When Bioelectrochemical Systems Meet Extremophiles, Possibilities and Challenges

Gaofeng Ni

Linnaeus University Dissertations [framsida] No 325/2018 Linnaeus University Dissertations No 325/2018 When extremophiles, and challenges systems meet bioelectrochemical possibilities Gaofeng Ni

[Huvudtitel]

Linnaeus University Press When bioelectrochemical systems

[rygg] meet extremophiles, possibilities and challenges [baksida]

Lnu.se

Lnu.se ISBN: 978-91-88761-82-8 (print) 978-91-88761-83-5 (pdf)

linnaeus university press

When bioelectrochemical systems meet extremophiles, possibilities and challenges

Linnaeus University Dissertations

No 325/2018

WHEN BIOELECTROCHEMICAL SYSTEMS MEET EXTREMOPHILES, POSSIBILITIES AND CHALLENGES

GAOFENG NI

LINNAEUS UNIVERSITY PRESS

Linnaeus University Dissertations

No 325/2018

WHEN BIOELECTROCHEMICAL SYSTEMS MEET EXTREMOPHILES, POSSIBILITIES AND CHALLENGES

GAOFENG NI

LINNAEUS UNIVERSITY PRESS

Abstract Ni, Gaofeng (2018). When bioelectrochemical systems meet extremophiles, possibilities and challenges, Linnaeus University Dissertations No 325/2018, ISBN: 978-91-88761-82-8 (print), 978-91-88761-83-5 (pdf). Written in English. Extremophiles are microorganisms live and thrive in extreme environments that are harsh and hostile to most forms of life on earth (e.g. low pH, low temperature, high pH and high salinity). They have developed strategies to obtain nutrients and conserve energy to sustain life under these adverse conditions. Such metabolic capabilities are valuable to be exploit for industrial applications such as the remediation of environmental pollutions, which typically bring about extreme physicochemical conditions. The advancing technology bioelectrochemical systems can utilize the microbial metabolism to oxidize a substrate while simultaneously recover electrical energy or produce a useful product in an electrochemical set-up. It enables the remediation of pollutions, and its integration with extremophiles has opened up a wide range of possibilities to tackle various industrial waste streams with extreme conditions in an environmentally friendly manner. Inorganic sulfur compounds such as tetrathionate, thiocyanate and sulfide that originate from mining, metal refinery and petroleum industries are toxic and hazardous to the recipient water body and human health if discharged untreated. The remediation of these three compounds with bioelectrochemical systems that incorporates extremophiles was investigated in three separate studies of this thesis. 16S rRNA gene amplicon sequencing, metagenomics and metatranscriptomics are utilized to profile the microbial communities, and to understand their metabolic potential and states. Tetrathionate degradation with acidophilic microorganisms in microbial fuel cells at pH 2 was demonstrated in the first study of this thesis. Electricity was When bioelectrochemical systems meet extremophiles, possibilities and produced from the oxidation of tetrathionate, facilitated by the anodic challenges microbiome. 16S rRNA gene amplicon sequencing showed that this community Doctoral Dissertation, Department of Biology and Environmental Science, was dominated by members of the genera Thermoplasma, Ferroplasma, Linnaeus University, Växjö, 2018 Leptospirillum, Sulfobacillus and Acidithiobacillus. Metagenomic analysis reconstructed genomes that were most similar to the genera Ferroplasma, ISBN: 978-91-88761-82-8 (print), 978-91-88761-83-5 (pdf) Acidithiobacillus, Sulfobacillus and Cuniculiplasma. Together with Published by: Linnaeus University Press, 351 95 Växjö metatranscriptomic analysis, it was indicated that this microbial community was Printed by: DanagårdLiTHO, 2018 metabolizing tetrathionate and other intermediate sulfur compounds via multiple pathways, the electrons released from oxidation were suggested to be transferred to the electrode via soluble electron shuttles. In addition, the Ferroplasma-like population in this study was suggested to be active in metabolising inorganic sulfur compounds and synthesizing soluble electron shuttles. Since characterized Ferroplasma species do not utilize inorganic sulfur compounds, the anodic compartment might have selected a novel Ferroplasma population. Abstract Ni, Gaofeng (2018). When bioelectrochemical systems meet extremophiles, possibilities and challenges, Linnaeus University Dissertations No 325/2018, ISBN: 978-91-88761-82-8 (print), 978-91-88761-83-5 (pdf). Written in English. Extremophiles are microorganisms live and thrive in extreme environments that are harsh and hostile to most forms of life on earth (e.g. low pH, low temperature, high pH and high salinity). They have developed strategies to obtain nutrients and conserve energy to sustain life under these adverse conditions. Such metabolic capabilities are valuable to be exploit for industrial applications such as the remediation of environmental pollutions, which typically bring about extreme physicochemical conditions. The advancing technology bioelectrochemical systems can utilize the microbial metabolism to oxidize a substrate while simultaneously recover electrical energy or produce a useful product in an electrochemical set-up. It enables the remediation of pollutions, and its integration with extremophiles has opened up a wide range of possibilities to tackle various industrial waste streams with extreme conditions in an environmentally friendly manner. Inorganic sulfur compounds such as tetrathionate, thiocyanate and sulfide that originate from mining, metal refinery and petroleum industries are toxic and hazardous to the recipient water body and human health if discharged untreated. The remediation of these three compounds with bioelectrochemical systems that incorporates extremophiles was investigated in three separate studies of this thesis. 16S rRNA gene amplicon sequencing, metagenomics and metatranscriptomics are utilized to profile the microbial communities, and to understand their metabolic potential and states. Tetrathionate degradation with acidophilic microorganisms in microbial fuel cells at pH 2 was demonstrated in the first study of this thesis. Electricity was When bioelectrochemical systems meet extremophiles, possibilities and produced from the oxidation of tetrathionate, facilitated by the anodic challenges microbiome. 16S rRNA gene amplicon sequencing showed that this community Doctoral Dissertation, Department of Biology and Environmental Science, was dominated by members of the genera Thermoplasma, Ferroplasma, Linnaeus University, Växjö, 2018 Leptospirillum, Sulfobacillus and Acidithiobacillus. Metagenomic analysis reconstructed genomes that were most similar to the genera Ferroplasma, ISBN: 978-91-88761-82-8 (print), 978-91-88761-83-5 (pdf) Acidithiobacillus, Sulfobacillus and Cuniculiplasma. Together with Published by: Linnaeus University Press, 351 95 Växjö metatranscriptomic analysis, it was indicated that this microbial community was Printed by: DanagårdLiTHO, 2018 metabolizing tetrathionate and other intermediate sulfur compounds via multiple pathways, the electrons released from oxidation were suggested to be transferred to the electrode via soluble electron shuttles. In addition, the Ferroplasma-like population in this study was suggested to be active in metabolising inorganic sulfur compounds and synthesizing soluble electron shuttles. Since characterized Ferroplasma species do not utilize inorganic sulfur compounds, the anodic compartment might have selected a novel Ferroplasma population. Next, thiocyanate degradation with psychrophilic microorganisms in microbial fuel cells at 8 °C was demonstrated for the first time. Electricity generation alongside with thiocyanate degradation facilitated by the anodic Investigation and dialogues are the best counters against fear microbiome was observed. 16S rRNA gene amplicon sequencing and metatranscriptomics suggested that Thiobacillus was the predominant and most of uncertainty. active population. mRNA analysis revealed that thiocyanate was metabolized primarily via the ‘cyanate’ degradation pathway; the resultant sulfide was oxidized; ammonium was assimilated; carbon dioxide was fixed as carbon source. It was also suggested by mRNA analysis that the consortium used multiple mechanisms to acclimate low temperature such as the synthesis of cold shock proteins, cold inducible proteins and molecular chaperones. Finally, sulfide removal with haloalkaliphilic microorganisms in microbial electrolysis cells operated at pH 8.8 to 9.5 and with 1.0 M sodium ion was “Life is nothing but an electron looking for a place to rest.” investigated. The anodic microbiome was hypothesized to facilitate current Albert Szent-Györgyi generation by the oxidation of sulfide and of intermediate sulfur compounds to sulfate, which was supported by chemical analysis and microbial profiling. Dominant populations from the anode had 16S rRNA gene sequences that aligned within the genera Thioalkalivibrio, Thioalkalimicrobium, and Desulfurivibrio, which are known for sulfide oxidation. Intriguingly, Desulfurivibrio dominated the electrode-attached community, possibly enriched by the electrode as a selecting pressure. This finding suggested a novel role of this organism to carry out sulfide oxidation coupled to electron transfer to the electrode. These three studies demonstrated the possibilities of utilizing extremophilic bioelectrochemical systems to remediate various inorganic sulfur pollution streams. The advancing molecular microbiological tools facilitated the investigation towards the composition and metabolic state of the microbial community. Challenges remain in a more thorough understanding regarding the metabolism of extremophiles (e.g. sulfur metabolism and extracellular electron transfer) and better energy recovery in bioelectrochemical systems.

Keywords: acidophiles, psychrophiles, haloalkaliphiles, bioelectrochemical systems, microbial sulfur metabolism, 16S rRNA gene amplicon sequencing, metagenomics, metatranscriptomics.

Next, thiocyanate degradation with psychrophilic microorganisms in microbial fuel cells at 8 °C was demonstrated for the first time. Electricity generation alongside with thiocyanate degradation facilitated by the anodic Investigation and dialogues are the best counters against fear microbiome was observed. 16S rRNA gene amplicon sequencing and metatranscriptomics suggested that Thiobacillus was the predominant and most of uncertainty. active population. mRNA analysis revealed that thiocyanate was metabolized primarily via the ‘cyanate’ degradation pathway; the resultant sulfide was oxidized; ammonium was assimilated; carbon dioxide was fixed as carbon source. It was also suggested by mRNA analysis that the consortium used multiple mechanisms to acclimate low temperature such as the synthesis of cold shock proteins, cold inducible proteins and molecular chaperones. Finally, sulfide removal with haloalkaliphilic microorganisms in microbial electrolysis cells operated at pH 8.8 to 9.5 and with 1.0 M sodium ion was “Life is nothing but an electron looking for a place to rest.” investigated. The anodic microbiome was hypothesized to facilitate current Albert Szent-Györgyi generation by the oxidation of sulfide and of intermediate sulfur compounds to sulfate, which was supported by chemical analysis and microbial profiling. Dominant populations from the anode had 16S rRNA gene sequences that aligned within the genera Thioalkalivibrio, Thioalkalimicrobium, and Desulfurivibrio, which are known for sulfide oxidation. Intriguingly, Desulfurivibrio dominated the electrode-attached community, possibly enriched by the electrode as a selecting pressure. This finding suggested a novel role of this organism to carry out sulfide oxidation coupled to electron transfer to the electrode. These three studies demonstrated the possibilities of utilizing extremophilic bioelectrochemical systems to remediate various inorganic sulfur pollution streams. The advancing molecular microbiological tools facilitated the investigation towards the composition and metabolic state of the microbial community. Challenges remain in a more thorough understanding regarding the metabolism of extremophiles (e.g. sulfur metabolism and extracellular electron transfer) and better energy recovery in bioelectrochemical systems.

Keywords: acidophiles, psychrophiles, haloalkaliphiles, bioelectrochemical systems, microbial sulfur metabolism, 16S rRNA gene amplicon sequencing, metagenomics, metatranscriptomics.

Table of Contents

List of papers ...... 3 Authors’ contributions to the papers ...... 4 Additional papers not included in the thesis ...... 5 List of abbreviations ...... 6 Introduction ...... 7 Microbe-mineral interactions and extracellular electron transfer ... 7 Bioelectrochemical Systems (BESs) ...... 8 Extremophiles ...... 10 Acidophiles ...... 11 Alkaliphiles and haloalkaliphiles ...... 12 Psychrophiles ...... 13 ISC remediation with extremophilic BESs ...... 14 Bioremediation of acidic inorganic sulfur compounds ...... 15 Biological thiocyanate removal at low temperature ...... 16 Biological desulfurization at high pH and salinity ...... 16 The molecular toolbox, pros and cons ...... 17 16S rRNA gene amplicon sequencing ...... 17 Metagenomics and metatranscriptomics ...... 19 Aims of this thesis ...... 21 Overview of methodology ...... 22 Operation and electrochemical characterization of the BESs ...... 22 Choice of inoculum ...... 23 16S rRNA gene amplicon sequencing ...... 24 Metagenomics ...... 24 Metatranscriptomics ...... 24 Overview of the experimental conditions and molecular methods ...... 26 Results and discussion ...... 27 The acidophilic MFC study (Papers I & II) ...... 27 Electrochemical characterization and 16S rRNA gene amplicon sequencing (Paper I) ...... 27 Taxonomy and relative abundance (Paper II) ...... 28 Unraveling metabolic processes (Paper II) ...... 28 Summary of the acidophilic MFC study (Papers I & II) ...... 30

1 Table of Contents

1 Psychrophilic MFC study (Paper III)...... 32 Electrochemical characterization and 16S rRNA gene amplicon sequencing List of papers ...... 32 Metatranscriptomics of the anodic psychrophilic microbiome ...... 33 Summary of the psychrophilic MFC study (Paper III)...... 34 I. Gaofeng Ni, Stephan Christel, Pawel Roman, Zhen Lim Wong, Martijn FM Bijmans, and Mark Dopson. "Electricity generation from an inorganic sulfur Haloalkaliphilic MEC study (Paper IV) ...... 36 compound containing mining wastewater by acidophilic microorganisms." Electrochemical characterization for sulfide removal ...... 36 Research in Microbiology 167 (2016): 568-575. Development of microbial community based on 16S rRNA gene amplicon sequencing ...... 37 II. Gaofeng Ni, Domenico Simone, Daniela Palma, Elias Broman, Xiaofen Wu, Post analysis of the electrode surface ...... 38 Stephanie Turner, and Mark Dopson. "A novel inorganic sulfur compound Summary of the haloalkaliphilic MEC study (Paper IV) ...... 38 metabolizing Ferroplasma-like population is suggested to mediate extracellular Overview of electrochemical performance (Papers I to IV) ...... 39 electron transfer via soluble shuttles." Manuscript.

General conclusions and future outlook ...... 41 III. Gaofeng Ni, Sebastian Canizales, Elias Broman, Domenico Simone, Viraja Acknowledgements ...... 43 R. Palwai, Daniel Lundin, Margarita Lopez-Fernandez, Tom Sleutels, and Mark Dopson. "Microbial community and metabolic activity in thiocyanate degrading References ...... 47 low temperature microbial fuel cells." Manuscript.

IV. Gaofeng Ni, Pebrianto Harnawan, Laura Seidel, Annemiek Ter Heijne, Tom Sleutels, Cees J. N. Buisman, and Mark Dopson. "Haloalkaliphilic microorganisms assist sulfide removal in a microbial electrolysis cell." Manuscript.

2 3 Psychrophilic MFC study (Paper III)...... 32 Electrochemical characterization and 16S rRNA gene amplicon sequencing List of papers ...... 32 Metatranscriptomics of the anodic psychrophilic microbiome ...... 33 Summary of the psychrophilic MFC study (Paper III)...... 34 I. Gaofeng Ni, Stephan Christel, Pawel Roman, Zhen Lim Wong, Martijn FM Bijmans, and Mark Dopson. "Electricity generation from an inorganic sulfur Haloalkaliphilic MEC study (Paper IV) ...... 36 compound containing mining wastewater by acidophilic microorganisms." Electrochemical characterization for sulfide removal ...... 36 Research in Microbiology 167 (2016): 568-575. Development of microbial community based on 16S rRNA gene amplicon sequencing ...... 37 II. Gaofeng Ni, Domenico Simone, Daniela Palma, Elias Broman, Xiaofen Wu, Post analysis of the electrode surface ...... 38 Stephanie Turner, and Mark Dopson. "A novel inorganic sulfur compound Summary of the haloalkaliphilic MEC study (Paper IV) ...... 38 metabolizing Ferroplasma-like population is suggested to mediate extracellular Overview of electrochemical performance (Papers I to IV) ...... 39 electron transfer via soluble shuttles." Manuscript.

