitation with inorganic salts present in bioreactors and C) metal sulphide

precipitation near the biomass. F-EEM spectroscopy results show that the DOM present

in bioreactors, identified as soluble microbial by-products, (Chen et al. 2003) complexes

with metals. This was observed by the notable decrease in the fluorescence signal after

metal addition. The complexation affects the crystallization and agglomeration of the

metal sulphides and thus, the size of the precipitates for recovery (Villa-Gomez et al.

2014). The role of DOM in metal binding has shown to be significant (Wu et al. 2012),

while the effect of DOM during the metal sulphide precipitation is a largely uncharted

area of research.

Although metal sulphide precipitation is thermodynamically more favourable than other precipitate forms (Lewis 2010), the formation of alternative precipitates that trigger metal sulphide purity has been reported in bioreactors when sulphide is below the stoichiometry. These include: brochantite (Cu4(OH)6SO4) (Mokone et al. 2010),

108

phosphate (Villa-Gomez et al. 2012) and hydroxide precipitation (Samaranayake et al. 2002; Neculita et al. 2008). Figure 1B shows the Zn K-edge XANES spectra of the Zn precipitates obtained when the metals where put in contact with biogenic sulphide at pH 3, 5 and 7. The spectroscopic similarities with sphalerite were highest at pH 5. In contrast, at pH 7 it can be observed that the features A and E suggest the presence of minor amounts of Zn-sorbed hydroxyapatite, Zn:Ca5(PO4)3•(OH) that could be due to the presence of phosphate added as micronutrient to the bioreactor, despite that the sulphide concentration was above the stoichiometry.

Finally, metal sulphide precipitation near the biomass has been identified as a pathway affecting metal recovery (Villa-Gomez et al. 2011). Figure 1C shows the metals concentration in the support material used in the IFB bioreactor operated at high and low sulphide concentration. At low sulphide concentration a readily precipitation where the sulphide is being produced occurs (Villa-Gomez et al. 2011). Biomass function as nucleation seeds, enhancing the crystal formation and growth of the metal sulphides (Bijmans et al. 2009). Metal precipitation in biofilm is of lesser importance at high sulphide concentrations, as a larger fraction of the supersaturation is in the bulk liquid, and thus the precipitation. Although the sulphide concentration ensures prior metal sulphide precipitation avoiding thus free metal ion toxicity, the tendency of the metal sulphides to precipitate within/near the biofilm causes inhibition and decreases microbial population (Utgikar et al. 2002; Villa Gomez et al. 2015).

Areas of interest for research

1. Study of the effect of the use of organics as substrates for BSR on metal

recovery. Full-scale BSR has been applied using hydrogen as electron donor, while its

application using organic waste streams is null (Zhuang et al. 2015). The later can be

cheaper, readily available and with the concomitant to sustain more resilient microbial

communities as compared with single substrates (Sánchez-Andrea et al. 2014; Liu et al.

2015). However, the use of organic compounds as an electron donor might leave

residues and stimulate microbial pathways that can generate exopolymeric substances.

This in turn affects the aggregation/settling of the metal precipitates (Villa-Gomez et al.

2014; Hennebel et al. 2015). Therefore, research on the effects of the use of organics as

substrates for BSR on metal recovery is higly relevant.

2. Study of the physicochemical properties that allow selective metal recovery in

sulphate reducing bioreactors. While selective metal recovery with chemicals is an

ongoing research, investigations in sulphate reducing bioreactors is insufficient.

Research in settling rates, metals complexation and sorption mechanisms in bioreactors

as a way to separate metals from mixtures and allow selective recovery is needed.

These studies will allow the design of sulphate reducing bioreactor configurations that

allow metal recovery. For instance, a bioreactor with different compartments separated in

length according to the different settling rates of the precipitates, as inspired by the

removal of suspended and colloidal materials from wastewater by gravity separation

(Metcalf & Eddy 2002).

3. Fundamental understanding of the microbe-metal interactions. In bioreactors,

interactions between metals and microorganisms are unavoidable and can occur on

extracellular or intracellular basis (Hennebel et al. 2015; Zhuang et al. 2015). The

particular physical-chemical properties conferred by substances associated to

microorganisms may be even deliberately tuned to enhance aggregation/settling

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behaviour for recovery needs (Hennebel et al. 2015). Therefore, its understanding is

essential for improvement of the technology.

Figure 1. Main pathways affecting metal sulphide recovery in the IFB bioreactor. A) F-EEM spectra of the

biogenic sulphide before and after metal addition and SEM image of the biofilm at 10 µm resolution. B) Zn K-

edge XANES spectra for selected models (c-ZnS sphalerite and Zn sorbed on apatite) as compared with the

precipitation experiments (Villa-Gomez et al. 2014). C) Metal precipitation with inorganic salts present in

bioreactors and C) Metal sulphide (MeS) precipitation outside the biofilm at high sulphide concentrations and

MeS precipitation in the biofilm at low sulphide concentrations (Villa-Gomez et al. 2011). Metal concentration

in the biofilm of both IFB reactors at the end of the experiment (Table).

References

Bijmans M. F. M., van Helvoort P.-J., Buisman C. J. N. and Lens P. N. L. (2009). Effect of the sulfide concentration on zinc bio-precipitation in a single stage sulfidogenic bioreactor at pH 5.5. Separation and Purification Technology 69(3), 243-8.

Chen W., Westerhoff P., Leenheer J. A. and Booksh K. (2003). Fluorescence Excitation−Emission Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environmental Science & Technology 37(24), 5701-10.

Hennebel T., Boon N., Maes S. and Lenz M. (2015). Biotechnologies for critical raw material recovery from primary and secondary sources: R&D priorities and future perspectives. New Biotechnology 32(1), 121-7.

Lewis A. E. (2010). Review of metal sulphide precipitation. Hydrometallurgy 104(2), 222-34. Liu Y., Zhang Y. and Ni B.-J. (2015). Zero valent iron simultaneously enhances methane production and

sulfate reduction in anaerobic granular sludge reactors. Water Research 75, 292-300. Metcalf and Eddy (2002). Wastewater Enginering: Treatment & Reuse. McGraw-Hill Education (India) Pvt

Limited. Mokone T. P., van Hille R. P. and Lewis A. E. (2010). Effect of solution chemistry on particle characteristics

during metal sulfide precipitation. Journal of Colloid and Interface Science 351(1), 10-8. Mudd G. M. (2007). Global trends in gold mining: Towards quantifying environmental and resource

sustainability. Resources Policy 32(1–2), 42-56. Neculita C.-M., Zagury G. J. and Bussière B. (2008). Effectiveness of sulfate-reducing passive bioreactors

for treating highly contaminated acid mine drainage: II. Metal removal mechanisms and potential mobility. Applied Geochemistry 23(12), 3545-60.

Pokhrel L. R. and Dubey B. (2013). Global Scenarios of Metal Mining, Environmental Repercussions, Public Policies, and Sustainability: A Review. Critical Reviews in Environmental Science and Technology 43(21), 2352-88.

Samaranayake R., Singhal N., Lewis G. and Hyland M. (2002). Kinetics of biochemically driven metal precipitation in synthetic landfill leachate. Remediation Journal 13(1), 137-50.

Sánchez-Andrea I., Sanz J. L., Bijmans M. F. M. and Stams A. J. M. (2014). Sulfate reduction at low pH to remediate acid mine drainage. Journal of Hazardous Materials 269, 98-109.

110

Utgikar V. P., Harmon S. M., Chaudhary N., Tabak H. H., Govind R. and Haines J. R. (2002). Inhibition of sulfate-reducing bacteria by metal sulfide formation in bioremediation of acid mine drainage. Environmental Toxicology 17(1), 40-8.

Villa-Gomez D., Ababneh H., Papirio S., Rousseau D. P. L. and Lens P. N. L. (2011). Effect of sulfide concentration on the location of the metal precipitates in inversed fluidized bed reactors. Journal of Hazardous Materials 192(1), 200-7.

Villa-Gomez D. K., Papirio S., van Hullebusch E. D., Farges F., Nikitenko S., Kramer H. and Lens P. N. L. (2012). Influence of sulfide concentration and macronutrients on the characteristics of metal precipitates relevant to metal recovery in bioreactors. Bioresource Technology 110(0), 26-34.