2 3 Authors’ contributions to the papers Additional papers not included in the thesis Paper I Concept and design: Mark Dopson and Martijn Bijmans Laboratory work: Gaofeng Ni, Stephan Christel, Zhen Lim Wong, and Pawel Roman Mark Dopson, Gaofeng Ni, and Tom HJA Sleutels. "Possibilities for Bioinformatic analysis: Gaofeng Ni extremophilic microorganisms in microbial electrochemical systems." FEMS Data analysis and interpretation: Gaofeng Ni and Mark Dopson Microbiology Reviews 40 (2015): 164-181. Manuscript drafting: Mark Dopson and Gaofeng Ni Vytautas Abromaitis, V. Racys, P. Van der Marel, Gaofeng Ni, Mark Dopson, Paper II A. L. Wolthuizen, and R. J. W. Meulepas. "Effect of shear stress and carbon Concept and design: Mark Dopson surface roughness on bioregeneration and performance of suspended versus Laboratory work: Gaofeng Ni and Stephanie Turner attached biomass in metoprolol-loaded biological activated carbon systems." Bioinformatic analysis: Gaofeng Ni, Domenico Simone, Daniela Palma, Chemical Engineering Journal 317 (2017): 503-511. Elias Broman, and Xiaofen Wu Data analysis and interpretation: Gaofeng Ni and Mark Dopson Elias Broman, Abbtesaim Jawad, Xiaofen Wu, Stephan Christel, Gaofeng Ni, Manuscript drafting: Gaofeng Ni, Mark Dopson, Domenico Simone, and Margarita Lopez-Fernandez, Jan-Eric Sundkvist, and Mark Dopson. "Low Stephanie Turner temperature, autotrophic microbial denitrification using thiosulfate or thiocyanate as electron donor." Biodegradation 28 (2017): 287-301. Paper III Concept and design: Mark Dopson and Gaofeng Ni Laboratory work: Gaofeng Ni, Sebastian Canizales, and Margarita Lopez- Fernandez Bioinformatic analysis: Gaofeng Ni, Domenico Simone, Elias Broman, Viraja R. Palwai, and Daniel Lundin Data analysis and interpretation: Gaofeng Ni, Mark Dopson, and Tom Sleutels Manuscript drafting: Gaofeng Ni, Mark Dopson, Domenico Simone, and Elias Broman

Paper IV Concept and design: Mark Dopson, Tom Sleutels, Annemiek Ter Heijne, and Gaofeng Ni Laboratory work: Gaofeng Ni, Pebrianto Harnawan and Laura Seidel Bioinformatic analysis: Laura Seidel and Gaofeng Ni Data analysis and interpretation: Gaofeng Ni, Tom Sleutels, Annemiek Ter Heijne, and Mark Dopson Manuscript drafting: Gaofeng Ni

4 5 Authors’ contributions to the papers Additional papers not included in the thesis Paper I Concept and design: Mark Dopson and Martijn Bijmans Laboratory work: Gaofeng Ni, Stephan Christel, Zhen Lim Wong, and Pawel Roman Mark Dopson, Gaofeng Ni, and Tom HJA Sleutels. "Possibilities for Bioinformatic analysis: Gaofeng Ni extremophilic microorganisms in microbial electrochemical systems." FEMS Data analysis and interpretation: Gaofeng Ni and Mark Dopson Microbiology Reviews 40 (2015): 164-181. Manuscript drafting: Mark Dopson and Gaofeng Ni Vytautas Abromaitis, V. Racys, P. Van der Marel, Gaofeng Ni, Mark Dopson, Paper II A. L. Wolthuizen, and R. J. W. Meulepas. "Effect of shear stress and carbon Concept and design: Mark Dopson surface roughness on bioregeneration and performance of suspended versus Laboratory work: Gaofeng Ni and Stephanie Turner attached biomass in metoprolol-loaded biological activated carbon systems." Bioinformatic analysis: Gaofeng Ni, Domenico Simone, Daniela Palma, Chemical Engineering Journal 317 (2017): 503-511. Elias Broman, and Xiaofen Wu Data analysis and interpretation: Gaofeng Ni and Mark Dopson Elias Broman, Abbtesaim Jawad, Xiaofen Wu, Stephan Christel, Gaofeng Ni, Manuscript drafting: Gaofeng Ni, Mark Dopson, Domenico Simone, and Margarita Lopez-Fernandez, Jan-Eric Sundkvist, and Mark Dopson. "Low Stephanie Turner temperature, autotrophic microbial denitrification using thiosulfate or thiocyanate as electron donor." Biodegradation 28 (2017): 287-301. Paper III Concept and design: Mark Dopson and Gaofeng Ni Laboratory work: Gaofeng Ni, Sebastian Canizales, and Margarita Lopez- Fernandez Bioinformatic analysis: Gaofeng Ni, Domenico Simone, Elias Broman, Viraja R. Palwai, and Daniel Lundin Data analysis and interpretation: Gaofeng Ni, Mark Dopson, and Tom Sleutels Manuscript drafting: Gaofeng Ni, Mark Dopson, Domenico Simone, and Elias Broman

4 5 List of abbreviations Introduction

BES: bioelectrochemical system Microbe-mineral interactions and extracellular CBB cycle: Calvin–Benson–Bassham cycle CE: Coulombic efficiency electron transfer CIP: cold-inducible proteins The fascinating interplay of microbe-mineral interactions via redox CSP: cold shock proteins transformations is a widely spread phenomenon through which microorganisms COD: chemical oxygen demand obtain energy and nutrients from their environment for growth while shaping DNA: deoxyribonucleic acid their surroundings. Knowledge of this process is valuable to unravel the EDS: energy dispersive X-ray spectroscopy microbial metabolism and is also fundamental to understand the cycling of EET: extracellular electron transfer elements such as iron and sulfur, which are often present in such minerals. For ISC: inorganic sulfur compound instance, minerals that contain ferric iron (Fe(III)) or manganese (Mn(IV)) MEC: microbial electrosynthesis cells or microbial electrolysis cells oxides are redox-active (Shi et al., 2016) and support microorganisms carrying MFC: microbial fuel cell out the dissimilatory reduction of these metals coupled to the oxidation of mRNA: messenger ribonucleic acid organic matter (Nealson et al., 2002). In addition, the moderate thermophile NGS: next-generation sequencing Sulfobacillus acidophilus is able to bring about the reductive dissolution of PCR: polymerase chain reaction ferric iron containing minerals such as ferric hydroxide, jarosite, and goethite RNA: ribonucleic acid (Bridge and Johnson, 1998). In these microorganisms, electrons derived from rRNA: ribosomal ribonucleic acid the oxidation of electron donors are transported from the quinone and quinol SEM: scanning electron microscope pool in the cytoplasmic membrane, across the cell envelope to extracellular XRD: X-ray diffraction minerals that function as electron sinks. This process is termed extracellular electron transfer (EET) (Lovley, 2008; Shi et al., 2016) and is a key to the microbe-mineral interactions. In the last three decades, EET has been extensively investigated among two microbial genera Shewanella and Geobacter, revealing a complex and yet not fully-understood process. Currently, there is evidence for three microbial EET mechanisms: 1) direct transfer via certain outer membrane multi-heme c-type cytochromes (Gorby et al., 2006; Holmes et al., 2006; Bretschger et al., 2007b; Richter et al., 2009; Shi et al., 2016), whose diverse functions include electron transfer (Shi et al., 2007); 2) the formation of electrically conductive pili that directly transfer electrons from the cell envelop to a solid surface (Reguera et al., 2005; Gorby et al., 2006; Bretschger et al., 2007b; Richter et al., 2009; Vargas et al., 2013); and 3) the secretion of electron carriers such as redox-active quinone or flavin molecules, which among other functions can mediate electron transport without the direct contact between the microorganisms and extracellular electron sinks (Newman and Kolter, 2000; Myers and Myers, 2004; Marsili et al., 2008). Although EET is a wide-spread phenomenon, other microorganisms are rarely investigated for EET mechanisms. These microorganism capable of carrying out EET are terms electrochemically-active microorganisms.

6 7 List of abbreviations Introduction

6 7 Bioelectrochemical Systems (BESs) solid anode (the only electron acceptor available). A variety of microorganisms Despite being wide-spread and long-existing, the first incidence of utilizing have been found in MFC anodes, particularly in the phyla Proteobacteria and microbial EET to generate an electrical current was reported in 1911, in which Firmicutes (Dopson et al., 2016). Apart from the extensively studied genera electrical energy was harvested using galvanic cells from the metabolism of Geobacter and Shewanella, other microbial populations such as Pseudomonas Saccharomyces and other microorganisms grown in nutrient media (Potter, (Rabaey et al., 2004), Rhodopseudomonas (Xing et al., 2008), Clostridium 1911). This finding gave birth to the research on microbial fuel cells (MFCs) or (Park et al., 2001), and Thermincola (Wrighton et al., 2008) were also identified in its broader sense bioelectrochemical system (BES) (Rabaey et al., 2007). This in MFC anodes. The electrons derived from the anodic oxidation flow through research field did not receive substantial attention until the 1990s when the an external wire to the cathode where reduction reactions take place. Common published scientific articles about MFCs grew exponentially (Fig. 1), due to its terminal electron acceptors include oxygen that is reduced to water or huge potential in energy recovery and sustainable treatment of various ferricyanide that is reduced to ferrocyanide (Logan et al., 2006). While the wastewaters (Pant et al., 2010; Li et al., 2014). cathodic reaction in MFCs are mostly abiotic, microbial populations being key players in cathodic compartments were also reported, including members of Sphingobacterium, Acinetobacter, Acidovorax (Rabaey et al., 2008), and Geobacter (Gregory et al., 2004). In many cases, the anodic and cathodic reactions are separated by an ion exchange membrane, which also prevents the crossover of components from anodic and cathodic electrolytes. Apart from recovering electrical energy, the uses of BESs are versatile. With additional energy input, BESs can also be used to drive processes that are not thermodynamically favorable, such as the production of ammonia (Kuntke et al., 2018), hydrogen (Call and Logan, 2008), methane (Geppert et al., 2016), acetate (Jourdin et al., 2015), and longer chain fatty acids (Raes et al., 2017). These systems were categorized as microbial electrosynthesis cells or microbial electrolysis cells (MECs, Fig. 2B) (Nevin et al., 2010).

Fig. 1 Published scientific studies from scholar.google.com using the keywords “bioelectrochemical system” and “microbial fuel cell”.

An MFC typically consists of an anodic and a cathodic compartment, separated by an ion exchange membrane (Fig. 2A) (Logan et al., 2006). All microorganisms gain energy from the oxidation of an electron donor (lower potential) to an electron acceptor (higher potential). To maximize their energy gain, microorganisms will, within their capacity, attempt to deliver electrons to the highest potential possible (Rabaey, 2010). In the anodic chamber of an MFC, microorganisms oxidize the electron donors and deliver the electrons to the Fig. 2 Schematic drawings of an MFC (A) and an MEC (B), two subtypes of BES.

8 9 Bioelectrochemical Systems (BESs) solid anode (the only electron acceptor available). A variety of microorganisms Despite being wide-spread and long-existing, the first incidence of utilizing have been found in MFC anodes, particularly in the phyla Proteobacteria and microbial EET to generate an electrical current was reported in 1911, in which Firmicutes (Dopson et al., 2016). Apart from the extensively studied genera electrical energy was harvested using galvanic cells from the metabolism of Geobacter and Shewanella, other microbial populations such as Pseudomonas Saccharomyces and other microorganisms grown in nutrient media (Potter, (Rabaey et al., 2004), Rhodopseudomonas (Xing et al., 2008), Clostridium 1911). This finding gave birth to the research on microbial fuel cells (MFCs) or (Park et al., 2001), and Thermincola (Wrighton et al., 2008) were also identified in its broader sense bioelectrochemical system (BES) (Rabaey et al., 2007). This in MFC anodes. The electrons derived from the anodic oxidation flow through research field did not receive substantial attention until the 1990s when the an external wire to the cathode where reduction reactions take place. Common published scientific articles about MFCs grew exponentially (Fig. 1), due to its terminal electron acceptors include oxygen that is reduced to water or huge potential in energy recovery and sustainable treatment of various ferricyanide that is reduced to ferrocyanide (Logan et al., 2006). While the wastewaters (Pant et al., 2010; Li et al., 2014). cathodic reaction in MFCs are mostly abiotic, microbial populations being key players in cathodic compartments were also reported, including members of Sphingobacterium, Acinetobacter, Acidovorax (Rabaey et al., 2008), and Geobacter (Gregory et al., 2004). In many cases, the anodic and cathodic reactions are separated by an ion exchange membrane, which also prevents the crossover of components from anodic and cathodic electrolytes. Apart from recovering electrical energy, the uses of BESs are versatile. With additional energy input, BESs can also be used to drive processes that are not thermodynamically favorable, such as the production of ammonia (Kuntke et al., 2018), hydrogen (Call and Logan, 2008), methane (Geppert et al., 2016), acetate (Jourdin et al., 2015), and longer chain fatty acids (Raes et al., 2017). These systems were categorized as microbial electrosynthesis cells or microbial electrolysis cells (MECs, Fig. 2B) (Nevin et al., 2010).

Fig. 1 Published scientific studies from scholar.google.com using the keywords “bioelectrochemical system” and “microbial fuel cell”.

8 9 Attempts have been made to demonstrate the potential of using MFCs to treat forms of life on earth. However, such chemically active environments offer municipal wastewater. For instance, two 4 L MFCs were operated in a energy and niches for a variety of extremophiles. municipal wastewater treatment plant for 400 days that removed 65-70% of the COD and achieved a positive energy balance (Zhang et al., 2013). However, the In addition to those described below, categories of extremophiles include technology is still in its infancy because challenges remain in reactor design and thermophiles which are dominated by archaea, that grow at temperatures above material cost; as well as in operational obstacles such as temperature fluctuation 45 °C; oligotrophs that grow in extremely nutrient-poor environments with and the fouling of the membrane and electrode; in addition, currently the extremely long generation times. They are also significant to the understanding understanding of the underlying microbiology is still inadequate (Logan and of the microbiota as well as offering inspirations to industrial applications. Regan, 2006). However, they are beyond the scope of this thesis.

Overall, by making use of microbial catalysis, BESs is a sustainable and Acidophiles promising technology in energy recovery. Due to the separation between the Acidophiles are a group of extremophilic microorganisms from all three oxidation and reduction reactions; the electrochemical manipulation; and the domains of life (Bacteria, Archaea, and Eukarya) that are defined as having an diverse microbial metabolism, BESs offer a wide range of combinations for optimum growth pH < 5, with extreme acidophiles having an optimum pH < 3 substrate oxidation (organic and inorganic) and the reduction/synthesis of end (Dopson et al., 2016). They are often found in places where sulfide is abundant, products (Rabaey et al., 2009; Dopson et al., 2016). In addition, the uses of such as sulfide mineral ores (Dopson et al., 2003), acid sulfate soil (Wu et al., electrodes that are in close contact with the microorganisms enables the 2013), or geothermal sulfur-rich locations e.g. sulfur cauldron at Yellowstone technology itself to be an excellent platform to investigate EET. U.S.A. (Johnson et al., 2003) or the highly acidic river “Rio Tinto” from Spain (Fig. 3). These locations are characterized by extremely high acidity because the oxidation of sulfide and other intermediate sulfur compounds produces Extremophiles protons. In order to grow, acidophiles must maintain circumneutral intracellular pH that results in a pH gradient of several pH units across the cellular membrane Extremophiles are intriguing microorganisms; they are able to live and thrive in (Baker-Austin and Dopson, 2007). Therefore, they have developed several harsh and hostile environments (e.g. extremely high or low pH or temperature, strategies to acclimate to such conditions. A passive mechanism is cytoplasmic extremely high salinity etc.). There are theories hypothesizing that buffering with amino acids (e.g. lysine, histidine and arginine) that sequester extremophiles are associated with early forms of life on earth. For instance, the protons (Baker-Austin and Dopson, 2007; Slonczewski et al., 2009). formation of pyrite from hydrogen sulfide and ferrous ion is an exergonic Acidophiles can also utilize a variety of active mechanisms including a highly reaction, allowing the formation of simple reduced carbon molecules, thus proton-impermeable membrane (Konings et al., 2002) that could even be altered offering possibilities to support autotrophic life forms. This is known as the to resist protons upon acid stress (Chao et al., 2008); the generation of inside “pyrite as the first energy source for life” hypothesis (Wächtershäuser, 1988; positive transmembrane electrical potential that reduces the influx of protons Russell et al., 1990) The positively charged surfaces of pyrite also offers (Baker-Austin and Dopson, 2007;Slonczewski et al., 2009); active proton catalyzing capacity for metabolism (Wachtershauser, 1990). Environments rich pumping that removes excess protons (Michels and Bakker, 1985; Tyson et al., in pyrite are commonly associated with high acidity because the oxidation of 2004); as well as the repair of DNA and protein damage from low pH and the sulfide is an acid generating reaction. In addition, if the released Fe(II) is stabilization of intracellular enzymes (reviewed in Baker-Austin and Dopson, exposed to oxygen, Fe(III) is formed that further oxidizes pyrite and thus, (2007)). Since acidophiles are often involved in the dissolution of metal- accelerates the production of acid. Sustaining life in such milieu requires containing minerals, these environments are often abundant in metals. strategies to copy with high acidity. Furthermore, high acidity favors metal dissolution and therefore, acidophiles need strategies to cope with high metal concentrations (Dopson and Holmes, Another hypothesis is that life originated from submarine hydrothermal vents 2014). Passive mechanisms for metal resistance include: the utilization of (Martin et al., 2008). Such environments are characterized by high temperature, sulfate anion to complex free metal cations (Dopson et al., 2014); and the use acidity, rich in dissolved transition metals such as Fe(II) and Mn(II), and high of extracellular polymeric substances in the biofilm to sorb metals (Harrison et concentrations of magmatic CO2, H2S, and dissolved H2. These conditions, e.g. al., 2007). Active metal resistance mechanisms include metal export systems extreme pH, hostile temperatures, and high salinity are inhospitable for most (Baker-Austin et al., 2005; Mangold et al., 2013); the use of inorganic

10 11 Attempts have been made to demonstrate the potential of using MFCs to treat forms of life on earth. However, such chemically active environments offer municipal wastewater. For instance, two 4 L MFCs were operated in a energy and niches for a variety of extremophiles. municipal wastewater treatment plant for 400 days that removed 65-70% of the COD and achieved a positive energy balance (Zhang et al., 2013). However, the In addition to those described below, categories of extremophiles include technology is still in its infancy because challenges remain in reactor design and thermophiles which are dominated by archaea, that grow at temperatures above material cost; as well as in operational obstacles such as temperature fluctuation 45 °C; oligotrophs that grow in extremely nutrient-poor environments with and the fouling of the membrane and electrode; in addition, currently the extremely long generation times. They are also significant to the understanding understanding of the underlying microbiology is still inadequate (Logan and of the microbiota as well as offering inspirations to industrial applications. Regan, 2006). However, they are beyond the scope of this thesis.

10 11 polyphosphate as an energy reservoir for metal detoxification as well as to machinery in order to acclimate high salinity (Lanyi, 1974; Oren, 2008); they sequestrate metals (Orell et al., 2012); the conversion of toxic metal species to can also synthesize and accumulate organic osmotic solutes to suppress the salt a less toxic form (Schelert et al., 2004); and the inside positive transmembrane influx (Oren, 2008). electrical potential to repel the influx of metal ions, the same mechanism that repels protons as discussed above.

Fig. 4 A soda lakes where haloalkaliphiles can be found. Source of image: Wikimedia Commons; author: Dracblau7; licensed under Creative Common (CC Fig. 3 The highly acidic river “Rio Tinto” that can support acidophilic BY-SA 2.0). microorganisms. Source of image: Wikimedia Commons; Author: Antonio; licensed under Creative Common (CC BY-SA 2.0). Psychrophiles Psychrophiles have an optimum growth temperature of <15 °C, with extreme Alkaliphiles and haloalkaliphiles examples of metabolic activities at -20 °C (Rivkina et al., 2000). They can be Alkaliphiles grow in pH values from 8.5 to 11 and are also identified from all found in the deep oceans, the Antarctic and the Arctic (Fig. 5), glacial ice, as three domains of life. They can be found in natural saline environments such as well as sediments of mining operations in the high extremes of latitude and soda lakes (Fig. 4) or the Red Sea (Banciu et al., 2004). Such environments are altitude (Deming, 2002; D'Amico et al., 2006; Liljeqvist et al., 2011). In general, often associated with high salinity (> 0.3 M Na+) and microorganisms that can low temperature negatively impacts biological reactions, such as by reducing survive and thrive in these dual extremes are termed haloalkaliphiles. To adapt reaction rates, altering the viscosity of the solvent, and changing the solubility to high pH, microorganisms can promote the activity of enzymes to capture and of proteins, salt, and gases that can ultimately lead to protein cold-denaturation retain protons; increase acid-generating metabolic processes and elevate (reviewed in Margesin et al., (2008)). To acclimate to low temperature, cytoplasmic proton retention by altering cell surface layers (Padan et al., 2005; psychrophiles utilize a number of strategies. The expression of cold shock Krulwich et al., 2011) and thereby, maintain an internal pH that is at least two proteins (CSPs) and cold-inducible proteins (CIPs) from psychrophiles were units lower than the external (Dopson et al., 2016). In order to cope with the observed during exposure to cold (Phadtare and Inouye, 2004; Barria et al., osmotic pressure from high salinity, microorganisms can accumulate molar 2013) and an example of CSPs and CIPs are chaperones that remove low concentrations of potassium and chloride to balance the internal and external temperature induced mRNA and DNA secondary structures and facilitate the osmotic pressure. This requires adaptations of the intracellular enzymatic function of ribosomes and RNA polymerases. Psychrophiles can also produce compatible solutes that protect against freezing (Casanueva et al., 2010) and

12 13 polyphosphate as an energy reservoir for metal detoxification as well as to machinery in order to acclimate high salinity (Lanyi, 1974; Oren, 2008); they sequestrate metals (Orell et al., 2012); the conversion of toxic metal species to can also synthesize and accumulate organic osmotic solutes to suppress the salt a less toxic form (Schelert et al., 2004); and the inside positive transmembrane influx (Oren, 2008). electrical potential to repel the influx of metal ions, the same mechanism that repels protons as discussed above.

12 13 alter their membrane structure to increase flexibility during cold stress containing organic components (reviewed in Pant et al., (2010)). A few (Chintalapati et al., 2004). In addition, psychrophiles can increase the flexibility exceptions include MFCs studies that coupled sulfide oxidation to electricity and activity of proteins as well as to utilize physiological changes in the production (Rabaey et al., 2006; Sun et al., 2009). These studies were conducted membrane to adapt to cold conditions (reviewed in De Maayer et al., (2014)). under relatively mild conditions (in terms of pH, temperature and salinity), and the utilized microorganisms were primarily neutrophiles. Because of the diverse range of conditions that extremophiles are able to live in, the integration of extremophiles in BES opened up possibilities to remediate pollution streams with more challenging conditions. The remediation of ISC-containing streams with extreme conditions for instance, are seldom investigated in BES. This was pushed forward in three separate studies within this thesis, they were: MFCs with acidophiles for the remediation of acidic, tetrathionate-containing wastewater; MFCs with psychrophiles for the remediation of low-temperature, thiocyanate-containing wastewater; and MECs with haloalkaliphiles for the remediation of sulfide-containing wastewater with high pH and salinity. Bioremediation of acidic inorganic sulfur compounds Metals such as copper and iron are crucial for modern industry. Conventional metal extraction from metal sulfide ores such as smelting is energy intensive and produces the toxic and acidifying sulfur dioxide gas. Acidophilic microorganisms can accelerate the dissolution of the metals from ores via Fig. 5 An Antarctic iceberg where psychrophilic microorganisms could inhabit. metabolizing inorganic sulfur compounds (ISC). Therefore, they are utilized to Source of image: Wikimedia Commons; author: Andrew Shiva; licensed under Creative Common (CC BY-SA 2.0). extract metals in a less energy-demanding manner and the process is termed ‘biooxidation’ or ‘bioleaching’ (Dopson et al., 2004; Dopson and Johnson, 2012). For instance, the chemolithotrophic microorganism Leptospirillum are ISC remediation with extremophilic BESs known to gain energy from the oxidation of ferrous iron (Fe(II)) to ferric iron (Fe(III)) (Rawlings et al., 1999) and the resulting Fe(III) is an oxidizing agent Sulfur is the 16th most abundant element of the lithosphere and accounts for that further accelerates the dissolution of metal sulfide. In addition, process 0.05 % of its weight; it is highly concentrated in components of earth’s crust, water and effluents from mining or sulfide-rich ores contains considerable such as metal sulfide ores, coal, and crude oil (Orr and Sinninghe Damsté, 1990; levels of ISCs and if this process is uncontrolled, the discharge of acid Dopson and Johnson, 2012). Human activities such as mining and the extraction generating ISCs and metals can severely endanger the environment as well as of fossil fuels has exploited nature to produce materials and energy to sustain human health. Again, the ability of acidophiles to metabolize ISCs in a humankind, and this also lead to large quantities of ISC-containing waste controlled setting to mitigate potential pollution was demonstrated in Liljeqvist streams. ISC such as tetrathionate, thiocyanate, and sulfide are examples of et al., (2011). Recently, acidophiles have been utilized in MFCs to remove 2- anthropogenic sulfur pollutants and their remediation is important to preserve tetrathionate (S4O6 ) while producing electricity (Sulonen et al., 2015). This the environment. Since these compounds contains chemical energy, the BES evidence shows that the technology is promising to incorporate sulfur technology is a good candidate for their remediation and to recover the available metabolizing acidophiles into the BES technology for the treatment of acidic energy. However, a complicating factor is that the waste-streams of these wastewater polluted with ISCs. In this thesis (Papers I & II), this approach was compounds can bring about extreme physicochemical conditions (e.g. high and taken one step closer to industrial application by asking the questions: is it low pH, low temperature, and high salinity, detailed in later sections) that possible to use an MFC to remediate tetrathionate from real (as opposed to hinders the use of conventional BES to remediate such pollution streams. synthetic) process water and how did the underlying microbiology contribute to such treatment? Prior to this thesis, the majority of substrates used in BESs are organic compounds such as acetate, glucose or various industrial wastewaters

14 15 alter their membrane structure to increase flexibility during cold stress containing organic components (reviewed in Pant et al., (2010)). A few (Chintalapati et al., 2004). In addition, psychrophiles can increase the flexibility exceptions include MFCs studies that coupled sulfide oxidation to electricity and activity of proteins as well as to utilize physiological changes in the production (Rabaey et al., 2006; Sun et al., 2009). These studies were conducted membrane to adapt to cold conditions (reviewed in De Maayer et al., (2014)). under relatively mild conditions (in terms of pH, temperature and salinity), and the utilized microorganisms were primarily neutrophiles. Because of the diverse range of conditions that extremophiles are able to live in, the integration of extremophiles in BES opened up possibilities to remediate pollution streams with more challenging conditions. The remediation of ISC-containing streams with extreme conditions for instance, are seldom investigated in BES. This was pushed forward in three separate studies within this thesis, they were: MFCs with acidophiles for the remediation of acidic, tetrathionate-containing wastewater; MFCs with psychrophiles for the remediation of low-temperature, thiocyanate-containing wastewater; and MECs with haloalkaliphiles for the remediation of sulfide-containing wastewater with high pH and salinity. Bioremediation of acidic inorganic sulfur compounds Metals such as copper and iron are crucial for modern industry. Conventional metal extraction from metal sulfide ores such as smelting is energy intensive and produces the toxic and acidifying sulfur dioxide gas. Acidophilic microorganisms can accelerate the dissolution of the metals from ores via Fig. 5 An Antarctic iceberg where psychrophilic microorganisms could inhabit. metabolizing inorganic sulfur compounds (ISC). Therefore, they are utilized to Source of image: Wikimedia Commons; author: Andrew Shiva; licensed under Creative Common (CC BY-SA 2.0). extract metals in a less energy-demanding manner and the process is termed ‘biooxidation’ or ‘bioleaching’ (Dopson et al., 2004; Dopson and Johnson, 2012). For instance, the chemolithotrophic microorganism Leptospirillum are ISC remediation with extremophilic BESs known to gain energy from the oxidation of ferrous iron (Fe(II)) to ferric iron (Fe(III)) (Rawlings et al., 1999) and the resulting Fe(III) is an oxidizing agent Sulfur is the 16th most abundant element of the lithosphere and accounts for that further accelerates the dissolution of metal sulfide. In addition, process 0.05 % of its weight; it is highly concentrated in components of earth’s crust, water and effluents from mining or sulfide-rich ores contains considerable such as metal sulfide ores, coal, and crude oil (Orr and Sinninghe Damsté, 1990; levels of ISCs and if this process is uncontrolled, the discharge of acid Dopson and Johnson, 2012). Human activities such as mining and the extraction generating ISCs and metals can severely endanger the environment as well as of fossil fuels has exploited nature to produce materials and energy to sustain human health. Again, the ability of acidophiles to metabolize ISCs in a humankind, and this also lead to large quantities of ISC-containing waste controlled setting to mitigate potential pollution was demonstrated in Liljeqvist streams. ISC such as tetrathionate, thiocyanate, and sulfide are examples of et al., (2011). Recently, acidophiles have been utilized in MFCs to remove 2- anthropogenic sulfur pollutants and their remediation is important to preserve tetrathionate (S4O6 ) while producing electricity (Sulonen et al., 2015). This the environment. Since these compounds contains chemical energy, the BES evidence shows that the technology is promising to incorporate sulfur technology is a good candidate for their remediation and to recover the available metabolizing acidophiles into the BES technology for the treatment of acidic energy. However, a complicating factor is that the waste-streams of these wastewater polluted with ISCs. In this thesis (Papers I & II), this approach was compounds can bring about extreme physicochemical conditions (e.g. high and taken one step closer to industrial application by asking the questions: is it low pH, low temperature, and high salinity, detailed in later sections) that possible to use an MFC to remediate tetrathionate from real (as opposed to hinders the use of conventional BES to remediate such pollution streams. synthetic) process water and how did the underlying microbiology contribute to such treatment? Prior to this thesis, the majority of substrates used in BESs are organic compounds such as acetate, glucose or various industrial wastewaters

14 15 Biological thiocyanate removal at low temperature 2008). On the other hand, biological processes have the potential to be operated Thiocyanate (SCN-) is a toxic ISC generated from operations such as gold under ambient temperature and atmospheric pressure, require minimal chemical recovery via cyanide (Kantor et al., 2015). Chemical remediation of SCN- in dosing, and can remove sulfide efficiently (Jensen and Webb, 1995; Kennes et principle involves using oxidative agents such as ozone (Soto et al., 1995), al., 2009). In addition, electrochemical oxidation of sulfide can effectively hydrogen peroxide (Wilson and Harris, 1960; Wilson and Harris, 1961), or remove sulfide and reduce the energy demand, but the deposition of elemental macroporous resins containing an oxidizing functional group (Kociołek- sulfur leads to electrode passivation that complicates long-term operation of this Balawejder, 1999). However, chemical techniques tend to be costly, ineffective, technique (Dutta et al., 2008). Paper IV of this thesis took advantage of both and produce toxic by-products (reviewed in Gould et al., (2012)). A the biological and electrochemical approaches and aimed at investigating the complicating factor for mining operations in high latitude locations (such as questions: is it possible to remove sulfide from a synthetic caustic solution in northern Sweden) is that the temperature is low, with water temperature an MEC and what are the dominant microbial populations involved in this approaching 0 °C during winter times (Liljeqvist et al., 2011). To reduce the process? cost from heating, remediation techniques are required to function at low temperatures. Biological treatments that utilize indigenous microbial species The molecular toolbox, pros and cons such as Thiobacillus are inexpensive and can effectively remove SCN- and coexisting contaminants such as CN- (Kantor et al., 2015). These populations A longstanding goal of microbial ecology is to identify the microorganisms of are reported to have acclimated to low temperature conditions and to carry out interest and to study their metabolic states (Blazewicz et al., 2013). Current denitrification using thiocyanate as electron donor (Broman et al., 2017). As molecular toolboxes are all established upon one foundation; that genes encode lowering the operational temperature negatively impacts on the microbial transcripts that induce protein synthesis that results in enzyme catalyzed metabolism, psychrophilic BESs are an unpopular direction of exploration. chemical reactions (Crick, 1958). As a result, the deciphering of genetic codes However, psychrophilic BES studies have demonstrated the capacity for a is crucial to answer such questions. In this thesis, the molecular approaches (Fig. potentially huge energy reserve when the MFCs are installed in marine sediment 6) identified the anodic microbial community and characterized their metabolic (Bond et al., 2002) and have shown improved Coulombic efficiencies (CE) (Xu states. Nevertheless, virtually all molecular techniques have limitations and et al., 2014). In this thesis (Paper III), psychrophilic and autotrophic boundaries (Blazewicz et al., 2013). While these limitations do not prevent their thiocyanate metabolizing microorganisms were introduced to MFCs operated at use, they require awareness, understanding, and caution (Holben, 1994). 8 °C to investigate the questions: is it possible to use an MFC to remove SCN- at low temperature and how did the underlying microbiology contribute to this The development of sequencing technologies went from the labor intensive process? ‘first-generation’ Sanger method first described in the 1970s (Sanger et al., 1977) to ‘next generation’ sequencing (NGS) epitomized by pyrosequencing from 454 Biological desulfurization at high pH and salinity Life Sciences (now Roche) (Margulies et al., 2005), the Illumina sequencers, Sulfide is another example of an ISC. For instance, it accounts for 0.05 to 6 wt% and the SOLiD sequencers. These later methods dramatically increased the (as sulfur) in crude oil as an impurity. Consequently, petroleum refineries throughput and reduced the costs per run (Pop and Salzberg, 2008). In addition, - 2- produce substantial quantities of sulfides (H2S, HS , and S ). Another the PacBio sequencing technologies are able to generate reads with length of considerable source of anthropogenic sulfides is the anaerobic digestion of several-kilo-bases (Eid et al., 2009). These technological advances facilitated protein rich substances for the production of biogas (Buisman et al., 1989). more in-depth investigations in molecular ecology. Sulfides can acidify the recipient water body as well as deplete its oxygen (Buisman et al., 1989); they are also very odorous, highly toxic to life, and 16S rRNA gene amplicon sequencing corrosive to materials (Ren et al., 2005). A common way to concentrate gaseous The 16S rRNA is a component of the prokaryotic ribosome that forms the sulfides into the liquid phase is by stripping with caustic solution (e.g. NaOH), translation machinery. It is ubiquitous in prokaryotes and crucial to protein resulting in spent caustics solutions (Alnaizy, 2008) that are characterized by synthesis. It has extremely conserved regions that are ideal to trace phylogeny, high pH, salinity, and sulfide content. Traditional physicochemical processes to and also contains less conserved regions that tend to be distinct for specific taxa, remediate the spent caustics streams include wet air oxidation, oxidation at high thus making the 16S rRNA gene an excellent biological marker to examine the temperature, or chemical dosing aided oxidation. However, these methods are phylogenetic profile of a microbial community. Together with the rapid energy intensive, costly, and high-maintenance (Sheu and Weng, 2001; Alnaizy, advances in sequencing technologies in terms of throughput, sequence length,