Villa-Gomez D. K., van Hullebusch E. D., Maestro R., Farges F., Nikitenko S., Kramer H., Gonzalez-Gil G. and Lens P. N. L. (2014). Morphology, Mineralogy, and Solid-Liquid Phase Separation Characteristics of Cu and Zn Precipitates Produced with Biogenic Sulfide. Environmental Science & Technology 48(1), 664-73.

Villa Gomez D. K., Enright A. M., Rini E. L., Buttice A., Kramer H. and Lens P. (2015). Effect of hydraulic retention time on metal precipitation in sulfate reducing inverse fluidized bed reactors. Journal of Chemical Technology and Biotechnology 90(1), 120-9.

Wu J., Zhang H., Shao L.-M. and He P.-J. (2012). Fluorescent characteristics and metal binding properties of individual molecular weight fractions in municipal solid waste leachate. Environmental Pollution 162(0), 63-71.

Zhuang W.-Q., Fitts J. P., Ajo-Franklin C. M., Maes S., Alvarez-Cohen L. and Hennebel T. (2015). Recovery of critical metals using biometallurgy. Current Opinion in Biotechnology 33, 327-35.

111

Ion-selective electrode for monitoring of cobalt in natural waters released into the environmental as a result of anaerobic

biotechnologies

Cecylia Wardak*, Malgorzata Grabarczyk*, Joanna Lenik*

*Department of Analytical Chemistry and Instrumental Analysis, Faculty of Chemistry, Maria Curie Sklodowska University, Maria Curie Sklodowska 5 Sq., 20-031 Lublin Poland

Keywords: bioproceses; cobalt determination; ion-selective electrode; potentiometry;

Many of trace elements play an important function in all organisms due to their functions in enzyme complexes. One of such element is cobalt which is component of vitamin B12 and enzymes such as carbonic anhydrases, alkaline phosphatases. They are essential by microorganisms involved in methanogenesis [Kirda et al., 2001]. It was shown that deficiency of trace element such as cobalt may result in process instability and decreased biogas production [Pobeheim et al., 2011]. Hence addition of cobalt, nickel and other trace elements to bioreactors is used to create optimal conditions for the microorganisms present in the digester and improve the capacity of biogas plant. On the other hand supplementation with trace metals could lead to an increase of these metals released into the environment. Therefore continuous monitoring of supplemented elements in particular environmental component is necessary.

A quick, simple and cheap analytical method facilitate direct determination of many elements in its ionic form is potentiomery with ion-selective electrode [De Marco et al. 2007; Wardak et al. 2016]. Obtainment of correct determination results requires the use of a sensor with an appropriate detection limit displaying selectivity towards interfering ions which may potentially occur in a tested sample. From a practical point of view, it is also important for the electrode to feature a short response time as well as stability and potential reproducibility.

In this work ion-selective electrode with solid contact (SCISE) sensitive to Co(II) ions is described. SCISE refer to a type of ISEs in which the electroactive membrane is in direct contact with the internal reference electrode and contains no internal solution. In comparison to classic electrodes with internal filling solution, this type of sensors is significantly cheaper, simpler to use and transport and more mechanically resistant. Moreover, they can work in any position, in special pressure conditions and in a wide range of temperatures. For preparation of ion-selective membrane as active substance bis(2,4,4-trimethylpentyl)phosphinic acid (Cyanex 272) was used. It is known as selective extractant for cobalt in the presence of nickel. So incorporation of such substance in polymeric membrane result in improvement of sensor selectivity in towards to nickel ions. The electrode with the membrane composition: Cyanex 272: PVC: plasticizer in the percentage ratio of (wt.) 5:33:62 exhibited the best performance, having a slope of 29.8 mV/decade in the concentration range 5×10-7-1×10-1 M. The limit of detection was 1.6×10-7 M. It had a fast response time of 5-7 s and exhibited stable and reproducible potential. The response of proposed sensor did not depend on pH in the range 2.0-8.0 and showed a good discriminating ability towards Co2+ ion in comparison with Ni2+ and other interfering ions. Proposed Co-SCISE was successfully applied for direct determination of cobalt in natural water samples including river, lake and ground waters. The water samples were collected from river and lake located near biogas plants. The analysis was performed using the standard addition technique. The obtained results were in good agreement with results obtained by adsorptive stripping voltammetry. Thus the proposed electrode provides a good alternative for the determination of cobalt in real samples.

112

References Kida, K., Shigematsu, T., Kijima, J., Numaguchi, M., Mochinaga, Y., Abe, N., Morimura, S., 2001. Influence

of Ni2+

and Co2+

on methanogenic activity and the amounts of coenzymes involved in methanogenesis. J. Biosci. Bioeng. 91, 590–595.

De Marco R., Clarke G., Pejcic B., 2007. Ion-selective electrode potentiometry in environmental analysis Electroanal. 19, 1987-2001.

Pobeheim, H., Munk, B., Johansson, J., Guebitz, G.M., 2010. Influence of trace elements on methane formation from a synthetic model substrate for maize silage. Bioresour. Technol. 101, 836–839.

Wardak C., Grabarczyk M., 2016. Analytical application of solid contact ion-selective electrodes for determination of copper and nitrate in various food products and waters. J. Environ. Sci. Heal. B 51, 519-525.

113

Impact of Nano-ZnO on Biogas Generation in Simulated Landfills

T.T. Onay*1, I. Temizel

1, N.K. Copty

1, B. Demirel

1, T. Karanfil

2

1T.T. Onay*, Boğaziçi University, Institute of Environmental Sciences, Istanbul, Turkey,

Mail: onayturg@boun.edu.tr, Phone: +90 212 3597257

2Environmental Engineering and Earth Science, College of Engineering and Sciences, Clemson University,

Clemson, South Carolina, 29634, USA

Keywords: Landfills; Nano-ZnO; Biogas; Waste stabilisation; Bioreactors

The extensive use of nanomaterials (NMs) in commercial consumer products and their

eventual release to the environment through various pathways have recently raised

concern about the potential impacts of these materials on the environment and human

health. It is estimated that 50 % of NMs used in cosmetics, health, electronic, textile and

water treatment sectors will ultimately be sent to landfills for final disposal after the end

of their useful lives (1). In particular, little data is available about how these materials

behave in landfills under changing environmental conditions during waste stabilization

(2). In this study, two simulated conventional reactors and two bioreactor landfills were

operated using real municipal waste samples at mesophilic temperature (35 ºC) for a

period of about one year to investigate the effect of nano-ZnO on waste stabilization and

biogas generation.

Real municipal solid waste (MSW) samples from a sanitary landfill near Izmit, Turkey

were used in the study. Uncoated powder form of Nano-ZnO was obtained from Sigma-

Aldrich with an average particle size of <100 nm. To accelerate waste stabilization, 1.4

liter of mesophilic anaerobic seed sludge with a dry total solids content of 6% was added

to conventional and bioreactors for microbial seeding. Reactor configurations were

selected as bioreactor with nano-ZnO (R1, Bio with Zn), bioreactor control (R2, Bio),

Conventional control (R3, Con), Conventional with nano-ZnO (R4, Con with Zn). 18 kg of

fresh MSW on wet basis was placed in each reactor. 100 mg nano-ZnO/g of dry waste

was added to the bioreactor and conventional reactors with ZnO addition.

While conventional and bioreactors with and without nano-ZnO showed similar

performance in terms of leachate pH, chemical oxygen demand (COD) and volatile fatty

acids (VFA) concentrations, cumulative gas generation data (Fig. 1) indicate that the

mode of landfill operation influences the rate of waste stabilization, with the bioreactors

starting to produce gas before conventional reactors as an indication of faster waste

stabilization. However, both reactors with nano-ZnO produced somewhat lower amount

of gas compared to the corresponding reactors without the addition of nano-ZnO. The

observed data suggested that the mode of landfill operation had a significant effect on

the waste degradation and biogas generation. Moreover, the presence of nano-ZnO

within the municipal solid waste resulted in approximately %15 less gas generation.