16 17 Biological thiocyanate removal at low temperature 2008). On the other hand, biological processes have the potential to be operated Thiocyanate (SCN-) is a toxic ISC generated from operations such as gold under ambient temperature and atmospheric pressure, require minimal chemical recovery via cyanide (Kantor et al., 2015). Chemical remediation of SCN- in dosing, and can remove sulfide efficiently (Jensen and Webb, 1995; Kennes et principle involves using oxidative agents such as ozone (Soto et al., 1995), al., 2009). In addition, electrochemical oxidation of sulfide can effectively hydrogen peroxide (Wilson and Harris, 1960; Wilson and Harris, 1961), or remove sulfide and reduce the energy demand, but the deposition of elemental macroporous resins containing an oxidizing functional group (Kociołek- sulfur leads to electrode passivation that complicates long-term operation of this Balawejder, 1999). However, chemical techniques tend to be costly, ineffective, technique (Dutta et al., 2008). Paper IV of this thesis took advantage of both and produce toxic by-products (reviewed in Gould et al., (2012)). A the biological and electrochemical approaches and aimed at investigating the complicating factor for mining operations in high latitude locations (such as questions: is it possible to remove sulfide from a synthetic caustic solution in northern Sweden) is that the temperature is low, with water temperature an MEC and what are the dominant microbial populations involved in this approaching 0 °C during winter times (Liljeqvist et al., 2011). To reduce the process? cost from heating, remediation techniques are required to function at low temperatures. Biological treatments that utilize indigenous microbial species The molecular toolbox, pros and cons such as Thiobacillus are inexpensive and can effectively remove SCN- and coexisting contaminants such as CN- (Kantor et al., 2015). These populations A longstanding goal of microbial ecology is to identify the microorganisms of are reported to have acclimated to low temperature conditions and to carry out interest and to study their metabolic states (Blazewicz et al., 2013). Current denitrification using thiocyanate as electron donor (Broman et al., 2017). As molecular toolboxes are all established upon one foundation; that genes encode lowering the operational temperature negatively impacts on the microbial transcripts that induce protein synthesis that results in enzyme catalyzed metabolism, psychrophilic BESs are an unpopular direction of exploration. chemical reactions (Crick, 1958). As a result, the deciphering of genetic codes However, psychrophilic BES studies have demonstrated the capacity for a is crucial to answer such questions. In this thesis, the molecular approaches (Fig. potentially huge energy reserve when the MFCs are installed in marine sediment 6) identified the anodic microbial community and characterized their metabolic (Bond et al., 2002) and have shown improved Coulombic efficiencies (CE) (Xu states. Nevertheless, virtually all molecular techniques have limitations and et al., 2014). In this thesis (Paper III), psychrophilic and autotrophic boundaries (Blazewicz et al., 2013). While these limitations do not prevent their thiocyanate metabolizing microorganisms were introduced to MFCs operated at use, they require awareness, understanding, and caution (Holben, 1994). 8 °C to investigate the questions: is it possible to use an MFC to remove SCN- at low temperature and how did the underlying microbiology contribute to this The development of sequencing technologies went from the labor intensive process? ‘first-generation’ Sanger method first described in the 1970s (Sanger et al., 1977) to ‘next generation’ sequencing (NGS) epitomized by pyrosequencing from 454 Biological desulfurization at high pH and salinity Life Sciences (now Roche) (Margulies et al., 2005), the Illumina sequencers, Sulfide is another example of an ISC. For instance, it accounts for 0.05 to 6 wt% and the SOLiD sequencers. These later methods dramatically increased the (as sulfur) in crude oil as an impurity. Consequently, petroleum refineries throughput and reduced the costs per run (Pop and Salzberg, 2008). In addition, - 2- produce substantial quantities of sulfides (H2S, HS , and S ). Another the PacBio sequencing technologies are able to generate reads with length of considerable source of anthropogenic sulfides is the anaerobic digestion of several-kilo-bases (Eid et al., 2009). These technological advances facilitated protein rich substances for the production of biogas (Buisman et al., 1989). more in-depth investigations in molecular ecology. Sulfides can acidify the recipient water body as well as deplete its oxygen (Buisman et al., 1989); they are also very odorous, highly toxic to life, and 16S rRNA gene amplicon sequencing corrosive to materials (Ren et al., 2005). A common way to concentrate gaseous The 16S rRNA is a component of the prokaryotic ribosome that forms the sulfides into the liquid phase is by stripping with caustic solution (e.g. NaOH), translation machinery. It is ubiquitous in prokaryotes and crucial to protein resulting in spent caustics solutions (Alnaizy, 2008) that are characterized by synthesis. It has extremely conserved regions that are ideal to trace phylogeny, high pH, salinity, and sulfide content. Traditional physicochemical processes to and also contains less conserved regions that tend to be distinct for specific taxa, remediate the spent caustics streams include wet air oxidation, oxidation at high thus making the 16S rRNA gene an excellent biological marker to examine the temperature, or chemical dosing aided oxidation. However, these methods are phylogenetic profile of a microbial community. Together with the rapid energy intensive, costly, and high-maintenance (Sheu and Weng, 2001; Alnaizy, advances in sequencing technologies in terms of throughput, sequence length,

16 17 and depth as well as the reduction in costs per base; 16S rRNA gene amplicon mutants, and heteroduplexes (Qiu et al., 2001); and the amplification efficiency sequencing has become a standard method to study culture-independent could be uneven for different genes (Suzuki and Giovannoni, 1996). In addition, microbial diversity (Klindworth et al., 2013)(Fig. 6). different taxa vary in the copy numbers of the 16S rRNA gene, which can also affect the analysis of relative abundance, as organisms with higher copy numbers of the 16S rRNA gene will be overrepresented (Větrovský and Baldrian, 2013). All these factors may compromise the accurate representation of the microbial community under investigation. Metagenomics and metatranscriptomics Culturing is a traditional technology to study the microbial community. However, the vast majority of microorganisms cannot be cultured in laboratories (Amann et al., 1995); furthermore, the cultivable populations do not necessarily represent the environment outside the laboratory from where they were sampled (Sharon and Banfield, 2013). As a result, culture- independent approaches are gaining increasing popularity.

Metagenomics is the direct extraction and characterization of DNA from a microbial community (Fig. 6). It is free from errors and biases induced by cultivation and PCR amplification. It is used to reveal the taxonomic profile as well as the metabolic potential on the community level (Sharon and Banfield, 2013). However, one should be reminded that the presence of certain genes shows that the microorganism could potentially carry out the related metabolic process; it does not guarantee the occurrence of the process. In addition, taxonomy-dependent metagenomic analysis would fail to classify reads from hitherto uncharacterized organisms while taxonomy-independent approaches can retrieve reads from novel organisms, yet such reads remain challenging for taxonomical characterization (Mande et al., 2012). Furthermore, compare to abundant populations within a metagenome, the coverage of genomes from less

Fig. 6 Overview of the molecular tools used in this thesis. abundant populations will be less efficient due to the limitation of sequencing depth.

All molecular techniques require the extraction of DNA or RNA, yet nucleic Metatranscriptomics is the extraction of community RNA and the profiling acid extraction methods can suffer from incomplete cell lysis, co-extraction of of its gene transcripts (Fig. 6). Compared to metagenomics, it is one-step further enzymatic inhibitors, as well as loss, degradation, and damage of DNA (as to elucidate the metabolism of the community by offering information about the reviewed in Miller et al., (1999). In addition, the unstable RNA molecules have genes that were actively transcribed at the time of cell harvest. The presence of short half-life once extracted (Brooks, 1998). These extraction-related rRNA and mRNA reads is a powerful index of certain metabolic processes constraints affect downstream analyses. In order to perform NGS, the extracted being carried out, because RNA directly links to protein synthesis, but it does nucleic acids are used to construct a library, which typically includes steps such not prove the occurrence of realized protein synthesis nor the biochemical as fragmentation of the DNA or RNA, size selection, and incorporating the reactions (Blazewicz et al., 2013; Rocca et al., 2015). Such confirmation DNA or RNA molecules with adapters that contain the necessary elements for requires further analysis such as quantitative reverse transcriptase-PCR (Franks, sequencing (van Dijk et al., 2014). These steps are also subject to biases (van 2010), enzymatic, proteomic, and chemical analyses. Dijk et al., 2014). In the case of the 16S rRNA gene amplicon sequencing, PCR amplification is a crucial step. However, PCR primers can be biased (Parada et al., 2016); the PCR reactions can generate undesired artifacts such as chimeras,

18 19 and depth as well as the reduction in costs per base; 16S rRNA gene amplicon mutants, and heteroduplexes (Qiu et al., 2001); and the amplification efficiency sequencing has become a standard method to study culture-independent could be uneven for different genes (Suzuki and Giovannoni, 1996). In addition, microbial diversity (Klindworth et al., 2013)(Fig. 6). different taxa vary in the copy numbers of the 16S rRNA gene, which can also affect the analysis of relative abundance, as organisms with higher copy numbers of the 16S rRNA gene will be overrepresented (Větrovský and Baldrian, 2013). All these factors may compromise the accurate representation of the microbial community under investigation. Metagenomics and metatranscriptomics Culturing is a traditional technology to study the microbial community. However, the vast majority of microorganisms cannot be cultured in laboratories (Amann et al., 1995); furthermore, the cultivable populations do not necessarily represent the environment outside the laboratory from where they were sampled (Sharon and Banfield, 2013). As a result, culture- independent approaches are gaining increasing popularity.

Fig. 6 Overview of the molecular tools used in this thesis. abundant populations will be less efficient due to the limitation of sequencing depth.

18 19 In general, the field of bioinformatics is a rapidly advancing area and software is in constant development and optimization (Pop and Salzberg, 2008). Aims of this thesis In addition, the taxonomical and functional annotation is only as good as the databases against which they were annotated (Bunse, 2017). Furthermore, omics techniques are hypothesis generating and the findings require further This thesis pioneered investigations for the remediation of tetrathionate, experimental confirmation. In this thesis, these techniques were combined in thiocyanate, and sulfide and the recovery electrical energy using extremophiles their use to minimize the biases from each technique (e.g. the coupling of 16S in BESs under various extreme conditions (detailed in Table 1). Molecular rRNA amplicon sequencing with metagenomics and/or metatranscriptomics to microbiological methods including 16S rRNA gene amplicon sequencing, profile the microbial community), also, they were coupled to chemical and metagenomics, and metatranscriptomics were utilized in conjunction with electrical analysis in order to have a more accurate representation of the BESs. chemical and electrical measurements to examine the microbial consortium within the anodic compartments of the extremophilic BESs. The use of these techniques aimed at understanding: • The community composition of the anodic microbiomes. • The genetic metabolic potential of the anodic microbiomes. • The active metabolic processes that were carried out.

Special attention was paid to: • Metabolism of ISCs. • Energy conservation and EET. • Inorganic carbon fixation. • Adaptation to the given extreme environments.

This thesis endeavors to integrate extremophiles into BESs, to generate microbiological insights of these communities, as well as to widen the application range for BESs. It serves as an example for BES characterization from both the microbiological and the electrochemical perspectives.

20 21 In general, the field of bioinformatics is a rapidly advancing area and software is in constant development and optimization (Pop and Salzberg, 2008). Aims of this thesis In addition, the taxonomical and functional annotation is only as good as the databases against which they were annotated (Bunse, 2017). Furthermore, omics techniques are hypothesis generating and the findings require further This thesis pioneered investigations for the remediation of tetrathionate, experimental confirmation. In this thesis, these techniques were combined in thiocyanate, and sulfide and the recovery electrical energy using extremophiles their use to minimize the biases from each technique (e.g. the coupling of 16S in BESs under various extreme conditions (detailed in Table 1). Molecular rRNA amplicon sequencing with metagenomics and/or metatranscriptomics to microbiological methods including 16S rRNA gene amplicon sequencing, profile the microbial community), also, they were coupled to chemical and metagenomics, and metatranscriptomics were utilized in conjunction with electrical analysis in order to have a more accurate representation of the BESs. chemical and electrical measurements to examine the microbial consortium within the anodic compartments of the extremophilic BESs. The use of these techniques aimed at understanding: • The community composition of the anodic microbiomes. • The genetic metabolic potential of the anodic microbiomes. • The active metabolic processes that were carried out.

Special attention was paid to: • Metabolism of ISCs. • Energy conservation and EET. • Inorganic carbon fixation. • Adaptation to the given extreme environments.

20 21 Overview of methodology

Operation and electrochemical characterization of the BESs

All the MFCs and MECs used in this study had a similar design (Fig. 7). In essence, the anode and cathode flow chambers were made from Plexiglas; the electrode materials included graphite paper, graphite felt, and Platinum-coated Titanium flat plate, depending on the situation; cation or anion exchange membranes were used to separate the anodic and cathodic liquid flows (termed the anolyte and the catholyte), which was maintained by peristaltic pumps; all these parts were pressed tightly together and were secured using screws. Reference electrodes (Ag/AgCl) were connected to both the anolyte and the catholyte. Electrical parameters were recorded in each study using either an in- line data logger or manually recorded using a multimeter when a data logger Fig. 7 Conceptual illustrations of the BESs used in this thesis. was not available. These parameters were cathode potential, as the potential difference between the cathode against the cathodic reference electrode (Eq. 1); In addition, concentrations of tetrathionate (Paper I) and thiocyanate (Paper the anode potential as the difference between the anode against the anodic III) were measured via cyanolysis (Kelly et al., 1969); sulfide concentration reference electrode (Eq. 2); the membrane potential as the difference between (Paper IV) was determined using a methylene blue method based commercial the cathodic and anodic reference electrodes (Eq. 3); and the cell voltage as the kit (LCK 653, Hach Lange); and anions (Paper IV) were measured using ion potential difference between the cathode against the anode (Eq. 4). The chromatography. Finally, surface structure and elemental composition of the production of current was either calculated as the cell voltage divided by graphite paper electrode was inspected using a scanning electron microscope resistance (Ohm’s law) or recorded by a data logger. Current was normalized to coupled with energy dispersive X-ray spectroscopy (SEM-EDS) (Paper IV); projected anode/membrane surface area for convenience and consistency. The X-ray diffraction (XRD) measurements were carried out to determine the CE calculation was based on total charge from the electrical current divided by crystalline structures of the precipitates on the electrode surface (Paper IV). the total charge from the oxidation of electron donors (Eq. 5). Choice of inoculum Eq. 1 The inocula for each study was chosen based on the physicochemical conditions of the wastewater and the ISC that was to be remediated (detailed in Table 1). !"#ℎ%&' )%#'*#+", = .%#'*#+", %/ 0"#ℎ%&' − .%#'*#+", %/ 0"#. 3'/ . Eq. 2 The inoculum for the acidophilic MFC study was from the sediment of an acidic multi-metal sulfide mine drainage (Liljeqvist et al., 2015). Its natural 4*%&' )%#'*#+", = .%#'*#+", %/ "*%&' − .%#'*#+", %/ "*%. 3'/. Eq. 3 environment is highly acidic and rich in ISCs, which selects microorganisms that are acclimated to such milieu and metabolize ISCs. The inoculum for the 5'673"*' )%#'*#+", = .%#'*#+", 0"#. 3'/. − .%#'*#+", %/ "*% . 3'/ . Eq. 4 psychrophilic MFC study was from a lab-scale low temperature stirred-tank reactor capable of degrading thiocyanate (Broman et al., 2017). Biomass- !',, 8%,#"9' = .%#'*#+", 0"#ℎ%&' − .%#'*#+", %/ "*%&' containing process water from the recirculation loop of an industrial sulfide D C Eq. 5 ∫D ?∙AB removal operation in Eerbeek, the Netherlands was used as the inoculum for the Where cat.ref. indicates the reference electrode that was connected to the !:(%) = [FGHFBIJBK]∙M∙N ∙ 100 haloalkaliphilic MEC to remove sulfide. This process water had high pH and catholyte; ano.ref. indicates the reference electrode that was connected to the salinity, which selects for haloalkaliphilic sulfide-oxidizing microorganisms. anolyte; Δ[substrate] was the decrease of substrate in mol during the period from timepoint 0 to timepoint t; n is the number of electrons per reaction mol, F is the faraday constant (96485 C/mol); and current (I) is expressed in A.

22 23 Overview of methodology

Operation and electrochemical characterization of the BESs

22 23 16S rRNA gene amplicon sequencing Anodic biomass was collected by either centrifugation or filtration with sterile In the psychrophilic MFC study (Paper III), community RNA was also 0.2 µm filters. DNA was extracted using commercially available DNA isolation extracted from the anodic biomass when the MFCs were generating a current. kits according to manufacturer’s instructions. Amplification of the V3-V4 The biomass was immediately mixed with RNA fix solution. The extracted region of 16S rRNA gene using PCR primers 341F and 805R (Hugerth et al., RNA was used to generate cDNA that was subsequently sequenced on an 2014) was performed as described in Lindh et al., (2015). DNA sequencing was Illumina HiSeq2500 platform. The obtained metatranscriptomic reads were conducted at the Science for Life Laboratory (Stockholm, Sweden; quality controlled and sorted for mRNA reads and 16S rRNA reads. In order to www.scilifelab.se) on an Illumina MiSeq platform. For the acidophilic MFC investigate the metabolic processes of interest, the mRNA reads were analyzed study and the psychrophilic MFC study (Papers I & III), the pair-end reads for taxonomic placement and functional annotation in conjunction with NCBI from amplicon sequencing were analyzed using the UPARSE pipeline (Edgar, NR and InterPro2GO databases. In addition, rRNA reads were taxonomically 2013), taxonomic annotation was performed using the SILVA database (SILVA placed to reveal the active members of the anodic microbial community. 119), and the final data handling was performed in Explicet 2.10.5 (Robertson et al., 2013). For the haloalkaliphilic MEC study (Paper IV), the amplicon reads were analyzed using the DADA2 pipeline (Callahan et al., 2016), annotated against the SILVA database version 132 (Quast et al., 2013) and visualized using the Rstudio package Tidyverse (Wickham, 2017). Metagenomics For the acidophilic MFC study (Paper II), community DNA for metagenomic sequencing was extracted in the same manner as for the 16S rRNA gene amplicon sequencing. Metagenomic sequencing was performed on an Illumina HiSeq 2500 platform. The obtained reads underwent a workflow of trimming, assembly into contigs, binning to obtain metagenome-assembled genomes (MAGs) as well as taxonomic and functional annotation. The bioinformatics was carried out with a software package including FastQC (Andrews, 2010), sickle (Joshi and Fass, 2011), Ray (Boisvert et al., 2010), Newbler, CONCOCT (Alneberg et al., 2014), CheckM (Parks et al., 2015), PhyloPhlAn (Segata et al., 2013)and Prokka (Seemann, 2014). The annotated reads were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa and Goto, 2000) and the MetaCyc (Caspi et al., 2014) databases. Metatranscriptomics In the acidophilic MFC study (Paper II), community RNA was extracted from the anodic biomass when the MFCs were generating an electrical current. Metatranscriptome sequencing was carried out on an Illumina HiSeq 2500 platform without rRNA depletion. The quality of the obtained metatranscriptomic reads was controlled followed by the identification of rRNA and mRNA reads. The mRNA reads were assembled into transcripts in order to assess their relative abundance and to perform taxonomical assignment plus functional annotation. The activity of the microbial populations was assessed in three complementary procedures: 1) mapping of mRNA reads to the MAGs; 2) performing a taxonomic assignment of assembled transcripts; and 3) phylogenetic placement of the 16S rRNA reads.