114

Figure 1.1. Cumulative Gas Production

This study investigated the long-term impact of nano-ZnO on the waste stabilization and

biogas production from simulated landfill reactors operated both in conventional and

bioreactor modes. The results of this study indicated that both conventional and

bioreactors with and without nano-ZnO showed similar performance in terms of leachate

pH, COD and VFA concentrations. Zn analysis of the leachate revealed that Zn was

mostly retained within the waste matrix. About 98 and 99% of the Zn was retained within

the bioreactor and conventional reactors, respectively. This suggests that the Zn will

remain in the waste for long periods of time and that any impact the nano-ZnO will have

on waste stabilization will persist in time. On the other hand, the experiments revealed

some differences in gas production. Both conventional and bioreactor landfills with nano-

ZnO produced lower gas compared to the control reactors. Gas generation data

indicated that the organic fraction of the waste started to be degraded in the bioreactor

faster than the conventional reactors due to positive effect of leachate recirculation in

bioreactors. Moreover, methane generation took place in bioreactors before and at a

higher rate compared to the conventional reactors. Even though the same amount of Zn

has been added to both reactor types, no impact was observed in the leachate indicator

parameters and leachate Zn concentrations. On the other hand, reactors without nano-

ZnO addition produced about 15% more gas than the reactors with nano-ZnO,

suggesting that the added 100 mg nano-ZnO/kg of dry waste did have some inhibitory

effects on waste stabilization.

Acknowledgements

The financial support for this research was provided by the Scientific and Technological

Research Council of Turkey (TUBITAK) through Project No 112Y322.

References

(1) Nano-tech Project: Nanotechnology now used in Nearly 500 Everyday Products Analysis; Project on

Emerging Nanotechnologies; Woodrow Wilson International Center for Scholars: Washington, DC,

2007;http://www.nanotechproject.org/process/assets/files/5987/051507nanotechnology_productinven05_

07.pdf

(2) Bolyard, S. C.; Reinhart, D. R.; Santra, S. Behavior of engineered nanoparticles in landfill leachate.

Environ. Sci. Technol. 2013, 47 (15), 8114-8122; DOI 10.1021/es305175e.

0

500

1000

1500

2000

2500

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300Cu

mu

lati

ve G

as P

rod

uct

ion

(L)

Days

Bio with Zn Bio Con Con with Zn

Seed

115

Removal of Trace Metals from Anaerobically Digested Sewage Sludge using Microwave Assisted Extraction with Chelants

Valdas Paulauskas*, Ernestas Zaleckas*, Klaus Fischer**

*Institute of Environment and Ecology, Aleksandras Stulginskis University, Lithuania **Department of Analytical and Ecological Chemistry, Trier University, Germany

Keywords: anaerobic digestion; sewage sludge; trace metals; micro-wave induced extraction; chelants

Alongside with energy crops various types of biowaste substrates can be used as

feedstock for anaerobic biogas production: sewage sludge, organic fraction of municipal

waste, organic waste from food processing industry, animal manures and slurries,

agricultural residues and by-products, etc. In most cases different types of biowaste are

co-digested and the qualitative characteristics, including trace metal (TM) content in

anaerobic digestate, are strongly dependent upon the feedstock used. After anaerobic

digestion TM can be released into the environment via different routes of entry, either

suspended/dissolved in effluents or in a form of biosolids. On-land application of

anaerobically digested sludge (ADS) improves physical, chemical as well as biological

properties of the soil, being a valuable source of plant nutrients, whereas agricultural

application of TM contaminated biosolids poses a great concern, because soil acts as a

transferor, and due to bioaccumulation TM can appear in a food chain (Benitez et al.,

2001; Castro et al., 2009).

Various methods for TM removal from biosolids have been extensively studied in order

to minimize the prospective health risks of biowaste on-land application. One of these is

biowaste washing that involves separation of contaminants from the solids by solubilising

them in a washing solution. Use of organic metal chelants in the wash formulation offers

the advantages of high potential extraction efficiencies (EE), homogeneous treatment of

the polluted matrix, specificity for metals, and low cost because residual wastes requiring

landfilling are minimized (Peters 1999). Aminopolycarboxylic acids and their salts are by

far the dominant group of substances used as chelating agents worldwide. EDTA is a

widely tested chelant and in most cases was highly effective in removal metals from

biosolids, but due to its poor biodegradability it is also very persistent in the environment

and can cause a possible long-term influence on speciation and bioavailability of TM.

Therefore, many investigators suggest that EDTA is unsuitable agent and stake on the

use of easily biodegradable ones (Takahashi et al. 1997; Luo et el., 2006; Zhang et al.,

2008).

In this study MW-assisted washing was applied, aiming to remove TM from ADS

(collected after centrifugation at local Kaunas WWTP) and ADS amended clay loam

(mixture A) and sandy loam (mixture B) soils (ratio 1:1, dry mass basis). “Total”, aqua

regia extractable, TM concentrations in ADS was (mg/kg d.m.): 3.6 Cd, 196.7 Cr, 201.7

Cu, 44.1 Ni, 56.0 Pb, 1503.0 Zn; and exceeded the limit values for on-land sludge

application according to LT normative document LAND 20-2005. Washing experiment

was performed with 0.1M solutions of different chelating agents: citric acid (CA), S-

carboxymethyl-L-cysteine (SCLC), ethylenediaminedisuccinic acid (EDDS),

methylglycinediacetic acid (MGDA) and ethylenediaminetetraacetic acid (EDTA).

Besides the extraction technique, the pH value, temperature and reaction time were test

116

variables. EDTA was used as trisodium salt Na3-EDTA while EDDS – as S,S-isomer,

because the R,R- and S,R-isomers are not so easy biodegradable. Batch extraction was

carried out at room temperature in an orbital shaker at 160 rpm for 8 h, at a solid:liquid

ratio 1:10. Temperature during MW-induced extraction was 50, 100 and 150°C; 500 W

MW power was applied; extraction duration – 15 and 60 min. Finally, either flame or

graphite furnace AAS was used for the determination of TM concentration in the

extraction solutions.

Figure 1. Cu extraction efficiency using 0.1M chelant solution: a – pH 4; b – pH 6; c – pH 10 (no data for

SCLC and EDDS at pH 4 because of precipitation)

Easier than EDTA biodegradable metal-complexing agents, such as EDDS and MGDA,

were found to be capable to extract high amounts of TM from the solids (Fig. 1). Ranking

order of the investigated chelants according to TM EE was as follows:

EDTA≥EDDS>MGDA>>SCLC>CA. Great differences between EE of the investigated

chelants can be explained by unequal strength of complexation with TM. Comparison of

TM removals between different solids showed, that ADS mixture with sandy soil in all

cases demonstrated higher removal efficiency than that from the mixture with higher clay

content. Furthermore, TM removal efficiency from soil-ADS mixtures was better than that

from ADS. This can be explained not only by different granulometric composition, but

also by markedly higher OM content in ADS. This factor is particularly important for Cu,

as data of TM fractionation showed (Östman et al., 2008), that Cu tends to form stable

complexes with soil/sludge organic matter.

Figure 2. Microwave-assisted Cd removal with 0.1M MGDA: a – mixture A; b – mixture B; c – ADS

MW-assisted washing (Fig. 2) revealed improved removal efficiency. Whereas, under

high-temperature conditions (150oC), the investigated aminopolycarboxylic acids seemed

to show lower chelating capability of TM ions, or even such metal-chelator complexes

becoming unstable, and consequently losing their ability to effectively extract TM.

Similarly to batch washing, MW-assisted metal EE from soil-ADS mixtures was also

higher than that from unmixed ADS.

117

References Benitez, E.; Romero, M.; Gomez, M.; Gallardolaro, F.; Nogales, R. (2001). Biosolid and Biosolid Ash as

Sources of Heavy Metals in Plant-Soil System. Water, Air and Soil Pollution, 132, 75-87. Castro, E.; Manas, P.; De las Heras, J. (2009). A Comparison of the Application of Different Waste Products

to Lettuce Crop: Effects on Plant and Soil Properties. Scientia Horticulturae, 123,148-155. Luo, C.; Shen, Z.; Lou, L.; Li, X. (2006). EDDS and EDTA-enhanced Phytoextraction of Metals from

Artificially Contaminated Soil and Residual Effects of Chelants. Environ. Pollution,144, 862-871. Östman, M.; Wahlberg, O.; Martensson A. (2008). Leachability and Metal-Binding Capacity in Ageing Landfill

Material. Waste Management, 28,142-150. Peters R. W. (1999). Chelant Extraction of Heavy Metals from Contaminated Soils. Journal of Hazardous

Materials, 66, 151-210. Takahashi, R.; Fujimoto, N.; Suzuki, M.; Endo, T. (1997). Biodegradabilities of EDDS and other Chelating

Agents.’’ Biosci. Biotech. Biochem., 61, 1957-1959. Zhang, L.; Zhu, Z.; Zhang, R.; Zheng, C.; Zhang, H.; Qiu, Y; Zhao, J. (2008). Extraction of Copper from

Sewage Sludge using Biodegradable Chelant EDDS. J of Environmental Sciences, 20, 970-974.

118

Modeling Landfill Gas Generation and Transport for Energy Recovery

Nadim K. Copty*, Didar Ergene* Turgut T. Onay*

*Institute of Environmental Sciences, Bogazici University, Istanbul, Turkey

Keywords: sanitary landfills; gas generation; anaerobic processes; waste stabilization; stochastic modelling

Large volumes of landfill gas (LFG) are generated as a result of the anaerobic decomposition of the organic fraction in municipal solid waste (MSW). In order to evaluate the potential for energy recovery, it is necessary to accurately estimate the amount of methane that would be generated and collected from the landfill. In practice, predicting the rate of LFG capture and its composition is difficult due to the difficulty in defining the factors influencing waste stabilization such as waste composition, moisture content, pH and the presence of heavy metals that can have inhibitory effects on microbial activity. Another important source of uncertainty is the capture efficiency of the gas collection system which is influenced by the permeability of the waste and soil cover, and landfill design. In this study a stochastic model for the LFG generation and collection is developed. The model uses a Monte Carlo approach to account for input parameter uncertainty, whereby multiple realizations of key input parameters are first generated. LFG generation and transport are then simulated for each realization and used to evaluate probabilistically the rates and efficiency of energy recovery. Because of the complex nature of the relationships describing LFG production and transport, the governing equations were solved numerically. For demonstration, the three-dimensional stochastic model is applied to the Kemerburgaz landfill in Istanbul, Turkey. Three key parameters, namely: the LFG production rate, and the permeabilities of the waste and soil cover, were identified as the main sources of uncertainty. The impact of heavy metal concentrations on the rate of LFG generation and the efficiency of the gas collection system was also evaluated. Results of this modelling effort show that the existing collection system is adequate for the capture of the generated LFG, but that lack of complete information on the factors controlling LFG production is the main source of uncertainty of the amount of energy recovery.

119

Effect of Sewage Sludge Co-Disposal on Waste Degradation in Anaerobic Landfill Bioreactors

Merve Harmankaya, Turgut T. Onay*, Ayşen Erdinçler*

* Boğaziçi University, Institute of Environmental Sciences, Bebek 34342 Istanbul –Turkey

Keywords: Co-disposal; anaerobic digestion; sewage sludge; landfills; municipal solid waste.

The treatment of the water and wastewater results in generation of biosolids which are

also known as sewage sludge. Like many other wastes, sewage sludge creates serious

disposal problems. Landfilling of sewage sludge has significant advantages such as

easier handling, accelerating waste stabilization rate and lower capital investment when

compared to other disposal techniques. Bioreactor landfills are a modification of

conventional landfill systems with the addition of leachate recirculation which promotes

optimum moisture content ensuring energy recovery in the form of biogas and sufficient

nutrients for the microorganisms during waste stabilization. Previous studies indicated

that co-disposal of municipal solid waste and sewage sludge in a single bioreactor landfill

offers potential cost savings, dilution of toxic compounds, increased digestion rate,

increased load of biodegradable organic matter, improved quality of leachate and

enhanced biogas yield.

From this point of view in this study, the impact of co-disposal of sewage sludge with

municipal solid waste was evaluated by using batch tests. To accomplish the objectives

of the study, dewatered sewage sludge samples taken from an advanced biological

wastewater plant, seed sludge samples obtained from the anaerobic digester of a yeast

factory and synthetically prepared municipal solid waste samples were mixed and

digested in 10L anaerobic reactors under mesophilic (32oC) conditions for 100 days. In

order to understand the effect of sludge addition on the efficiency of waste stabilization

process and biogas/methane production and to determine the most promising sludge to

solid waste ratio, COD removal rate, total biogas production, methane yield, and removal

of heavy metals, were investigated for different sludge to waste ratios of 1:4, 1:7, 1:10, in

separate reactors. Two reactors were prepared to be the control reactors, one containing

only sludge and the other only solid waste

At the end of 100-day digestion period, the highest methane yield of 0.38 L CH4/g

CODremoved was obtained in control reactor containing sewage sludge only. The methane

yields in co-digestion rectors with sludge to solid waste ratio of 1:1, 1:4 and 1:7 were

found to be 0.13 L CH4/g CODremoved, 0.3 L CH4/g CODremoved, and 0.1 L CH4/g CODremoved

respectively. The yield was 0.11 L CH4/g CODremoved in reactor containing solid waste

only. The removal efficiency of organics, heavy metals and TKN were also the highest in

the reactor having sludge to solid waste ratio of 1:4. The results of this comperative

study revealed that the co-disposal of sewage sludge in bioreactors is a very promising

technique and 1:4 sludge to waste ratio appears to be the optimum ratio for co-

stabilization of solid wastes and sewage sludges.

120

Thermoelectric Fly Ash, application for the improvement in

Anaerobic Digestion: there are some effect in hydrolytic or

methanogenic stages?

Guerrero L.*, Barahona A.*, Montalvo S.**, Huiliñir C.**, Carvajal A.* and Toledo M.*

* Department of Chemical and Environmental Engineering, Federico Santa Marıa Technical University, Ave. España 1680, Valparaıso, Chile. (lorna.guerrero@usm.cl)

** Department of Chemical Engineering, Santiago de Chile University, Ave. Lib. Bernardo O’Higgins 3363, Santiago, Chile.

Keywords: Anaerobic Contact Reactor; Anaerobic Digestion; Biogas; Thermoelectric Fly Ash.

The use of trace amounts of iron, nickel, cobalt and molybdenum maximize anaerobic biodegradability (Lo et al., 2012), although, the high cost of these salts limits their use. On the other hand, the fly ash, a residue from thermoelectric power plants, contains the aforementioned elements, which makes it interesting to study their application in the anaerobic digestion of sludge (Soto 2013), the objective of this work. First, the optimum concentration of ash is determined via an anaerobic biodegradability test, which is applied to the purge of an activated sludge plant that treats effluents from a poultry slaughterhouse. Subsequently, this optimum concentration is applied to an anaerobic contact reactor (ACR) to determine the maximum organic loading rate (OLR) that the system tolerates. This is then compared to the process without ash. The optimum ash concentration was found to be 100 [mg/L], achieving a 79.4% of biodegradability compared with 59.9% obtained in the sample without ash. With regards to the ACR, the maximum OLR at which the reactor was operable was 6 [kgCOD/m3/d], where a COD reduction of 92.2% was obtained. With ash, the maximum OLR was 10 [kgCOD/m3/d] and a 96.5% of COD reduction was achieved. The influence of ash during the hydrolytic and methanogenic stages of anaerobic digestion was also studied, resulting in only the latter being improved with the addition of 100 [mg/L] of fly ash.

Table 1. Maximum biodegradability of sludge from the purge of an aerobic system, utilizing concentrations

between 10 and 100 [mg/L].

Ash concentration,

[mg/L]

Particle size, [mm]

Maximum biodegradability

[%]

Time*, [d]

0 0.12 – 0.2 58.7 14 0.80 – 1.2 59.9 14

10 0.12 – 0.2 59.2 14 0.80 – 1.2 59.2 14

50 0.12 – 0.2 59.0 14 0.80 – 1.2 58.3 14

100 0.12 – 0.2 79.1 14 0.80 – 1.2 78.9 12

*Refers to the time that it takes to reach maximum biodegradability.

121

Figure 1. Production of methane and the variation in organic matter concentration, measured as COD,

during the batch start-up of the anaerobic contact reactor.

Figure 2. Applied OLR to the anaerobic reactor and removal percentage of COD obtained, operating with

and without ash.

Figure 3. Degradation of suspended solids in the hydrolytic reactor without ash (A), and with 100 [mg/L] of

ash at the different HRT studied.

Table 2. Percentage of increase of VFA in reactors with and without ash, at different HRT.

Reactor HRT [h]

36 30 24 18 12 6

Without ash 24.95% 7.65% 59.84% 67.91% 20.33% 11.67%

With ash, 100 mg/L 4.98% 10.48% 10.75% 40.46% 58.15% 9.33%

interesante. Puede situar el cuadro de texto en

cualquier lugar del documento. Use la ficha

Herramientas de dibujo para cambiar el formato

del cuadro de texto de la cita.]