24 25 16S rRNA gene amplicon sequencing Anodic biomass was collected by either centrifugation or filtration with sterile In the psychrophilic MFC study (Paper III), community RNA was also 0.2 µm filters. DNA was extracted using commercially available DNA isolation extracted from the anodic biomass when the MFCs were generating a current. kits according to manufacturer’s instructions. Amplification of the V3-V4 The biomass was immediately mixed with RNA fix solution. The extracted region of 16S rRNA gene using PCR primers 341F and 805R (Hugerth et al., RNA was used to generate cDNA that was subsequently sequenced on an 2014) was performed as described in Lindh et al., (2015). DNA sequencing was Illumina HiSeq2500 platform. The obtained metatranscriptomic reads were conducted at the Science for Life Laboratory (Stockholm, Sweden; quality controlled and sorted for mRNA reads and 16S rRNA reads. In order to www.scilifelab.se) on an Illumina MiSeq platform. For the acidophilic MFC investigate the metabolic processes of interest, the mRNA reads were analyzed study and the psychrophilic MFC study (Papers I & III), the pair-end reads for taxonomic placement and functional annotation in conjunction with NCBI from amplicon sequencing were analyzed using the UPARSE pipeline (Edgar, NR and InterPro2GO databases. In addition, rRNA reads were taxonomically 2013), taxonomic annotation was performed using the SILVA database (SILVA placed to reveal the active members of the anodic microbial community. 119), and the final data handling was performed in Explicet 2.10.5 (Robertson et al., 2013). For the haloalkaliphilic MEC study (Paper IV), the amplicon reads were analyzed using the DADA2 pipeline (Callahan et al., 2016), annotated against the SILVA database version 132 (Quast et al., 2013) and visualized using the Rstudio package Tidyverse (Wickham, 2017). Metagenomics For the acidophilic MFC study (Paper II), community DNA for metagenomic sequencing was extracted in the same manner as for the 16S rRNA gene amplicon sequencing. Metagenomic sequencing was performed on an Illumina HiSeq 2500 platform. The obtained reads underwent a workflow of trimming, assembly into contigs, binning to obtain metagenome-assembled genomes (MAGs) as well as taxonomic and functional annotation. The bioinformatics was carried out with a software package including FastQC (Andrews, 2010), sickle (Joshi and Fass, 2011), Ray (Boisvert et al., 2010), Newbler, CONCOCT (Alneberg et al., 2014), CheckM (Parks et al., 2015), PhyloPhlAn (Segata et al., 2013)and Prokka (Seemann, 2014). The annotated reads were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa and Goto, 2000) and the MetaCyc (Caspi et al., 2014) databases. Metatranscriptomics In the acidophilic MFC study (Paper II), community RNA was extracted from the anodic biomass when the MFCs were generating an electrical current. Metatranscriptome sequencing was carried out on an Illumina HiSeq 2500 platform without rRNA depletion. The quality of the obtained metatranscriptomic reads was controlled followed by the identification of rRNA and mRNA reads. The mRNA reads were assembled into transcripts in order to assess their relative abundance and to perform taxonomical assignment plus functional annotation. The activity of the microbial populations was assessed in three complementary procedures: 1) mapping of mRNA reads to the MAGs; 2) performing a taxonomic assignment of assembled transcripts; and 3) phylogenetic placement of the 16S rRNA reads.

24 25

omics

Results and discussion Molecular methods metatranscriptomics The acidophilic MFC study (Papers I & II) Paper I set out to investigate if the MFC technology could be used to remove etagenomics, and and metatranscript etagenomics, 16S rRNA gene amplicon sequencing amplicon 16S gene rRNA 16S rRNA gene amplicon sequencing, sequencing, amplicon 16S gene rRNA m tetrathionate from acidic real (as opposed to synthetic) process water while 16S rRNA gene amplicon sequencing and sequencing amplicon 16S gene rRNA producing electricity with an acidophilic inoculum. Electrochemical

- measurements coupled to 16S rRNA gene amplicon sequencing were utilized

2 - 6

O to characterize the electrochemical performance as well as to profile the anodic 4 HS ISC SCN S microbial community. In Paper II, a combined metagenomic and metatranscriptomic investigation was carried out to examine the microbial

, .

metabolism that was responsible for tetrathionate degradation, the oxidation of

. intermediate ISC, EET, the fixation of carbon dioxide as well as how the

. community adapted to an acidic environment.

Sweden temperature temperature -

iment , Electrochemical characterization and 16S rRNA gene amplicon

sed sequencing (Paper I) degrading bioreactor degrading

Kalmar, Sweden Kalmar, scale low - The inoculum originated from the sediment of an acid mine drainage (AMD). - Origin of inoculum Acid mine drainage drainage mine Acid Kristineberg, operation process water process operation

A tetrathionate-containing synthetic media was supplied to the anodic chambers Industrial desulfurization desulfurization Industrial Eerbeek, The Netherlands The Eerbeek, Lab SCN of MFCs, which was subsequently stepwise replaced with real mining process

water added with the same concentration of tetrathionate. This had no adverse

effect on the bioelectrochemical performance, suggesting that the microbial consortium is able to acclimate to real process water. Electricity was generated 8 °C

pH 2 2

pH 8.5, at a maximum of 275 mA/m (normalized to the projected anode surface area), 1M Na Condition while the highest degradation rate of tetrathionate was 3.35 mM/day. The CE

operational conditions, source of inoculum, remediated ISCs and applied molecular methods of each study each study of methods molecular applied and ISCs remediated inoculum, of source conditions, operational

ranged from 0.08% to 11.8%. An abiotic system was operated in addition to the

biotic MFCs revealing that: 1) cyclic voltammetry scans of the biotic operation showed more current than the abiotic control; 2) abiotic operation generated

y electricity but it decreased more rapidly compared to biotic operation; 3)

philic MEC subsequent inoculation of the abiotic system with the same AMD culture used Stud in the MFCs resulted in increased cell voltage. Although the disproportionation of tetrathionate does not yield electrons (Eq. 6). This comparison strongly

Acidophilic MFC

Psychrophilic MFC Psychrophilic Haloalkali Overview of experimental the conditions and molecular methods Table 1. Overview of 26 conducted in this thesis. this in conducted

27 Results and discussion

The acidophilic MFC study (Papers I & II) Paper I set out to investigate if the MFC technology could be used to remove tetrathionate from acidic real (as opposed to synthetic) process water while producing electricity with an acidophilic inoculum. Electrochemical measurements coupled to 16S rRNA gene amplicon sequencing were utilized to characterize the electrochemical performance as well as to profile the anodic microbial community. In Paper II, a combined metagenomic and metatranscriptomic investigation was carried out to examine the microbial metabolism that was responsible for tetrathionate degradation, the oxidation of intermediate ISC, EET, the fixation of carbon dioxide as well as how the community adapted to an acidic environment.

Electrochemical characterization and 16S rRNA gene amplicon sequencing (Paper I) The inoculum originated from the sediment of an acid mine drainage (AMD). A tetrathionate-containing synthetic media was supplied to the anodic chambers of MFCs, which was subsequently stepwise replaced with real mining process water added with the same concentration of tetrathionate. This had no adverse effect on the bioelectrochemical performance, suggesting that the microbial consortium is able to acclimate to real process water. Electricity was generated at a maximum of 275 mA/m2 (normalized to the projected anode surface area), while the highest degradation rate of tetrathionate was 3.35 mM/day. The CE ranged from 0.08% to 11.8%. An abiotic system was operated in addition to the biotic MFCs revealing that: 1) cyclic voltammetry scans of the biotic operation showed more current than the abiotic control; 2) abiotic operation generated electricity but it decreased more rapidly compared to biotic operation; 3) subsequent inoculation of the abiotic system with the same AMD culture used in the MFCs resulted in increased cell voltage. Although the disproportionation of tetrathionate does not yield electrons (Eq. 6). This comparison strongly

27 indicated that the acidophilic microbial consortium contributed to the oxidation attributed to the Ferroplasma-like populations and was the highest expressed of intermediate ISC that were produced from tetrathionate degradation and gene in this study (Paper II, Fig. 3). Sulfur could also be oxidized to sulfide by carried out EET. 16S rRNA gene amplicon sequencing of the microbial heterodisulfide reductase (Hdr) and its gene was present in the Archaea-like consortia resulted in sequences that aligned within the acidophilic genera populations and its RNA transcripts were dominated by the Ferroplasma-like Thermoplasma, Ferroplasma, Leptospirillum, Sulfobacillus and MAGs. Thiosulfate was suggested to be oxidized to sulfite via the Sox complex Acidithiobacillus. Results from this study suggested that tetrathionate present in the Acidithiobacillus-like populations, solely expressed by the A. degradation with acidophiles coupled to electricity generation could be a caldus-like population. Finally, the oxidation of sulfite to sulfate involves ATP potential bioremediation method. sulfurylase (Sat), its gene was present in the Acidithiobacillus-like populations and the S. thermosulfidooxidans-like MAGs. Overall, this consortium was able Eq. 6 to carry out the complete transformation of tetrathionate to sulfate, while the %& %& %& 1 highest activity was observed regarding the sulfide oxidation to sulfur as well " $ % (-) % / " Taxonomy! # + ) # and => relative! + ! #abundance+ !# + (Paper2) II) as sulfur oxidation to sulfite, attributed to the Ferroplasma-like population Five metagenome-assembled genomes (MAGs) representing acidophilic (Paper II, Fig. 3). microorganisms were obtained from each replicate MFC (when fed with synthetic media, namely S1 and S2) that were most similar to the Archaea All the MAGs were equipped with a suite of standard genes coding for Ferroplasma spp., and Cuniculiplasma divulgatum as well as the Bacteria respiration, indicating the presence of electron transfer chains and the ability of Acidithiobacillus caldus, Acidithiobacillus ferrivorans, and Sulfobacillus the microbial consortium to obtain energy via oxidative phosphorylation as well thermosulfidooxidans. This was in agreement with the 16S rRNA gene as to transport electrons from the oxidation of ISC to the quinone pool (Dopson amplicon sequencing (Paper I) and also showed similarity with another and Johnson, 2012). Due to the lack of EET mechanisms described in tetrathionate-degrading MFC study that contained Acidithiobacillus spp. and acidophiles, the EET mechanisms from Geobacter and Shewanella were ‘Ferroplasma acidarmanus’ (Sulonen et al., 2015). These evidences suggested screened in the MAGs from this study. No genes for the abovementioned c-type the potential role of these microorganisms to degrade tetrathionate and carry out cytochromes were found and instead, various electron shuttle-related genes EET to the electrode. were present in all MAGs (Paper II, Fig. 3). RNA transcripts for menaquinone biosynthesis genes were dominated by the Ferroplasma-like population while According to the mapping of metagenome reads to the MAGs, the riboflavin biosynthesis transcripts were dominated by the Cuniculiplasma Acidithiobacillus-like populations were the most abundant in the consortium divulgatum-like population. Although these redox-active compounds can have that were followed by the Ferroplasma-like, the S. thermosulfidooxidans-like, other functions within the cell, the high number of transcripts suggested EET and the C. divulgatum-like populations. The highest proportion of rRNA and was mediated by electron shuttles. Furthermore, although the known EET mRNA reads were assigned to the Ferroplasma genus indicating this population mediating conductive pili genes were missing, the Acidithiobacillus-like was the most active at the time of sampling despite being the second most populations had the genes for type-II secretion system, while all the Bacteria- abundant population (Paper II, Fig. 2). like populations had type-IV pili formation genes, that could potentially contribute to energy conservation through EET via conductive pili. The Unraveling metabolic processes (Paper II) presence of the EET mechanisms of Geobacter and Shewanella require further Tetrathionate was suggested to be disproportionated to sulfate, thiosulfate, and validation if they also function in the populations from this study. In addition, elemental sulfur mediated by tetrathionate hydrolase (tetH), and its gene was it cannot be ruled out that novel uncharacterized pathways were responsible for present in the A. caldus-like MAGs from both S1 and S2. Its encoding gene was carrying out EET. expressed at a low level, potentially due to the depletion of tetrathionate in the anolyte or the A. caldus-like MAGs were limited by the availability of carbon Mining process and waste streams are characterized with low organic carbon, dioxide for cellular growth. The produced sulfur was likely disproportionated and high acidity and metal content. To thrive in such environments, acidophilic to sulfide and sulfite, catalyzed by sulfur oxygenase reductase coded by sor that microorganisms have developed mechanisms for carbon dioxide fixation to was present in all MAGs except for the C. divulgatum-like population. However, sustain biomass growth; to maintain a near-neutral intracellular pH for sor gene transcripts were only observed in the A. caldus-like population. Sulfide bioenergetics as well as the function of proteins (Baker-Austin and Dopson, could be oxidized to sulfur via sulfide:quinone oxidoreductase (Sqr) that was 2007; Krulwich et al., 2011); and to resist soluble metal ions that are harmful

28 29 indicated that the acidophilic microbial consortium contributed to the oxidation attributed to the Ferroplasma-like populations and was the highest expressed of intermediate ISC that were produced from tetrathionate degradation and gene in this study (Paper II, Fig. 3). Sulfur could also be oxidized to sulfide by carried out EET. 16S rRNA gene amplicon sequencing of the microbial heterodisulfide reductase (Hdr) and its gene was present in the Archaea-like consortia resulted in sequences that aligned within the acidophilic genera populations and its RNA transcripts were dominated by the Ferroplasma-like Thermoplasma, Ferroplasma, Leptospirillum, Sulfobacillus and MAGs. Thiosulfate was suggested to be oxidized to sulfite via the Sox complex Acidithiobacillus. Results from this study suggested that tetrathionate present in the Acidithiobacillus-like populations, solely expressed by the A. degradation with acidophiles coupled to electricity generation could be a caldus-like population. Finally, the oxidation of sulfite to sulfate involves ATP potential bioremediation method. sulfurylase (Sat), its gene was present in the Acidithiobacillus-like populations and the S. thermosulfidooxidans-like MAGs. Overall, this consortium was able Eq. 6 to carry out the complete transformation of tetrathionate to sulfate, while the %& %& %& 1 highest activity was observed regarding the sulfide oxidation to sulfur as well " $ % (-) % / " Taxonomy! # + ) # and => relative! + ! #abundance+ !# + (Paper2) II) as sulfur oxidation to sulfite, attributed to the Ferroplasma-like population Five metagenome-assembled genomes (MAGs) representing acidophilic (Paper II, Fig. 3). microorganisms were obtained from each replicate MFC (when fed with synthetic media, namely S1 and S2) that were most similar to the Archaea All the MAGs were equipped with a suite of standard genes coding for Ferroplasma spp., and Cuniculiplasma divulgatum as well as the Bacteria respiration, indicating the presence of electron transfer chains and the ability of Acidithiobacillus caldus, Acidithiobacillus ferrivorans, and Sulfobacillus the microbial consortium to obtain energy via oxidative phosphorylation as well thermosulfidooxidans. This was in agreement with the 16S rRNA gene as to transport electrons from the oxidation of ISC to the quinone pool (Dopson amplicon sequencing (Paper I) and also showed similarity with another and Johnson, 2012). Due to the lack of EET mechanisms described in tetrathionate-degrading MFC study that contained Acidithiobacillus spp. and acidophiles, the EET mechanisms from Geobacter and Shewanella were ‘Ferroplasma acidarmanus’ (Sulonen et al., 2015). These evidences suggested screened in the MAGs from this study. No genes for the abovementioned c-type the potential role of these microorganisms to degrade tetrathionate and carry out cytochromes were found and instead, various electron shuttle-related genes EET to the electrode. were present in all MAGs (Paper II, Fig. 3). RNA transcripts for menaquinone biosynthesis genes were dominated by the Ferroplasma-like population while According to the mapping of metagenome reads to the MAGs, the riboflavin biosynthesis transcripts were dominated by the Cuniculiplasma Acidithiobacillus-like populations were the most abundant in the consortium divulgatum-like population. Although these redox-active compounds can have that were followed by the Ferroplasma-like, the S. thermosulfidooxidans-like, other functions within the cell, the high number of transcripts suggested EET and the C. divulgatum-like populations. The highest proportion of rRNA and was mediated by electron shuttles. Furthermore, although the known EET mRNA reads were assigned to the Ferroplasma genus indicating this population mediating conductive pili genes were missing, the Acidithiobacillus-like was the most active at the time of sampling despite being the second most populations had the genes for type-II secretion system, while all the Bacteria- abundant population (Paper II, Fig. 2). like populations had type-IV pili formation genes, that could potentially contribute to energy conservation through EET via conductive pili. The Unraveling metabolic processes (Paper II) presence of the EET mechanisms of Geobacter and Shewanella require further Tetrathionate was suggested to be disproportionated to sulfate, thiosulfate, and validation if they also function in the populations from this study. In addition, elemental sulfur mediated by tetrathionate hydrolase (tetH), and its gene was it cannot be ruled out that novel uncharacterized pathways were responsible for present in the A. caldus-like MAGs from both S1 and S2. Its encoding gene was carrying out EET. expressed at a low level, potentially due to the depletion of tetrathionate in the anolyte or the A. caldus-like MAGs were limited by the availability of carbon Mining process and waste streams are characterized with low organic carbon, dioxide for cellular growth. The produced sulfur was likely disproportionated and high acidity and metal content. To thrive in such environments, acidophilic to sulfide and sulfite, catalyzed by sulfur oxygenase reductase coded by sor that microorganisms have developed mechanisms for carbon dioxide fixation to was present in all MAGs except for the C. divulgatum-like population. However, sustain biomass growth; to maintain a near-neutral intracellular pH for sor gene transcripts were only observed in the A. caldus-like population. Sulfide bioenergetics as well as the function of proteins (Baker-Austin and Dopson, could be oxidized to sulfur via sulfide:quinone oxidoreductase (Sqr) that was 2007; Krulwich et al., 2011); and to resist soluble metal ions that are harmful

28 29 for the microorganisms (Dopson and Holmes, 2014). The Acidithiobacillus-like answer such questions, future studies should focus on using each one of the MAGs and the S. thermosulfidooxidans-like MAGs were suggested to be ISCs for electricity generation. capable of fixing carbon dioxide via the Calvin-Benson-Bassham (CBB) cycle. However, these genes were not expressed, indicating that carbon dioxide was limited at the time of cell harvest for metatranscriptomic analysis. The Archaea MAGs lacked genes for carbon dioxide fixation, these populations might grow chemoorganotrophically utilizing organic carbon secreted by the autotrophs. One pH homeostasis mechanism acidophiles utilize is to maintain an inside positive membrane potential (by accumulation of K+ ions) as a chemiosmotic barrier against the influx of protons (Baker-Austin and Dopson, 2007). Such genes were present in the MAGs (Paper II, Fig. 3). Furthermore, the proton consuming enzyme glutamate decarboxylase (Gut et al., 2006) and cell permeability modifying spermidine synthase (Samartzidou et al., 2003) were also present in the MAGs (Paper II, Fig. 3). However, RNA transcripts were only identified for decarboxylase and potassium transporters as well as spermidine synthase, suggesting that the cells were not under heavy pH stress. In addition, a variety of metal resistance mechanisms to arsenate, copper, silver, cadmium, cobalt, and zinc were present in the MAGs, reflecting the multi-metal containing mining waste stream environment. RNA transcripts for these genes were also scarce, suggesting the cells were not under the stress from heavy metals in the MFCs containing synthetic media lacking high concentrations of metals (Paper II, Fig. 3). Summary of the acidophilic MFC study (Papers I & II) This study demonstrated the possibility to simultaneously remediate tetrathionate and generate an electrical current at low pH. The anodic microbiome was dominated by acidophilic microorganisms and in particular, the Ferroplasma-like population was shown to be the most active and grew via metabolizing ISCs. EET transfer was suggested to be facilitated by redox-active electron shuttles synthesized by the Ferroplasma-like population and the Cuniculiplasma divulgatum-like population. In addition, the organic carbon produced by autotrophs might support the heterotrophs via mutualistic interactions within the community. Finally, the use of real wastewater in this study has shown the industrial potential of using acidophilic BES to remediate acidic ISC containing pollutions.

A complicating factor in this study was that the degradation of tetrathionate yielded multiple ISCs, e.g. sulfide, sulfur, and thiosulfate. This constrained the interpretation of data and in particular, to answer the question: which ISC(s) was(were) responsible for the release of electrons that fueled EET? Based on the high activity of sulfide:quinone oxidoreductase (Sqr), it was speculated that sulfide oxidation was primarily responsible for donating electrons. However, this is insufficient to conclude that sulfide was the major EET contributor. To

30 31 for the microorganisms (Dopson and Holmes, 2014). The Acidithiobacillus-like answer such questions, future studies should focus on using each one of the MAGs and the S. thermosulfidooxidans-like MAGs were suggested to be ISCs for electricity generation. capable of fixing carbon dioxide via the Calvin-Benson-Bassham (CBB) cycle. However, these genes were not expressed, indicating that carbon dioxide was limited at the time of cell harvest for metatranscriptomic analysis. The Archaea MAGs lacked genes for carbon dioxide fixation, these populations might grow chemoorganotrophically utilizing organic carbon secreted by the autotrophs. One pH homeostasis mechanism acidophiles utilize is to maintain an inside positive membrane potential (by accumulation of K+ ions) as a chemiosmotic barrier against the influx of protons (Baker-Austin and Dopson, 2007). Such genes were present in the MAGs (Paper II, Fig. 3). Furthermore, the proton consuming enzyme glutamate decarboxylase (Gut et al., 2006) and cell permeability modifying spermidine synthase (Samartzidou et al., 2003) were also present in the MAGs (Paper II, Fig. 3). However, RNA transcripts were only identified for decarboxylase and potassium transporters as well as spermidine synthase, suggesting that the cells were not under heavy pH stress. In addition, a variety of metal resistance mechanisms to arsenate, copper, silver, cadmium, cobalt, and zinc were present in the MAGs, reflecting the multi-metal containing mining waste stream environment. RNA transcripts for these genes were also scarce, suggesting the cells were not under the stress from heavy metals in the MFCs containing synthetic media lacking high concentrations of metals (Paper II, Fig. 3). Summary of the acidophilic MFC study (Papers I & II) This study demonstrated the possibility to simultaneously remediate tetrathionate and generate an electrical current at low pH. The anodic microbiome was dominated by acidophilic microorganisms and in particular, the Ferroplasma-like population was shown to be the most active and grew via metabolizing ISCs. EET transfer was suggested to be facilitated by redox-active electron shuttles synthesized by the Ferroplasma-like population and the Cuniculiplasma divulgatum-like population. In addition, the organic carbon produced by autotrophs might support the heterotrophs via mutualistic interactions within the community. Finally, the use of real wastewater in this study has shown the industrial potential of using acidophilic BES to remediate acidic ISC containing pollutions.

30 31 Psychrophilic MFC study (Paper III) Metatranscriptomics of the anodic psychrophilic microbiome Community RNA was extracted from both the planktonic cells and those attached to the graphite felt (anode) from the replicate MFCs (termed MFC A Previously, a psychrophilic autotrophic denitrifying culture was shown capable anode, MFC A anolyte, MFC B anode, MFC B anolyte). A total of 170 million of degrading thiocyanate (Broman et al., 2017). This culture was inoculated into read pairs were obtained, of which 78% was kept after quality trimming. Eight MFCs in Paper III to investigate if it could facilitate thiocyanate degradation percent of the trimmed reads were classified as 16S rRNA; 4% of the trimmed that is coupled to electricity generation. 16S rRNA gene amplicon sequencing reads were classified as mRNA, of which 88% was assigned to a protein based as well as metatranscriptomic analysis was carried out to profile the dominant on the InterPro database. In agreement with 16S amplicon sequencing, the microbial species within the anode and to study the active metabolic processes dominant 16S rRNA transcripts (between 63% and 76%) were attributed to the of this psychrophilic consortium. Thiobacillus genus and primarily to T. denitrificans across the four samples (Paper III, Fig. 3). This suggested that the Thiobacillus spp. was the most Electrochemical characterization and 16S rRNA gene amplicon abundant and active population at the time of DNA and RNA extraction. An sequencing abundance of mRNA reads was not taxonomically assigned with better Electricity generation was observed after 118 days and the long start-up time precision than Bacteria (7%) or Proteobacteria (49%), yet these populations was also observed in other low temperature BES studies (Bond et al., 2002; were responsible for major metabolic processes. Due to the dominance and Reimers et al., 2006). In general, low temperature negatively impacts on the activeness of the Thiobacillus genus, it would be possible that these populations functions of enzymes and reduces electrochemical reaction rates (Dopson et al., belonged to an unclassified Thiobacillus population. 2016) and as psychrophilic microorganisms have longer doubling times compared to mesophiles (Ferroni et al., 1986), these factors may explain the lag A model for thiocyanate degradation together with the metabolism of sulfur, time before electricity production took place. The maximum current densities carbon and nitrogen plus potential EET mechanisms was proposed (Paper III, normalized to the projected anode/membrane area were 28 and 26 mA/m2 for Fig. 4). The substrate thiocyanate was suggested to be degraded via the ‘cyanate’ the duplicated MFCs A and B, respectively; the CE values were 2.5% and 1.0% pathway and mRNA reads for the key gene of this pathway, cyanase (cyn), was for MFCs A and B, respectively. The electrical current and CE were relatively primarily attributed to unclassified Proteobacteria and Betaproteobacteriales. low compared to other BES studies (reviewed in Dopson et al., (2015)). This Fewer numbers of mRNA reads for another ‘COS’ pathway (COS designates could have several explanations: firstly, the autotrophic populations within the carbonyl sulfide) for thiocyanate degradation (encoded by scn for thiocyanate anodic microbial consortium (described below) fix carbon dioxide for biomass hydrolase) were primarily attributed to unclassified Bacteria and unclassified growth that restricted the available electrons that could be harvested as current; Proteobacteria. The product of thiocyanate degradation is sulfide, carbon secondly, trace levels of oxygen (a competing electron acceptor) can diffuse dioxide, and ammonium. Sulfide could be oxidized to sulfite via dissimilatory into the MFC via rubber tubing and graphite plates despite the continuous N2 sulfite reductase (Dsr) and mRNA reads for dsr were primarily attributed to gas flushing in the anolyte; and thirdly, competing energy conservation unclassified Bacteria and unclassified Proteobacteria. The resultant sulfite can reactions may have occurred (discussed below). be oxidized to sulfate via adenylylsulphate reductase (Apr) and dissimilatory adenylyl-sulfate reductase (Sat) (Kappler and Dahl, 2001). mRNA reads for apr 16S rRNA gene amplicon sequencing of the MFC A anodic biomass revealed and sat were primarily attributed to unclassified Proteobacteria. In addition, that the community was dominated by the genus Thiobacillus (relative mRNA reads for an alternative sulfide oxidation pathway involving a truncated abundance 76.8 %), followed by the family Methylophilaceae (6.5%), the sox complex (soxABCXY) were primarily attributed to unclassified family Rhizobiaceae (3.7%), the order Corynebacteriales (2.8%), the genus Proteobacteria and unclassified Bacteria, which could oxidize sulfide Ferritrophicum (2.2%), and the families Flavobacteriaceae (1.0%) and generating either sulfur or sulfate (Kantor et al., 2017). Xanthomonadaceae (0.9%). The presence of Thiobacillus was in accordance with the inoculum (Broman et al., 2017), as well as a metagenomic study in Evidence was lacking for known EET mechanisms such as c-type which Thiobacillus dominated a thiocyanate degrading bioreactor (Kantor et al., cytochromes or conductive pili from the mRNA data. Instead, mRNA reads for 2015). menaquinone biosynthesis were primarily found in the family Propionibacteriaceae; mRNA reads for riboflavin biosynthesis were mostly identified from unclassified Proteobacteria. In addition, mRNA reads for type

32 33 Psychrophilic MFC study (Paper III) Metatranscriptomics of the anodic psychrophilic microbiome Community RNA was extracted from both the planktonic cells and those attached to the graphite felt (anode) from the replicate MFCs (termed MFC A Previously, a psychrophilic autotrophic denitrifying culture was shown capable anode, MFC A anolyte, MFC B anode, MFC B anolyte). A total of 170 million of degrading thiocyanate (Broman et al., 2017). This culture was inoculated into read pairs were obtained, of which 78% was kept after quality trimming. Eight MFCs in Paper III to investigate if it could facilitate thiocyanate degradation percent of the trimmed reads were classified as 16S rRNA; 4% of the trimmed that is coupled to electricity generation. 16S rRNA gene amplicon sequencing reads were classified as mRNA, of which 88% was assigned to a protein based as well as metatranscriptomic analysis was carried out to profile the dominant on the InterPro database. In agreement with 16S amplicon sequencing, the microbial species within the anode and to study the active metabolic processes dominant 16S rRNA transcripts (between 63% and 76%) were attributed to the of this psychrophilic consortium. Thiobacillus genus and primarily to T. denitrificans across the four samples (Paper III, Fig. 3). This suggested that the Thiobacillus spp. was the most Electrochemical characterization and 16S rRNA gene amplicon abundant and active population at the time of DNA and RNA extraction. An sequencing abundance of mRNA reads was not taxonomically assigned with better Electricity generation was observed after 118 days and the long start-up time precision than Bacteria (7%) or Proteobacteria (49%), yet these populations was also observed in other low temperature BES studies (Bond et al., 2002; were responsible for major metabolic processes. Due to the dominance and Reimers et al., 2006). In general, low temperature negatively impacts on the activeness of the Thiobacillus genus, it would be possible that these populations functions of enzymes and reduces electrochemical reaction rates (Dopson et al., belonged to an unclassified Thiobacillus population. 2016) and as psychrophilic microorganisms have longer doubling times compared to mesophiles (Ferroni et al., 1986), these factors may explain the lag A model for thiocyanate degradation together with the metabolism of sulfur, time before electricity production took place. The maximum current densities carbon and nitrogen plus potential EET mechanisms was proposed (Paper III, normalized to the projected anode/membrane area were 28 and 26 mA/m2 for Fig. 4). The substrate thiocyanate was suggested to be degraded via the ‘cyanate’ the duplicated MFCs A and B, respectively; the CE values were 2.5% and 1.0% pathway and mRNA reads for the key gene of this pathway, cyanase (cyn), was for MFCs A and B, respectively. The electrical current and CE were relatively primarily attributed to unclassified Proteobacteria and Betaproteobacteriales. low compared to other BES studies (reviewed in Dopson et al., (2015)). This Fewer numbers of mRNA reads for another ‘COS’ pathway (COS designates could have several explanations: firstly, the autotrophic populations within the carbonyl sulfide) for thiocyanate degradation (encoded by scn for thiocyanate anodic microbial consortium (described below) fix carbon dioxide for biomass hydrolase) were primarily attributed to unclassified Bacteria and unclassified growth that restricted the available electrons that could be harvested as current; Proteobacteria. The product of thiocyanate degradation is sulfide, carbon secondly, trace levels of oxygen (a competing electron acceptor) can diffuse dioxide, and ammonium. Sulfide could be oxidized to sulfite via dissimilatory into the MFC via rubber tubing and graphite plates despite the continuous N2 sulfite reductase (Dsr) and mRNA reads for dsr were primarily attributed to gas flushing in the anolyte; and thirdly, competing energy conservation unclassified Bacteria and unclassified Proteobacteria. The resultant sulfite can reactions may have occurred (discussed below). be oxidized to sulfate via adenylylsulphate reductase (Apr) and dissimilatory adenylyl-sulfate reductase (Sat) (Kappler and Dahl, 2001). mRNA reads for apr 16S rRNA gene amplicon sequencing of the MFC A anodic biomass revealed and sat were primarily attributed to unclassified Proteobacteria. In addition, that the community was dominated by the genus Thiobacillus (relative mRNA reads for an alternative sulfide oxidation pathway involving a truncated abundance 76.8 %), followed by the family Methylophilaceae (6.5%), the sox complex (soxABCXY) were primarily attributed to unclassified family Rhizobiaceae (3.7%), the order Corynebacteriales (2.8%), the genus Proteobacteria and unclassified Bacteria, which could oxidize sulfide Ferritrophicum (2.2%), and the families Flavobacteriaceae (1.0%) and generating either sulfur or sulfate (Kantor et al., 2017). Xanthomonadaceae (0.9%). The presence of Thiobacillus was in accordance with the inoculum (Broman et al., 2017), as well as a metagenomic study in Evidence was lacking for known EET mechanisms such as c-type which Thiobacillus dominated a thiocyanate degrading bioreactor (Kantor et al., cytochromes or conductive pili from the mRNA data. Instead, mRNA reads for 2015). menaquinone biosynthesis were primarily found in the family Propionibacteriaceae; mRNA reads for riboflavin biosynthesis were mostly identified from unclassified Proteobacteria. In addition, mRNA reads for type

32 33 II secretion were primarily assigned to unclassified Proteobacteria and BES under low temperatures. Improving the precision of the mRNA reads Thiobacillus. These findings suggested that the anodic microbial community taxonomic placement and the validation of potential EET mechanisms from the might carry out EET via soluble electron shuttles. Since these results are based genus Thiobacillus are required for future studies. on the genetic information of model EET organisms such as Geobacter and Shewanella, they require further validation to check if such mechanisms can function in the non-model organisms from this study. It is also possible that uncharacterized mechanisms were responsible for EET in these organisms.

The autotrophic Thiobacillus genus is known for fixing carbon dioxide via the Calvin-Benson-Bassham (CCB) cycle (Boden et al., 2017;Kantor et al., 2017). However, despite the presence of seven genes within the CBB cycle primarily attributed to unclassified Proteobacteria, mRNA reads for the key enzyme ribulose biphosphate carboxylase (RuBisCO) were absent. Furthermore, the key enzymes ATP-citrate lyase from the reductive TCA cycle and carbon monoxide dehydrogenase from the Wood-Ljungdahl pathway were also identified in unclassified Bacteria, suggesting that several carbon fixation pathways were present in this consortium. Finally, mRNA reads suggested that ammonium was assimilated via Type I glutamine synthetase, mostly assigned to unclassified Proteobacteria followed by Microbacteriaceae (Paper III, Fig. 4).

Psychrophilic microorganisms utilize a variety of mechanisms to cope with low temperature, including producing CSPs and CIPs. These proteins have a variety of functions including to assist ribosomes and RNA polymerases to function at low temperature (Horn et al., 2007;Margesin et al., 2008;Barria et al., 2013); to produce compatible solutes that can protect against freezing (Casanueva et al., 2010); and to alter the membrane structure to increase flexibility of the cells (Chintalapati et al., 2004). mRNA reads for CSPs and CIPs were identified primarily from unclassified Proteobacteria; from unclassified Proteobacteria and Actinobacteria for compatible solutes; and from unclassified Proteobacteria and Thiobacillus for membrane alteration. The identification of mRNA reads for CSPs, CIPs and cold adaptation proteins suggested the anodic microbiomes were under cold stress due to the low temperature and these energy-consuming adaptations may have lowered the CE. Summary of the psychrophilic MFC study (Paper III) This study successfully demonstrated the use of a psychrophilic microbial consortium to remove thiocyanate in conjunction with electricity generation under low temperature for the first time. This consortium was dominated by the genus Thiobacillus. Metatranscriptomic analysis revealed that thiocyanate degradation, ISC oxidation, EET, inorganic carbon fixation, and adaptation to cold temperature were primarily associated with unclassified Bacteria and unclassified Proteobacteria populations, potentially attributed to Thiobacillus. Findings from this study can contribute to the industrial ISC remediation using

34 35 II secretion were primarily assigned to unclassified Proteobacteria and BES under low temperatures. Improving the precision of the mRNA reads Thiobacillus. These findings suggested that the anodic microbial community taxonomic placement and the validation of potential EET mechanisms from the might carry out EET via soluble electron shuttles. Since these results are based genus Thiobacillus are required for future studies. on the genetic information of model EET organisms such as Geobacter and Shewanella, they require further validation to check if such mechanisms can function in the non-model organisms from this study. It is also possible that uncharacterized mechanisms were responsible for EET in these organisms.

34 35 Haloalkaliphilic MEC study (Paper IV) the abiotic and biotic experiments suggested that the anodic microbial consortium facilitated current production from sulfide oxidation.