D

E

G

R

A

D

A

T

I

O

N

%

SS

[g/L

]

SS

[g/L

]

A B

122

Table 3. Specific Methanogenic Activity in reactors with and without ash.

Reactor VSS [g/L]

Velocity of methane production [mL CH4/h]

SMA [gCOD/gVSS/d]

Without ash 3.08 3.89 0.79

With ash 3.04 5.89 1.21

References Lo, H. M., Chiu, H. Y., Lo, S. W., & Lo, F. C. (2012). Effects of micro-nano and non micro-nano MSWI ashes

addition on MSW anaerobic digestion. Bioresource technology, 114, 90-94. Soto P. (2013) Efecto de cenizas de termoeléctrica y escorias de acería como micronutrientes en la

digestión anaerobia de lodos. Memoria para Ingeniero Civil Químico, UTFSM, Valparaíso, Chile.

123

Effect of Selected Trace Elements on Methane Productivity

at Anaerobic Digestion of Maize and Grasses

S. Ustak, J. Munoz, J. Sinko

Crop Research Institute, Drnovska 507/73, 16106 Prague 6 – Ruzyne, Czech Republic

Keywords: anaerobic digestion; trace metals; maize; grasses; methane productivity.

Introduction

Anaerobic digestion (AD) is an attractive processing technology for biogas production, biological waste and waste-water treatment. It is the result of an incomplete stepwise conversion of biodegradable compounds, whose final products are mainly CH4 and CO2, and in lesser extent water-soluble NH3 and H2S. This conversion involves the syntrophic interaction between fermentative, acetogenic and methanogenic bacteria, whose growth and activity are dependent on an optimum supply of macro- and micro-nutrients. However, the doses of nutrients are specific to microorganism physiological groups. Therefore, it should be dedicated great attention to optimal nutrients dose into biogas reactor, since poor microbiological activity threats AD process stability, causing a decrease in biogas and methane production.

The main problem of raw materials and additive substrates, containing different trace elements concentrations (sometimes hazardous), is to determine the allowable range of their content in view of economically efficient and ecologically safe use at biogasification. It is especially actual for additive substrates designed to improve AD process, for biogas yield increase at simultaneously ensuring the required digestate quality for fertilizer use. This does necessary a content balance analysis of monitored trace elements to achieve optimal parameters of input raw materials and output products according to the regulations and limits in environmental protection.

Material and methods

The solution focused on additive effect of eight selected trace elements (Fe, Mn, Cu, Ni, Zn, Co, Mo, Se) on methane productivity at AD for two main plant raw materials used by biogas production – maize and grasses. The determination of additive quantity was based on existing limit values in the Czech Republic for digestates from biogas stations (see elements and doses in mg/kg of digestate dry matter: copper 150, nickel 50, zinc 600, molybdenum 20 and cobalt 30). For other elements doses were determined by the literature data (selenium 10, iron 2000 and manganese 500). Those were the same in dose mixtures at individual elements.

It is well known that biological tests using additives, to verify the efficiency increase, are mainly more evaluable using less productive substrates, for better expression differences. Therefore, it was specifically chosen a devalued year old maize silage for expecting about 1/3 of lower biogas yield (after experimentally confirmed). Further, as test substrate was selected dry grass meal mixture from various perennial grasses (red-canary grass, ryegrass and fescue). Then were established and conducted extensive biogasification trials with those substrates in 13 different additive variants, either individually (variants No. 2-8) or in mixtures (variants No. 9-14). For comparison was used a control variant without any additives (variant No. 1). The repetitions were at individual variants from 4 to 6.

Biogasification laboratory trials were performed on the assembly of 48 three-liter glass anaerobic fermenters heated at mesophilic temperature (37±1 °C) and stirred 15 minutes every two hours. The biogas and methane production testing was carried out according to the methodology VDI-4630. The input sample ratio of organic solids to inoculum was about 3:10. The inoculum was an adapted fermented substrate from

124

operating biogas station (using manure, maize and grass silage - ratio 40:40:20). The total fermentation period was set at 35 days. The chemical analyzes were carried out by common methodological procedures.

Results and Discussion

It was monitored biogas and methane yield and digestate quality for fertilizer requirements. The effect of the tested additives significantly varied by the uniform raw materials. The tested doses did not have an appreciable effect on the suppression of AD, and in many cases contrarily this activity promoted in grasses. Maize had an increase of methane yield only in Mo (21 %) and Fe (16 %), whereas at grasses in all variants of single elements (except in Se), wherein increased from 10 % in Cu up to 23-24 % in Co, Mo and Zn. In the rest (from 10 to 14) representing mixed additives, showed lower averages than in the control variant, even always statistically inconclusive.

Table 1. Specific methane production (LN / kg of dry matter) of maize silage and grasses

in different variants of chemical additives

Vari-ants Additive

Maize silage (devalued) Perennial grasses

Average (LN/kg suš.)

Standard error SE

(LN/kg suš.)

The ratio of average to control

Average (LN/kg suš.)

Standard error SE

(LN/kg suš.)

The ratio of average to control

1 Control 182 8 1,00 220 11,7 1,00

2 Fe 211 10 1,16 271 6,0 1,23

3 Mn 160 6 0,88 263 6,2 1,19

4 Cu 165 12 0,91 243 18,2 1,11

5 Ni 171 6 0,94 279 4,2 1,27

6 Zn 165 12 0,91 286 4,2 1,30

7 Co 170 5 0,93 290 13,6 1,32

8 Mo 221 5 1,21 285 4,8 1,30

9 Se 183 4 1,00 206 9,0 0,94

10 Fe-Mn 166 6 0,91 190 5,5 0,86

11 Cu-Ni-Zn 152 5 0,84 199 8,5 0,90

12 Co-Mo-Se 168 10 0,92 211 5,1 0,96

13 Cu-Ni-Zn-Co-Mo-Se 176 7 0,97 197 3,4 0,89

14 Fe-Mn-Cu-Ni-Zn-Co-Mo-Se 176 6 0,97 172 10,8 0,78

The largest decrease (29 %) was observed in the variant 14, at mixtures of all monitored elements. Apparently, this decline was caused by interactions of elements mixtures between each other and by the fermented substrate. The effect on the bioavailability of individual variants at individual elements based on their proportion of solubility showed a highest bioavailability of selected micro-nutrients by Co and Mo (average of 37 % and 34 % at soluble part), and the lowest by Cu and Zn (average of 17 % and 18 % at soluble part). The identified bioavailability for all elements was sufficient because it was not proven a significant impact of the increased methane production. A greater influence on the methane yield was exhibited by the type and content of individual elements.

Acknowledgment

The article was developed within the project of Czech Ministry of Education No. LD15164 and the Czech Ministry of Agriculture No. RO0416.

125

Improvement of the microbal activity by cobalt suplementation on the biomethanization of olive mill solid waste

F. Pinto-Ibieta*, A. Serrano**, D. Jeison***, R. Borja**, F.G. Fermoso**

* Escuela de Procesos Industriales, Facultad de Ingeniería, Universidad Católica de Temuco, Casilla 15-D,

Temuco, Chile

**Instituto de la Grasa (C.S.I.C.). Seville, Spain

***Departamento de Ingeniería Química, Universidad de La Frontera, Casilla 54-D, Temuco, Chile

Keywords: Trace metal; Methane production; Microbial activity; Cobalt; Olive Mill Solid Waste

Due to the low trace metals concentration in the Olive Mill Solid Waste (OMSW), a

proposed strategy to improve its biomethanization is the supplementation of key metals

to enhance the microorganism activity. Among essential trace metals, cobalt has been

reported to have a crucial role in anaerobic degradation, given its relevance for different

reactions within the acetogenic and methanogenic steps (Fermoso et al. 2008). Wide

ranges of total cobalt concentration, from 0.05 up to 3.0 mg/kg, have been reported as

recommended for the anaerobic process (Pobeheim et al. 2010; Demirel and Scherer

2011). Such differences could be explained by the fact that most of the authors only

report the total concentration provided, and not refer to the amount of cobalt that is

bioavailable for the microorganisms.

The objective of this study was to evaluate the effect of cobalt supplementation to

OMSW, as a way to improve its anaerobic degradation to biomethane. This research is

mainly focused on the connection between fractionation of cobalt and the biological

response presented in a digester. The influence of cobalt on the response of the

microorganisms involved in anaerobic degradation process was evaluated by

biochemical methane potential (BMP) tests. The BMP tests were supplemented with nine

increasing concentrations of cobalt (total added cobalt concentrations: 0, 0.18, 0.30, 0.6,

2.9, 5.9, 17.7, 29.5 and 58.9 mg Co2+/L). These concentration values were selected in

order to study a range from limiting to toxic cobalt levels.

Figure 1. Concentration of dissolved and total cobalt against the concentration of added cobalt.

The use of added cobalt concentration as a control parameter to assess the digester

performance is hindered by the involved physicochemical processes, which could entail

a wrong estimate of the required cobalt. This makes necessary to study the correlation

between the biological response of the microorganisms and easily measurable fractions

of cobalt, such as dissolved and total. Figure 1 shows the relationship between added

cobalt versus the dissolved and total cobalt present at the end of the BMP tests. Total

cobalt has been defined as the sum of the added cobalt and cobalt already contained in

Total cobalt (mg/L)

0 10 20 30 40 50 60 70

Dis

solv

ed c

ob

alt

(m

g/L

)

0.0

0.2

0.4

0.6

0.8

1.0

Ad

ded

co

ba

lt (

mg

/L)

0

10

20

30

40

50

60

70

Dissolved Cobalt

Added Cobalt

126

the OMSW and anaerobic inoculum. Concentration of dissolved cobalt reaches a

maximum value of 0.32 mg/L, at a total cobalt concentration of 29.5 mg/L (Figure 1).

Further addition of cobalt to the BMP test did not result in higher dissolved cobalt

concentrations.

The addition of cobalt, in the studied concentration range, entailed an improvement in

the methane yield coefficient from 9% up to 30%, when compared to the reactor without

addition of cobalt. The highest methane yield coefficient was obtained for a dissolved

cobalt concentration of 0.028 mg/L (0.35 mg total cobalt/L), reaching a value of 460 ± 40

mL/g VS (Figure 2A). Figure 2B shows the values of methane production rate (Rm)

against the dissolved and total cobalt concentration for each set of the BMP tests.

Dissolved cobalt in a range from 0.018 to 0.035 mg/L (0.24 - 0.65 mg total cobalt/L)

entailed a marked improvement of Rm, of around 25%, compared to the value obtained in

the test without cobalt addition (0.013 and 0.06 mg/L of dissolved and total cobalt,

respectively) (Figure 2B). The accumulation of dissolved cobalt at values higher than

0.16 mg/L (17.7 mg total cobalt/L) affected to the process decreasing Rm up to values

14% and 30% lower than the experiment without addition of cobalt and the test with the

highest observed methane production rate, respectively (Figure 4). The enhancement

could be explained due to cobalt activates enzymes essential for the microorganisms

involved in methanogenesis, such as vitamin B12, coenzyme A, and enzyme methyl-

H4SPT: CoM methyltransferase (Florencio et al. 1994; Pobeheim et al. 2011; Nordell et

al. 2016). These enzymes are known to play a key role in the degradation of volatile fatty

acids (Florencio et al. 1994). The validity of the obtained values of dissolved cobalt

concentrations depends on maintaining a constant chemical environment of the system.

If relevant variations happened, the relation between dissolved cobalt concentration and

the biological response must be redefined, although the trends in this relation should not

be affected.

Figure 2. (A) Variation of the maximum methane production, and (B) variation of the maximum methane production rate for the different concentrations of total and dissolved cobalt.

References Demirel, B.; Scherer, P. (2011). Trace element requirements of agricultural biogas digesters during biological

conversion of renewable biomass to methane. Biomass Bioenerg. 35(3), 992-998. Fermoso, F.G.; Collins, G.; Bartacek, J.; O'Flaherty, V.; Lens, P. (2008). Acidification of methanol-fed

anaerobic granular sludge bioreactors by cobalt deprivation: Induction and microbial community dynamics. Biotechnol. Bioeng. 99(1), 49-58.

Florencio, L.; Field, J.A.; Lettinga, G. (1994). Importance of cobalt for individual trophic groups in an anaerobic methanol-degrading consortium. Appl. Environ. Microb. 60(1), 227-234.

Total cobalt (mg/L)0.01 0.1 1 10 100

mL

ST

P C

H4

/g V

S

250

300

350

400

450

500

550

Dissolved cobalt (mg/L)

0.01 0.1 1

Total cobalt

Dissolved cobalt

Total cobalt (mg/L)

0,01 0,1 1 10 100

Rm

(m

L C

H4

/g V

S·d

)

60

70

80

90

100

110

Dissolved cobalt (mg/L)

0,01 0,1 1

Total cobalt

Dissolved cobalt

127

Nordell, E.; Nilsson, B.; Nilsson Påledal, S.; Karisalmi, K.; Moestedt, J. (2016). Co-digestion of manure and industrial waste – The effects of trace element addition. Waste Manage. 47, 21-27.

Pobeheim, H.; Munk, B.; Lindorfer, H.; Guebitz, G.M. (2011). Impact of nickel and cobalt on biogas production and process stability during semi-continuous anaerobic fermentation of a model substrate for maize silage. Water Res. 45(2), 781-787.

128

Zinc as additive in table olive fermentation

J. Bautista-Gallego*, F. Rodríguez-Gomez, V. Romero-Gil, A. Benítez-Cabello, A., B. Calero-

Delgado, R. Jiménez Díaz, Garrido-Fernández, F.N. Arroyo-López

*Food Biotechnology Department, Instituto de la Grasa, Agencia Estatal Consejo Superior de Investigaciones Científicas, Seville, Spain

Keywords: Preservation; Table olive packaging; Zinc

BACKGROUND: Zinc chloride has been used previously as a preservative in directly

brined olives with promising results in different varieties of table olives (Bautista-Gallego

et al., 2011 and 2013). In addition, zinc salts have also demonstrated an ability to retain

the green colour of thermally processed fruits and vegetables, due to the formation of

zinc-chlorophyll-derivative complexes with a stable, highly bright green colour (Gallardo-

Guerrero et al., 2013). The aim of this work was to evaluate the possible addition of

different concentrations of ZnCl2 to fully fermented Spanish-style Manzanilla green olives

and to improve this traditional fermentation process.

RESULTS: The presence of ZnCl2 affected the physico-chemical characteristics of the

products; the presence of the Zn led to lower pH and titratable acidity values than the

control (Figure 1A and 1B) but did not produce clear trends in the colour parameters

(data not shown).

Figure 1. Changes in pH and titratable acidity during shelf life.

Regarding to the microbial dynamics, no Enterobacteriaceae were found in any of the

treatments evaluated. At the different ZnCl2 concentrations, the lactic acid bacteria

reached slight higher levels than control while its presence showed a similar effect than

potassium sorbate against the yeast population (Figure 2).

With respect to organoleptic characteristics, the presentations containing ZnCl2 were not

differentiated from the traditional product and displayed a similar performance than the

industrial treatment (Figure 3).

129

Figure 2. Changes in LAB and yeasts during shelf life.

Figure 3. Organoleptic profile of all treatments at the end of the shelf life.

CONCLUSION: The metallic ion Zinc had a similar trend than potassium sorbate as a

yeast inhibitor in green Spanish-style olives, showing clear presentation style dependent

behaviour for this property. Its presence produced significant changes in chemical

parameters but scarcely affected colour or sensory characteristics. Therefore. the use of

this trace metal could be a good option to substitute potassium sorbate on table olive

industry.

References Bautista-Gallego, J.; Arroyo-López, F.N.; Romero-Gil, V.; Rodríguez-Gómez, F.; Garrido Fernández A.

(2011). Evaluating the effect on Zn chloride as a preservative in cracked table olive packing. J. Food Protect., 74, 2169–2176.

Bautista-Gallego, J.; Moreno-Baquero, J.M.; Garrido-Fernández, A.; López-López, A. (2013). Development of a novel Zn fortified table olive product. LWT–Food Sci. Technol. 50, 264-271.

Gallargo-Guerrero, L.; Gandul-Rojas, B.; Moreno-Baquero, J.M.; López-López, A.; Bautista-Gallego, J.; Garrido-Fernández, A. (2013). Pigment, physicochemical, and microbiological changes related to the freshness of cracked table olives. J. Agric. Food Chem., 61, 3737-3747.