At the time of writing, haloalkaliphilic sulfide oxidizing microorganisms had Development of microbial community based on 16S rRNA gene not been tested in a BES for the removal of sulfide. This was undertaken in amplicon sequencing Paper IV of thesis, which monitored the electrochemical performance as well DNA was extracted from the inoculum, the planktonic cells at the end of the as profiled the dominant microorganisms in the anodic compartments using 16S first biotic batch experiment and from both the planktonic and electrode- rRNA gene amplicon sequencing. attached populations at the end of the second and third biotic batch experiments. The dominant populations from the inoculum had 16S rRNA gene sequences Electrochemical characterization for sulfide removal that aligned within the family Izimaplasmataceae (42.5%) and the genera An MEC was constructed to investigate if electrical current could be generated Thioalkalivibrio (24.1%) and Thioalkalimicrobium (5.9%) (Paper IV, Fig. 3). from sulfide oxidation bioelectrochemically and three batches of biotic Izimaplasmataceae is a poorly documented family and its role is yet unclear. By experiments were conducted (Paper IV, Fig. 2). During the first biotic batch the end of the first biotic batch experiment, populations with 16S rRNA gene experiment, sulfide was repeatedly spiked into the anolyte circulation where it sequences that aligned within Thioalkalimicrobium dominated the anodic was depleted. Electrical current was produced concurrently with the community (60.7%), followed by Alkalilimnicola (10.7%), Izimaplasmataceae degradation of sulfide, suggesting the electrons released from sulfide oxidation (6.1%), and Thioalkalivibrio (5.7%) (Paper IV, Fig. 3). Populations within the contributed to current generation (Paper IV, Fig. 2A). The second biotic batch genera Thioalkalivibrio and Thioalkalimicrobium are haloalkaliphilic, obligate experiment resulted in an average of a 10-fold increase in current (Paper IV, Fig. chemolithoautotrophs known for the utilization of sulfide (Sorokin et al., 2001; 2B) and was due to the complete oxidation of sulfide to sulfate supported by Ahn et al., 2017) and their presence was in agreement with a previous sulfate concentration measurements. Since sulfate is an undesired product that characterization of the same microbial community from an industrial requires further reductive treatment to elemental sulfur, an adjustment in anode desulfurization operation (Roman et al., 2016). In addition, the genus potential was made in the third biotic batch experiment intending to reduce Thioalkalimicrobium is known for relatively fast growth (Sorokin et al., 2002), sulfate reduction. This strategy was suggested by the redox potential used in an which could potentially explain its succession over the Thioalkalivibrio industrial desulfurization operation that is highly specific at sulfide-to-sulfur populations. conversion (Janssen et al., 1998). However, the effectiveness of this approach was unclear since the sulfide-to-sulfate conversion ratio from the third biotic By the end of the second biotic operation, populations with 16S rRNA gene batch was similar to the fifth spike of the second batch, but lower than the sequences that aligned within Desulfurivibrio (46.1%) and previous spikes. Thioalkalimicrobium (23.4%) dominated the planktonic consortium while Desulfurivibrio dominated the electrode-attached community (84.4%). After Abiotic control experiments produced less current and could be maintained the third biotic operation, the dominating populations in the planktonic fraction for a shorter period of time. In addition, abiotic current production decreased to had 16S rRNA gene sequences that aligned within the Marinobacter (58.5%), zero while sulfide was still present in the anolyte (Paper IV, Fig. 2D main figure) Bacteroidia (6.7%), and Desulfurivibrio (4.8%). In contrast, the electrode- whereas, during biotic operations the current decreased to zero only when attached community was continued to be dominated by Desulfurivibrio (94.1%) sulfide was exhausted, and current production could be resumed when sulfide (Paper IV, Fig. 3). Marinobacter are halophilic populations capable of was spiked into the anolyte. This evidence suggested that a portion of the energy hydrocarbon degradation (Gauthier et al., 1992; Martin et al., 2003). However, from sulfide could be extracted as electricity abiotically while the anodic their sulfur metabolism is poorly understood and their role in the MEC anolyte microbiome contributed to a higher current production for a longer period of was unclear. Desulfurivibrio is known for their anaerobic haloalkaliphilic time. In addition, graphite felt was also tested in an abiotic system to investigate lifestyle, growing through sulfur disproportionation (Poser et al., 2013). D. if the electrochemical sulfide oxidation was surface limited. Compared to alkaliphiles was shown able to obtain energy via sulfide oxidation to sulfate graphite paper, the felt-electrode system produced much higher current and (Thorup et al., 2017). Such metabolic capacity could explain the increase of degraded sulfide more rapidly (Paper IV, Fig. 2D inset), suggesting that the sulfate production during the second biotic batch experiment. In addition, pilin process was limited by surface area of the electrode. The comparison between biosynthesis genes of D. alkaliphiles AHT2T was shown to facilitate EET in Geobacter sulfurreducens; while ‘cable bacteria’ from groundwater that could

36 37 Haloalkaliphilic MEC study (Paper IV) the abiotic and biotic experiments suggested that the anodic microbial consortium facilitated current production from sulfide oxidation.

36 37 carry out long distance electron transfer have 16S rRNA gene sequences similar Overview of electrochemical performance (Papers I to D. alkaliphiles AHT2T (Muller et al., 2016). Due to anodic current production being facilitated by this mixed microbial consortium, EET must to IV) have been carried out in this study. The Desulfurivibrio-like population could At the time of writing, published records for extremophilic BES studies utilizing be a novel candidate for EET and the electrode functioned as a selecting ISC as substrates with available current density and CE values are scarce. As a pressure to enrich this population. However, this speculation needs to be result, BESs operated at extremophilic conditions utilizing various organic and validated in future studies using molecular tools such as genomics, inorganic substrates were compared in Table 2 in terms of operational transcriptomics, and proteomics. conditions, the choice of inocula, the choice of electron donors, as well as Post analysis of the electrode surface electrochemical parameters.

In a separate abiotic experiment, electrical impedance measurement was Both the acidophilic MFC study (Papers I & II) and the study conducted by carried out that showed the electrical resistance of the graphite paper electrode Sulonen and colleagues (2015) used an inoculum from mining operations to increased by 37.8% from 0.92 Ω to 1.26 Ω over 6 hours. SEM imaging revealed remediate tetrathionate in MFCs. The maximum current density and CE values that the inspected area on the electrode was covered by precipitates (Fig 14A) achieved in both studies were relatively low compared to the other studies listed and EDS analysis showed that the major elements were carbon (68.2%) and in Table 2. This implies that not all the electrons released in the oxidation of sulfur (30.1%). The precipitates from the electrode were analyzed by XRD tetrathionate were recovered as electrical current. Potential electron competing showing its composition to be elemental octasulfur (S8). In addition, the processes included the fixation of carbon dioxide, maintaining pH homeostasis graphite paper electrode used in the third biotic batch experiment was also (Paper II), or trace level of oxygen that diffused into the anodic compartment inspected using SEM, revealing curved rod-shaped bacterial-like components. that consumed electrons (Paper I and Sulonen et al., 2015). The psychrophilic These finding suggested that elemental sulfur deposition during the abiotic MFC study (Paper III) produced lower current density and CE compared to the batch experiments passivates the graphite paper electrode. In contrast, the study performed by Catal and colleagues (2011). This was expected because the electrode was inhabited by microorganisms that might have contributed to metabolism of the inorganic thiocyanate required the autotrophic population to sulfide oxidation and electron transfer to the electrode (Paper IV, Fig. 4B). fix inorganic carbon for biomass growth, which needed additional energy and compromised the CE (Jurtshuk, 1996). Compared to the higher temperature of Summary of the haloalkaliphilic MEC study (Paper IV) 14 °C chosen by Catal and colleagues (2011), the temperature of 8 °C in the This study demonstrated the possibility to oxidize sulfide under high pH and psychrophilic MFC study might also explain the lower current density. high salinity conditions. The biotic current was higher than the abiotic current Compared to the study conducted by Rousseau et al., (2013) that utilizes a suggesting the ability of microorganisms to oxidize sulfide to sulfate. The highly saline anolyte (0.8 M Na+) as well as the study conducted by Badalamenti anodic microbiome was dominated by sulfide-metabolizing microorganisms et al., (2013) at pH 9.3, the haloalkaliphilic MEC study achieved considerable and in particular, a Desulfurivibrio population dominated the electrode surface. current density and CE, potentially due to the enhanced electrical conductivity This might suggest a novel candidate to oxidize sulfide and carry out EET in by the high salinity. It is noticeable that despite the substrate being inorganic BES. Although adjusting the anode potential did not result in the reduction of (sulfide), relatively high current density and CE was still possible to achieve. sulfate, it is worthwhile investigating this approach in more details in future studies. Findings from this study offer a new direction to remediate sulfide Although the maximum current and CE values achieved in this thesis were bioelectrochemically, which is more energy efficient and more environmentally sub-optimal compared to the other listed studies, they serve as the proof-of- friendly. principle for ISC remediation using the BES technology.

38 39 carry out long distance electron transfer have 16S rRNA gene sequences similar Overview of electrochemical performance (Papers I to D. alkaliphiles AHT2T (Muller et al., 2016). Due to anodic current production being facilitated by this mixed microbial consortium, EET must to IV) have been carried out in this study. The Desulfurivibrio-like population could At the time of writing, published records for extremophilic BES studies utilizing be a novel candidate for EET and the electrode functioned as a selecting ISC as substrates with available current density and CE values are scarce. As a pressure to enrich this population. However, this speculation needs to be result, BESs operated at extremophilic conditions utilizing various organic and validated in future studies using molecular tools such as genomics, inorganic substrates were compared in Table 2 in terms of operational transcriptomics, and proteomics. conditions, the choice of inocula, the choice of electron donors, as well as Post analysis of the electrode surface electrochemical parameters.

38 39

2013) s ( 2015) 2013) ( 2011) ( ( Paper III Paper Paper IV Paper Reference al., Catal et al., Paper Sulonen et et Sulonen al., Badalamenti et Rousseau al., Rousseau et

2 3 23 1 48 31 95 < 5 Max.

CE (%) General conclusions and future outlook

) 2

Extremophiles live and thrive in numerous hostile conditions on earth and their

3 3 7 85 60 survival depends on their biological capabilities to metabolize multiple sources 0. 0.08 0.03 of nutrients (e.g. carbon, sulfur, nitrogen etc.,) coupled to different energy Max. Current Current Max.

density (A/m density conservation strategies. These metabolic potentials make them candidates for the remediation of industrial waste streams with extreme conditions that could

tremendously profit the humankind. In this thesis, three extremophilic microbial s consortiums that previously have not been shown electrochemically-active were

- - - s, maximum current density and maximum CE and maximum density current maximum s,

2 2 introduced into BESs. Experimental results revealed evidences for ISC - 6 6

O O degradation and the occurrence of EET, showing the potential of extremophilic 4 4 HS SCN S S Acetate mixture Ethanol

BES for the remediation of ISC-containing wastewaters.

Electron donor Electron acid, and xylose xylose and acid, Glucose, acetate, In the acidophilic MFC study, the Ferroplasma-like population was galactose, glucuronic glucuronic galactose, suggested to be the most active and grew via metabolizing ISCs while

synthesizing redox-active molecules that could potentially mediate EET.

Metatranscriptomic data suggested a complex multi-species tetrathionate

metabolism to support the growth and facilitate EET of the anodic biomass. It

was also speculated that the organic carbon produced by autotrophs supported

degradation degradation

- heterotrophs, suggesting mutualistic interactions within the community. The plant

process water process psychrophilic MFC study demonstrated the first utilization of an autotrophic Inoculum bioreactor microbial consortium in MFCs that could couple thiocyanate degradation to ferrihydriticus Geoalkalibacter Geoalkalibacter electricity generation at low temperature. 16S rRNA gene amplicon sequencing Salt marsh sediment - scale SCN Mining Mining Domestic waste water and the analysis of RNA transcripts suggested that Thiobacillus was the most Industrial desulfurization desulfurization Industrial Lab Acid mine drainage sediment drainage mine Acid treatment plant mixed culture mixed plant treatment abundant population. mRNA analysis suggested that the degradation of thiocyanate; the oxidation of reduced sulfur compounds; EET to the anode via

soluble shuttles; the fixation of carbon dioxide; and cold adaptation processes + +

9.5,

- 2.5 were mostly associated with unclassified Bacteria and unclassified °C °C Proteobacteria populations, potentially attributed to the Thiobacillus genus. In 8 pH 2 14 pH 9.3 pH both acidophilic MFC study and psychrophilic MFC study, the achieved CE 1.0 M Na 0.8 M Na pH 1.2 pH Conditions pH 8.8 - 8.8 pH values were relatively low, potentially due to electron-competing metabolic

Comparison of operational conditions, inocula, electron donor electron inocula, conditions, operational of Comparison processes such as the fixation of carbon dioxide and the adaptation to the 2 .

extreme conditions. Data from the haloalkaliphilic MEC study revealed that the

BES MFC MFC MFC MFC MFC MFC MEC Table Table between this thesis and a selectionextremophilic of BES studies. 40 Type of of Type

41 General conclusions and future outlook

Extremophiles live and thrive in numerous hostile conditions on earth and their survival depends on their biological capabilities to metabolize multiple sources of nutrients (e.g. carbon, sulfur, nitrogen etc.,) coupled to different energy conservation strategies. These metabolic potentials make them candidates for the remediation of industrial waste streams with extreme conditions that could tremendously profit the humankind. In this thesis, three extremophilic microbial consortiums that previously have not been shown electrochemically-active were introduced into BESs. Experimental results revealed evidences for ISC degradation and the occurrence of EET, showing the potential of extremophilic BES for the remediation of ISC-containing wastewaters.

In the acidophilic MFC study, the Ferroplasma-like population was suggested to be the most active and grew via metabolizing ISCs while synthesizing redox-active molecules that could potentially mediate EET. Metatranscriptomic data suggested a complex multi-species tetrathionate metabolism to support the growth and facilitate EET of the anodic biomass. It was also speculated that the organic carbon produced by autotrophs supported heterotrophs, suggesting mutualistic interactions within the community. The psychrophilic MFC study demonstrated the first utilization of an autotrophic microbial consortium in MFCs that could couple thiocyanate degradation to electricity generation at low temperature. 16S rRNA gene amplicon sequencing and the analysis of RNA transcripts suggested that Thiobacillus was the most abundant population. mRNA analysis suggested that the degradation of thiocyanate; the oxidation of reduced sulfur compounds; EET to the anode via soluble shuttles; the fixation of carbon dioxide; and cold adaptation processes were mostly associated with unclassified Bacteria and unclassified Proteobacteria populations, potentially attributed to the Thiobacillus genus. In both acidophilic MFC study and psychrophilic MFC study, the achieved CE values were relatively low, potentially due to electron-competing metabolic processes such as the fixation of carbon dioxide and the adaptation to the extreme conditions. Data from the haloalkaliphilic MEC study revealed that the

41 microorganisms facilitated the removal of sulfide. The bioelectrochemically produced current was greater than electrochemically produced current due to Acknowledgements the ability of microorganisms to carry out complete oxidation from sulfide to sulfate. Finally, the Desulfurivibrio population enriched on the electrode surface “A little learning is a dang'rous thing; drink deep, or taste not in this study was suggested to be a novel candidate to carry out EET. the Pierian spring: there shallow draughts intoxicate the brain,

The process of EET is the foundation of BES. Since current molecular and drinking largely sobers us again.” knowledge of EET mechanisms from the organisms present in this thesis were Alexander Pope not available, EET mechanisms from the model organisms Geobacter and Shewanella were used as benchmarks instead. And this approach was I have grown a lot during my PhD, as an academic but more importantly as a complemented by electrochemical measurements. It also implies the necessity person. I want to thank these people because this would be impossible without to carry out more research to discover and validate EET mechanisms from non- their help. model organisms in the future. Evidence for EET facilitated by the three types of extremophilic anodic microbiomes were observed, showing the wide range Firstly, I want to thank my main supervisor Mark Dopson, for offering me of extremophilic BES applications. Furthermore, the three studies offer insights this PhD opportunity and having faith in me, for countless guidance, for the such as the cycling of sulfur, carbon and nitrogen as well as microbial adaptation huge amount of freedom and strong support. It has been a great pleasure mechanisms to extreme conditions; such knowledge could benefit future working with you, and I have learnt a lot from you. I deeply appreciate your bioremediation using extremophilic BESs as well as enrich the current help and understanding whenever issues arise. To my supervisor Tom Sleutels, understandings of microbial metabolism of these elements. In addition, these you always ask good questions that can makes me feel a bit unease, but stimulate studies demonstrated that the use of high throughput multi-omics techniques is me to think more and work in a scientific manner. This is very rewarding for powerful to profile microbial community composition; to unravels genetic me. And also thank you for being such a gezellig mentor. To my supervisor potential of the microbiome under investigation as well as to examine the active Annemiek Ter Heijne, I learnt from your critical way of thinking and your open metabolic processes being undertaken. An excellent example is the finding that mind, your guidance at work was very helpful, and also you have been very the Ferroplasma-like population that was suggested to metabolize ISCs and kind and caring for me, which make it a very nice experience working with you. carry out EET via soluble electron shuttles. However, one should be aware of To Samual Hylander, thank you for being my co-supervisor. To Jonas the limitations from individual techniques and use multiple approaches that Waldenström, thank you for being my examiner and for all the help I received complement each other in order to accurately represent the study subject. from you during my PhD.

I started my PhD as part of the EU research project BioelectroMET, and I Finally, this thesis demonstrated that BESs are a promising technology that not only can recover electrical energy from the oxidation of waste or toxic want to acknowledge my collaborators from this project, Adriaan Jeremiasse, compounds, but also provide an excellent platform that allow us to investigate Aino-Maija Lakaniemi, Francisco Fabregat-Santiago, Elorri Igos, Martijn the process of EET. Compared to conventional treatments for ISCs such as Bijmans, Michel Saakes, Mira Sulonen, Pau Rodenas Motos, Roel Meulepas chemical dosing, BESs are cost-effective and renewable (as the catalyzing and Steve Tennison for the nice and enjoyable collaboration. During my PhD, I microorganisms can renew themselves). Admittedly, the current state of BESs spent eighteen months as a guest PhD student at Wetsus, I want to thank Cees isn’t mature enough to be implemented on an industrial scale. This will require Buisman, Johannes Boonstra and also members of the Resource Recovery more research to understand the microbiology, as well as the optimization of theme that I was part of, Caroline Plugge, Casper Borsje, Fons Stams, Leire the engineering aspects. However, the combination of extremophiles and BESs Caizan, Marianna Rodriguez, Monir Mollaei, Paula van den Brink, Peer has opened new possibilities to remediate “extreme” pollution streams in a more Timmers, Philipp Kuntke, Pieter Hack, Renata van der Weijden, Sam Molenaar, environmentally friendly manner. and Steffen Georg. Last but not the least, I want to thank Anke de Graaf, Linda van der Ploeg and Nynke Broekens from Wetsus, and Catherine Legrand, Christian Andersson, Christina Esplund-Lindquist, Ian Nicholls, Margaretha Bjerker, Per Lundberg, Per-Eric Betzholtz, Sofia Jönsson Ekström, and Ymke Mulder from Linnaeus University for all the help I received from you.