0

5

10Colour

Odour

Acid

Salty

Bitter

Hardness

Fibrousness

Cruchiness Control

0.050%

0.075%

0.100%

130

Monitoring of Co(II) content which can cause harm to the environment after anaerobic processes

M. Grabarczyk, C. Wardak

Department of Analytical Chemistry and Instrumental Analysis, Chemical Faculty, Maria Curie-Sklodowska University, Lublin, POLAND

Keywords: cobalt, trace concentration; determination; voltammetry; environmental water samples

Trace metals are essential for enhanced metabolism in anaerobic processes,

nevertheless in practice they are often added to anaerobic digesters in excessive

amounts. On the other hand, trace metals should only be dosed below the inhibitory or

toxic level as an excessive metal content in the sludge will cause harm to the

environment, and minimize the quality and applicability of anaerobic products in

agriculture, e.g. digestate used as fertilizer [Thanh P.M. et al., 2015]. Trace metals

considered the most essential in anaerobic digestion are transition metals i.e. Fe, Ni, and

Co [Oleszkiewicz and Sharma,1990], so their effect on environment as the harming

element should be taken into account. This, in turn, means that the appropriate

procedures to determination of these elements in different natural samples are wanted.

One of the greatest problems associated with trace metal determination in such samples

is matrix containing the organic substances like surface active compounds and humic

substances. Surface active substances enter to the system mainly as a result of humic

activity, humic substances are formed during the decay of plant and animal remains in

soil.

Electrochemical methods such as adsorptive stripping voltammetry (AdSV), have been

used to measure the ‘free’ metal ion concentration in rainwater, river water, and

wastewater. The AdSV technique is relatively simple, fast, and inexpensive. However,

the limitations of this technique are due to interference from organic matrix of samples. In

this communication the proposition of the very sensitive and simple procedure of Co(II)

determination in environmental water samples is given. In the literature data a number of

AdSV procedures for trace cobalt determination have been developed, however the

most significant drawback of this method is their susceptibility to organic substances,

such as surfactants and humic substances [Korolczuk M. et al., 2004; Korolczuk M. et

al., 2005]. In order to eliminate the mentioned above interferences in the proposed

procedure the Amberlite XAD-7 resin was exploited. The whole measurement was based

on the following scheme:

- to the 10 mL of analysed water samples with added acetic buffer (pH = 4.6) the 0.5 g of

resin is introduced and whole solution is mixed for 5 min. At that time the organic matrix

is adsorbed on the resin while the cobalt ions remain in the solution.

- the 5 mL of the prepared sample and supporting electrolyte (containing

dimethylglyoxime (DMG) – complexing agent and ammonium buffer pH = 8.8) are

introduced to voltammetric cell and deaerated during the 5 min.

- the AdSV measurement was performed through adsorption of the previously formed

complexes Co(II)-DMG on working electrode at the -0.7 V for 30 s, and next registration

131

of voltammetric signal by changing potential from -0.7 V to -1.2 V. The intensity of the

obtained peak on the voltamperogram is directly proportional to the concentration of

Co(II) in the sample.

The proposed method was successfully applied for determination of Co(II) in estuarine

water certified reference material and river water samples collected in eastern areas of

Poland, from the rivers Bystrzyca and Czerniejowka.

References Thanh P.M.; Ketheesan B.; Yan Z.; Stuckey D. (2015). Tracemetal speciation and bioavailability in anaerobic

digestion: A review. Biotechnol. Adv., 34, 122-136. Oleszkiewicz, J.A.; Sharma, V.K.; (1990). Stimulation and inhibition of anaerobic processes by heavy

metals—a review. Biol. Wastes, 31, 45–67. Korolczuk M.; Moroziewicz A.; Grabarczyk M.; Kutyła R. (2004). Adsorptive stripping voltammetric

determination of cobalt in the presence of dimethylglioxime and cetyltrimethylammonium bromide, Anal. Bioanal. Chem., 380, 141-145.

Korolczuk M.; Moroziewicz A.; Grabarczyk M.; Paluszek K. (2005). Detremination of Traces of Cobalt in the Presence of Nioxime and Cetyltrimethylammonium bromide by Adsorptive Stripping Voltammetry, Talanta, 65, 1003-1007.

132

Determination in river water samples of trace Ni(II) which can get to environment from the residue after anaerobic processes

M. Grabarczyk, C. Wardak, J. Wasąg

Department of Analytical Chemistry and Instrumental Analysis, Chemical Faculty, Maria Curie-Sklodowska University, Lublin, POLAND

Keywords: nickel, trace concentration; determination; voltammetry; river water samples

Trace metals are essential for the growth of anaerobic microorganisms, and their roles in

anaerobic processes have been studied extensively. A lack of deep understanding of the

relationship between trace metal speciation and bioavailability of metals in an aerobic

digestion can result in practice that the trace metals are often supplemented in excess

quantities to support microbial growth in aerobic processes. On the other hand, trace

metals should only be dosed below the inhibitory or toxic level as an excessive metal

content in the sludge will cause pollution of the environment, and minimize the quality

and applicability of anaerobic products in agriculture, e.g. through digestate used as

fertilizer [Thanh P.M. et al., 2015].

One of the trace metals considered as the most essential in anaerobic digestion is nickel

as this element can get to environment from the residue after anaerobic processes

[Oleszkiewicz J.A. and Sharma V.K., 1990]. In the proposed communication the

procedure of Ni(II) determination in environmental water samples is described. The main

objective was drawing attention to interferences related to the presence of complicate

matrix of environmental samples. The constituents of such matrix which presence

distorts the determination of metal ions are surface active substances and humic

substances. In the proposed method of the trace Ni(II) determination in environmental

water samples the adsorptive stripping voltammetry was exploited and in order to

eliminate organic matrix the adsorptive properties of Amberlite XAD-7 resin have been

used. The measurement is composed of two steps. In the first step the preliminary

mixing analysed sample with resin in the acidic conditions was performed. In this step

the organic matrix is adsorbed on the resin while the nickel ions remains quantitatively in

the solution. During the second step the solution after mixing with resin is introduced to

voltammetric cell and the adsorptive stripping measurement is performed as follows:

- 5 min. deaeration of solution containing analysed sample, dimethylglyoksime (DMG)

added as a complexing agent and ammonium buffer pH = 7.4

- accumulation of Ni(II)-DMG complexes on the working electrode at the -0.675 V for 60

s.

- registration of the voltammetric signal by changing potential from -0.675 V to -1.0 V.

The intensity of the obtained peak on the voltammogram is directly proportional to the

concentration of Ni(II) in the sample.

The obtained data are presented in Fig. 1 and show the influence of organic substances

on voltammetric signal of nickel using the procedure with preliminary mixing with resin

and without mixing with resin. As you can see, thanks to the preliminary mixing with resin

we are able to obtain an undisturbed signal of Ni(II) even in the presence of 10 mg L −1

133

biosurfactant (Rhamnolipids) and 7 mg L−1 of humic substances (HS). Without mixing

with the resin, the total decay of signal of Ni(II) in the presence of 2 mg L −1 surfactant

(Triton X-100) and 3 mg L−1 humic substances (HS) was observed.

0 2 4 6 8 10

c / mg L-1

0

20

40

60

80

100

rela

tive s

ign

al o

f N

i(II

) / %

ab

c

d

Figure 1. The influence of Rhamnolipids (b, c) and HS (a, d) on relative signal of 1 × 10−7

mol L−1

Ni(II) using

the procedure without (a, b) and with (c, d) preliminary mixing with resin.

References Thanh P.M.; Ketheesan B.; Yan Z.; Stuckey D. (2015). Tracemetal speciation and bioavailability in anaerobic

digestion: A review. Biotechnol. Adv., 34, 122-136. Oleszkiewicz, J.A.; Sharma, V.K.; (1990). Stimulation and inhibition of anaerobic processes by heavy

metals—a review. Biol. Wastes, 31, 45–67.

134

Anaerobic digestion of animal wastes after rendering in a CSTR–

experimental and modeling results

A. Spyridonidis, Th. Skamagkis, L. Lambropoulos and K. Stamatelatou*

Democritus University of Thrace, Department of Environmental Engineering, Vas. Sofias 12, 67132 Xanthi,

Greece

*Author for correspondence: tel:+302541079315, e-mail: astamat@env.duth.gr

Rendering is a thermal treatment process under pressure (140οC, 4-5 bar for 20 min)

applied for the hygienization of slaughterhouse by-products prior to disposal. The

product of rendering, even after the largest part of fats is removed, contains lipids and

proteins which result in a high COD (150 to 250 gCOD/l), making it an ideal biogas

feedstock. However, the high content of lipids and protein could make the anaerobic

digestion process prone to inhibition. A system consisting of a mesophilic (38-39οC)

continuous stirred tank reactor (CSTR) has been operated at a hydraulic retention time

of 21.5±2.14 d under increasing organic loading (50.0-149.6 g COD/L) to study the

dynamics of the process. Kinetic experiments were performed to study the kinetics of

volatile fatty acids using the anaerobic digestion model (ADM1). The model (adjusted

under an organic loading rate (OLR) of 2.32 gCOD/L/d) was used to predict the dynamic

behavior of the CSTR successfully, confirming that the anaerobic digestion system was

stable even at a high organic loading rate (6.96 gCOD/L/d).

135

Simulation of biogas production from animal wastes after

rendering in an anaerobic contact process

A. Spyridonidis and K. Stamatelatou*

Democritus University of Thrace, Department of Environmental Engineering, Vas. Sofias 12, 67132 Xanthi, Greece

*Author for correspondence: tel:+302541079315, e-mail: astamat@env.duth.gr

Slaughterhouse by-products must be hygienized prior to disposal and this is commonly

achieved by treating them thermally under pressure (140οC, 4-5 bar for 20 min). After

rendering, the largest part of fats is removed and is utilized to produce biodiesel. The

residual is homogenized mixture of lipids and proteins where lipids have been emulsified

yielding fast kinetics. The organic load is high (COD: 150 to 250 gCOD/l) making it an

ideal biogas feedstock. To compensate for a potential inhibition due to the high level of

lipids and ammonia nitrogen, recirculation of mixed anaerobic liquor after settling was

applied to a continuous stirred tank reactor (CSTR) operated at mesophilic (38-39οC)

conditions at a hydraulic retention time of 21.5±2.14 d under increasing organic loading

(50.0-149.6 g COD/L). The anaerobic digestion model (ADM1) has been already adjusted

based on kinetic experiments performed on a single CSTR and was used in this study to

verify the dynamic response of the anaerobic contact process (CSTR with mixed liquor

recirculation after settling). The model successfully predicted the anaerobic contact

process in terms of biogas production, and concentrations of COD, ammonia nitrogen

and volatile fatty acids.

136

Effect of anaerobic digestate dosage over sunflower germination

A. Serrano*, F.G. Fermoso*, R. Borja*, R. Arias-Calderón**

*Instituto de la Grasa (C.S.I.C.). Seville, Spain.

**INIAV - Instituto Nacional de Investigação Agrária e Veterinária, I. P. Elvas, Portugal

Keywords: anaerobic digestate; germination test; olive mill solid waste; sunflower seed; phytotoxicity

Anaerobic digestion is a microbial process where an organic substrate is degraded to

methane and a stabilized digestate in absence of oxygen. The digestate obtained after

the anaerobic process is mainly composed by microorganisms, partially stabilized

substrate and water. Due to its content of carbon, nitrogen, and phosphorus, digestate

has been proposed as an organic amendment for agricultural applications. The

agricultural use of digestate should be considered as a recovery process instead of a

simple disposal method (Alburquerque et al., 2012a). Nevertheless, anaerobic digestate

can produce disgusting smells, and its viscosity and high humidity could complicate its

application to soils (Tchobanoglous and Kreith, 2002, Abdullahi et al., 2008). Moreover,

digestate can cause an extensive range of deleterious effects on crops, such as

prevention or delay of seed germination, plant death or marked reductions in growth

(Alburquerque et al., 2012a).

Due to the complex composition of digestate, many factors could produce the

undesirable effects. The main aim of this research was to determinate the effect of

digestate dosage over the germination of sunflower seeds. Moreover, different assays

were carried out to identify the factors from the digestate that were related to the effect of

the digestate over the germination.

Table 1.1. Main analytical characterization of OMSW digestate.

pH 8.55 ± 0.05 TC mg C/L 2934 ± 25 C5 mg/L 118 ± 5

Alkalinity mg CaCO3/L 3850 ± 50 TOC mg C/L 2020 ± 25 i- C5 mg/L 216 ± 5

Soluble COD mg O2/L 7555 ± 225 C2 mg/L 141 ± 5 C6 mg/L nd

BOD5 mg O2/L 1700 ± 75 C3 mg/L 751 ± 5 i- C6 mg/L nd

TS mg/L 10,195 ± 85 C4 mg/L 74 ± 5

VS mg/L 4935 ± 125 i- C4 mg/L 56 ± 5

nd, non-detected

The used digestate was obtained from an anaerobic reactor CSTR treating Olive Mill

Solid Waste (OMSW) (Tables 1 and 2). Germination assays were carried out by placing

30 seeds in Petri dishes. 40 ml of OMSW digestate, at different concentrations, were

added to each petri dish. In addition to digestate, four factors were assayed, i.e. salinity,

acids concentration, metal content and 3,4-Dihydroxyphenylglycol (DHG). Petri dishes

were unlit incubated for 48 hours at 25ºC. After this period, the length of the radicles of

each seed was measured. Positive germinations were considered when the radicle

reached more than 5 mm.

137

Table 2. Trace elements content (mg/L) in OMSW digestate

Al 3.38 Ca 126 Cu 0.27 Mg 34.7 Ni 0.15 Sn 1.04

As 0.23 Cd 0.01 Fe 12.1 Mn 0.27 P 68.3 Sr 0.40

B 63.4 Co 0.12 Hg nd Mo nd Pb nd V nd

Ba 0.12 Cr 0.11 K 381 Na 2388 S 51.5 Zn 1.96

nd, non-detected

Figure 1A shows the variation of the length of the radicle for the different dosages of

OMSW digestate. Concentrations in a range from 0.1% to 5% of OMSW digestate

resulted in a short positive effect over the length of the radicle. Higher OMSW digestate

concentration entailed a marked decrease of the length of the radicle. Figure 1B shows

the percentage of germinated seeds for the different dosages of OMSW digestate. As

can be seen, germination percentage presented values higher than 80% for OMSW

digestate concentrations up to 50%. Higher concentrations resulted in a decreased of the

germination percentage.

Figure 1 (A) Variation of the length of the radicule (%) and (B) percentage of germinated seeds (%) against

to the concentration of OMSW digestate

Figure 2 shows the correlation coefficients obtained by comparing the results of the

germinated seeds percentage and the variation of the length of the radicle for the studied

factors and the OMSW digestate assays. As can be seen, the variation of the length of

the radicle was directly related with the factor Acids, which presented a correlation

coefficient of 0.9641. Germinated seeds percentages were related to the factors salinity

and acids, although the relation is not so clear than the obtained for the variation of the

length. Metals and DHG factors seem not to be related to the effect of the OMSW

digestate over the germination of the sunflower seeds.

138

Correlation coefficient (length of the radicle)

-1,0 -0,5 0,0 0,5 1,0Co

rrela

tio

n c

oeff

icie

nt

(G

erm

inate

d s

eed

s)

-1,0

-0,5

0,0

0,5

1,0

Salinity

DHG

Acids

Metals

Figure 2. Correlation coefficients of the germinated seeds and the length of the radicle for the studied factors and the OMSW digestate.

References Abdullahi, Y.A.; Akunna, J.C.; White, N. A.; Hallett, P.D.; Wheatley, R. (2008). Investigating the effects of

anaerobic and aerobic post-treatment on quality and stability of organic fraction of municipal solid waste as soil amendment. Bioresource Technol., 99, 8631-8636.

Alburquerque, J.A.; de la Fuente, C.; Ferrer-Costa, A.; Carrasco, L.; Cegarra, J.; Abad, M.; Bernal, M.P. (2012a). Assessment of the fertiliser potential of digestates from farm and agroindustrial residues. Biomass Bioenerg., 40, 181-189.

Alburquerque, J.A.; de la Fuente, C.; Bernal, M. P. (2012b). Chemical properties of anaerobic digestates affecting C and N dynamics in amended soils. Agric. Ecosyst. Environ., 160, 15-22.

Tchobanoglous, G.; Kreith, F. (2002). Handbook of Solid Waste Management, Mcgraw-hill.