42 43 microorganisms facilitated the removal of sulfide. The bioelectrochemically produced current was greater than electrochemically produced current due to Acknowledgements the ability of microorganisms to carry out complete oxidation from sulfide to sulfate. Finally, the Desulfurivibrio population enriched on the electrode surface “A little learning is a dang'rous thing; drink deep, or taste not in this study was suggested to be a novel candidate to carry out EET. the Pierian spring: there shallow draughts intoxicate the brain,

42 43 My friends and colleagues at Linnaeus University has made my life in Tedesco, Natascha Pfaff, Olivier Schaetzle, Paweł Roman, Philipp Wilfert, Kalmar much more enjoyable. First, to my colleagues at the Systems Biology Prashanth Kumar, Pebrianto Harnawan, Rik de Vries, Sandra Drusova, Shuyana of Microorganisms research group that I was part of, it was great pleasure Heredia, Sławomir Porada, Stan Willems, Suyash Gupta, Swarupa Chatterjee, working with you guys. Elias, you are cool in many things you do, respect! Tania Mubita, Terica Sinclair, and Thomas Prot, for the great Wetsus Margarita, it was a lot of fun working with you, I miss you and wish you all the experience I had. best in Germany! Xiaofen, thank you for all the help I received from you especially during the beginning of my PhD! Many thanks to Abbtesaim, And a special thank you to Harm van der Kooi, Bianca de Vries, Ernst Panstra, Antoine, Domenico, Jimmy, Laura, Magnus, Stephan, and Stephanie, for being Gerben Breidenbach-Visser, Gerrit Visser, Heleen Sombekke, Ilse Gerrits, Jan very nice colleagues to me and for the good times. Jurjen Salverda, Janneke Tempel, Jannie Mulder, Jan-Willem Schoonen, Jelmer Dijkstra, Mieke Kersaan-Haan, Rienk Smit, Riet Bouwer, Ton van der Zande, I also want to thank Anders Månsson, Alexis Avril, Camilla Kalsson, Carina Wiebe Kingma, and Wim Borgonje for the great support I got from you. Gerben Bunse, Caroline Littlefield Kalsson, Charlotte Marchand, Clara Pérez Martínez, and Riet, you made the Wetsus kitchen lovely! Clara Vidal Carulla, Conny Tolf, Daniel Lundin, Daniela Palma, Daniela Polic, Dennis Amnebrink, Diego Brambilla, Elin Lindehoff, Emil Fridolfsson, Fabio I also thank Anke de Graaf, Gerben Breidenbach-Visser, Gonçalo Macedo, Kaczala, Fredrik Svensson, Hanna Berggren, Håkan Johansson, Henrik Flink, Jan de Groot, Karine Kiragosyan, Maarten van de Griend, Marianne Heegstra, Henrik Hallberg, Jacintha van Dijk, Javi Alegria, Jenny Olofsson, Joacim Paulina Sosa Fernandez, Raquel Barbosa and Rebeca Pallares for a very nice Rosenlund, Joanne Chapman, Johanna Sunde, Josanne, Karin Holmfeldt, PV experience. Kimberly Berglöf, Laura Ferrans, Leon Norén, Lina Mattsson, Mariëlle van Toor, Maurice Hirwa, Michelle Wille, Mireia Bertos-Fortis, Oscar Nordahl, You guys helped to start my life in Leeuwarden, and made it into something Rahaf Arabi, Richard Mutafela, Richard Williams, Sara Gunnarsson, Sarala beautiful, thank you Alfred Zwikstra, Alperen Sevinc, Assoumya Soumi Devi, Sina Shahabi Ghahfarokhi, Terese Uddh Söderberg, Viraja Viru, Yahya Bouderbala, Marysia Sliwczynska, Pamela Bukowska, Sonja Willemse, and Jani, and Yeserin Yildirim. It was a really nice experience to work with all of Guillaume Parmentier. you at Linnaeus University. My dear friends from China, Bingnan Song, Chen Zhang, Dong Peng, Furong Dear mentee buds, Anu, Ben, Christofer, Emelie and Eva, MP7 brought us Li, Jingyang Zhang, Ling Gao, Lingni Li, Mengyi Du, Qian Jia, Qingdian Shu, closely together, we have all learned and grown much during MP7, this Qiuhong Wang, Ruijun Pan, Runwen Zhu, Shan Huang, Shenghong Ma, Shiyu experience is precious to me. I also want to thank my mentor from MP7, Carina Yan, Yanyue Feng, Yang Lei, Yin Ye, Yuchen Liu, Zexin Qian, Wanrong Liu, Pålsson, for your help, your time and kindness. It was great to have met you, Xiaolu Li, Yingdi Zhang, and Zhichao Zheng, I am very happy to have met you, and our time together brought me much clarity and new perspectives. the time we had together was really nice and made me feel closer to home.

My dear office mates at Wetsus, Deepika Lakshmi Ramasamy, Elias Bodner, Thank you, Rowena Jansson, it was wonderful to be your student and later Ettore Virga, Hanieh Bazyar, Jan Willem van Egmond, Janneke Dickhout, Mark your friend. It was really kind of you to have my father and me at your house Roghair, Mithun Chowdhury, Sebastian Canizales, Sofia Semitsoglou Tsiapou, and we had delightful times together. Tim van der Lans, and Victor Torres Serrano, thank you all for the good times, I wish you all best of luck! And of course, our office rocks. Ingrid Weurman, thank you for your kindness, your wise words and advices. I have learnt much from you, and I am grateful for your help, as well as for the I also thank my friends and colleagues at Wetsus: Adam Wexler, Agnieszka nice discussions we had. Tomaszewska, Andrew Shamu, Antoine Karengera, Caspar Geelen, Chris Schott, Diego Pintossi, Diniz Emanuel, Doekle Yntema, Enas Othman, Erwin To Arne Sjöberg, thank you for opening your door for me, for being a very Tuithof, Fabian Ruhnau, Gerwin Steen, Gijs Doornbusch, Hakan Kandemir, good friend, and for all the nice time we spent together. Hector Hernandez Delgadillo, Ilse Verburg, Jaap Dijkshoorn, Jan Klok, Joeri de Valenca, Joana Carvalho, Jorge Ricardo Cunha, Jorrit Hillebrand, Jouke Dykstra, Luis Legrand, Maarten Biesheuvel, Malgorzata Nowak, Michele

44 45 My friends and colleagues at Linnaeus University has made my life in Tedesco, Natascha Pfaff, Olivier Schaetzle, Paweł Roman, Philipp Wilfert, Kalmar much more enjoyable. First, to my colleagues at the Systems Biology Prashanth Kumar, Pebrianto Harnawan, Rik de Vries, Sandra Drusova, Shuyana of Microorganisms research group that I was part of, it was great pleasure Heredia, Sławomir Porada, Stan Willems, Suyash Gupta, Swarupa Chatterjee, working with you guys. Elias, you are cool in many things you do, respect! Tania Mubita, Terica Sinclair, and Thomas Prot, for the great Wetsus Margarita, it was a lot of fun working with you, I miss you and wish you all the experience I had. best in Germany! Xiaofen, thank you for all the help I received from you especially during the beginning of my PhD! Many thanks to Abbtesaim, And a special thank you to Harm van der Kooi, Bianca de Vries, Ernst Panstra, Antoine, Domenico, Jimmy, Laura, Magnus, Stephan, and Stephanie, for being Gerben Breidenbach-Visser, Gerrit Visser, Heleen Sombekke, Ilse Gerrits, Jan very nice colleagues to me and for the good times. Jurjen Salverda, Janneke Tempel, Jannie Mulder, Jan-Willem Schoonen, Jelmer Dijkstra, Mieke Kersaan-Haan, Rienk Smit, Riet Bouwer, Ton van der Zande, I also want to thank Anders Månsson, Alexis Avril, Camilla Kalsson, Carina Wiebe Kingma, and Wim Borgonje for the great support I got from you. Gerben Bunse, Caroline Littlefield Kalsson, Charlotte Marchand, Clara Pérez Martínez, and Riet, you made the Wetsus kitchen lovely! Clara Vidal Carulla, Conny Tolf, Daniel Lundin, Daniela Palma, Daniela Polic, Dennis Amnebrink, Diego Brambilla, Elin Lindehoff, Emil Fridolfsson, Fabio I also thank Anke de Graaf, Gerben Breidenbach-Visser, Gonçalo Macedo, Kaczala, Fredrik Svensson, Hanna Berggren, Håkan Johansson, Henrik Flink, Jan de Groot, Karine Kiragosyan, Maarten van de Griend, Marianne Heegstra, Henrik Hallberg, Jacintha van Dijk, Javi Alegria, Jenny Olofsson, Joacim Paulina Sosa Fernandez, Raquel Barbosa and Rebeca Pallares for a very nice Rosenlund, Joanne Chapman, Johanna Sunde, Josanne, Karin Holmfeldt, PV experience. Kimberly Berglöf, Laura Ferrans, Leon Norén, Lina Mattsson, Mariëlle van Toor, Maurice Hirwa, Michelle Wille, Mireia Bertos-Fortis, Oscar Nordahl, You guys helped to start my life in Leeuwarden, and made it into something Rahaf Arabi, Richard Mutafela, Richard Williams, Sara Gunnarsson, Sarala beautiful, thank you Alfred Zwikstra, Alperen Sevinc, Assoumya Soumi Devi, Sina Shahabi Ghahfarokhi, Terese Uddh Söderberg, Viraja Viru, Yahya Bouderbala, Marysia Sliwczynska, Pamela Bukowska, Sonja Willemse, and Jani, and Yeserin Yildirim. It was a really nice experience to work with all of Guillaume Parmentier. you at Linnaeus University. My dear friends from China, Bingnan Song, Chen Zhang, Dong Peng, Furong Dear mentee buds, Anu, Ben, Christofer, Emelie and Eva, MP7 brought us Li, Jingyang Zhang, Ling Gao, Lingni Li, Mengyi Du, Qian Jia, Qingdian Shu, closely together, we have all learned and grown much during MP7, this Qiuhong Wang, Ruijun Pan, Runwen Zhu, Shan Huang, Shenghong Ma, Shiyu experience is precious to me. I also want to thank my mentor from MP7, Carina Yan, Yanyue Feng, Yang Lei, Yin Ye, Yuchen Liu, Zexin Qian, Wanrong Liu, Pålsson, for your help, your time and kindness. It was great to have met you, Xiaolu Li, Yingdi Zhang, and Zhichao Zheng, I am very happy to have met you, and our time together brought me much clarity and new perspectives. the time we had together was really nice and made me feel closer to home.

44 45 AA specialspecial thankthank youyou toto BengtBengt Fredriksson.Fredriksson. BecauseBecause ofof youryour help,help, II waswas ableable toto anchoranchor myselfmyself duringduring thethe upsups andand downsdowns ofof PhD.PhD. II havehave learntlearnt muchmuch fromfrom References youyou andand alsoalso aboutabout myself,myself, thisthis experienceexperience isis somethingsomething II cancan alwaysalways cherish.cherish.

AndAnd toto mymy deardear homehome group,group, Andreas,Andreas, Anna,Anna, Bukky,Bukky, Brian,Brian, Femke,Femke, Haniel,Haniel, Ahn, A.C., Meier-Kolthoff, J.P., Overmars, L., Richter, M., Woyke, T., Sorokin, Hellen,Hellen, Jane,Jane, Jesse,Jesse, Johanna,Johanna, Lea,Lea, Lydia,Lydia, Markus,Markus, Nahom,Nahom, Nimmy,Nimmy, Rahel,Rahel, Sina,Sina, D.Y., and Muyzer, G. (2017). Genomic diversity within the SonjaSonja andand Victor,Victor, II missmiss youyou aa lot.lot. HomeHome groupgroup hashas beenbeen aa bigbig partpart ofof mymy lilifefe inin haloalkaliphilic genus Thioalkalivibrio. PloS One 12, e0173517. Leeuwarden,Leeuwarden, II lovelove you.you. Alnaizy, R. (2008). Economic analysis for wet oxidation processes for the treatment of mixed refinery spent caustic. Environmental Progress 27, ��9;@O;PPRR -(1L/";<5# 295-301. �� Alneberg, J., Bjarnason, B.S., De Bruijn, I., Schirmer, M., Quick, J., Ijaz, U.Z., (:G&4Q?C,%B I$KTJ!( Lahti, L., Loman, N.J., Andersson, A.F., and Quince, C. (2014). �� NH;,L +F-(=0 *)- 3S Binning metagenomic contigs by coverage and composition. Nature �� Methods 11, 1144-1146. ��()A-2;DM%B>(� 377;8.9T ;'E-+ Amann, R.I., Ludwig, W., and Schleifer, K.H. (1995). Phylogenetic 6;T(9 T Identification and in-Situ Detection of Individual Microbial-Cells without Cultivation. Microbiological Reviews 59, 143-169.

Andrews, S. (2010). FastQC: a quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc Badalamenti, J.P., Krajmalnik-Brown, R., and Torres, C.I. (2013). Generation of high current densities by pure cultures of anode-respiring Geoalkalibacter spp. under alkaline and saline conditions in microbial electrochemical cells. MBio 4, e00144-00113. Baker-Austin, C., and Dopson, M. (2007). Life in acid: pH homeostasis in acidophiles. Trends in Microbiology 15, 165-171. Baker-Austin, C., Dopson, M., Wexler, M., Sawers, R.G., and Bond, P.L. (2005). Molecular insight into extreme copper resistance in the extremophilic archaeon 'Ferroplasma acidarmanus' Fer1. Microbiology 151, 2637-2646. Banciu, H., Sorokin, D.Y., Galinski, E.A., Muyzer, G., Kleerebezem, R., and Kuenen, J.G. (2004). Thialkalivibrio halophilus sp. nov., a novel obligately chemolithoautotrophic, facultatively alkaliphilic, and extremely salt-tolerant, sulfur-oxidizing bacterium from a hypersaline alkaline lake. Extremophiles 8, 325-334. Barria, C., Malecki, M., and Arraiano, C.M. (2013). Bacterial adaptation to cold. Microbiology 159, 2437-2443. Blazewicz, S.J., Barnard, R.L., Daly, R.A., and Firestone, M.K. (2013). Evaluating rRNA as an indicator of microbial activity in environmental communities: limitations and uses. The ISME Journal 7, 2061-2068. Boden, R., Hutt, L.P., and Rae, A.W. (2017). Reclassification of Thiobacillus aquaesulis (Wood & Kelly, 1995) as Annwoodia aquaesulis gen. nov., comb. nov., transfer of Thiobacillus (Beijerinck, 1904) from the Hydrogenophilales to the Nitrosomonadales, proposal of

46 46 47 A special thank you to Bengt Fredriksson. Because of your help, I was able to anchor myself during the ups and downs of PhD. I have learnt much from References you and also about myself, this experience is something I can always cherish.

And to my dear home group, Andreas, Anna, Bukky, Brian, Femke, Haniel, Ahn, A.C., Meier-Kolthoff, J.P., Overmars, L., Richter, M., Woyke, T., Sorokin, Hellen, Jane, Jesse, Johanna, Lea, Lydia, Markus, Nahom, Nimmy, Rahel, Sina, D.Y., and Muyzer, G. (2017). Genomic diversity within the Sonja and Victor, I miss you a lot. Home group has been a big part of my life in haloalkaliphilic genus Thioalkalivibrio. PloS One 12, e0173517. Leeuwarden, I love you. Alnaizy, R. (2008). Economic analysis for wet oxidation processes for the treatment of mixed refinery spent caustic. Environmental Progress 27, �� 295-301. �� Alneberg, J., Bjarnason, B.S., De Bruijn, I., Schirmer, M., Quick, J., Ijaz, U.Z., Lahti, L., Loman, N.J., Andersson, A.F., and Quince, C. (2014). �� Binning metagenomic contigs by coverage and composition. Nature �� Methods 11, 1144-1146. �� Amann, R.I., Ludwig, W., and Schleifer, K.H. (1995). Phylogenetic Identification and in-Situ Detection of Individual Microbial-Cells without Cultivation. Microbiological Reviews 59, 143-169. Andrews, S. (2010). FastQC: a quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc Badalamenti, J.P., Krajmalnik-Brown, R., and Torres, C.I. (2013). Generation of high current densities by pure cultures of anode-respiring Geoalkalibacter spp. under alkaline and saline conditions in microbial electrochemical cells. MBio 4, e00144-00113. Baker-Austin, C., and Dopson, M. (2007). Life in acid: pH homeostasis in acidophiles. Trends in Microbiology 15, 165-171. Baker-Austin, C., Dopson, M., Wexler, M., Sawers, R.G., and Bond, P.L. (2005). Molecular insight into extreme copper resistance in the extremophilic archaeon 'Ferroplasma acidarmanus' Fer1. Microbiology 151, 2637-2646. Banciu, H., Sorokin, D.Y., Galinski, E.A., Muyzer, G., Kleerebezem, R., and Kuenen, J.G. (2004). Thialkalivibrio halophilus sp. nov., a novel obligately chemolithoautotrophic, facultatively alkaliphilic, and extremely salt-tolerant, sulfur-oxidizing bacterium from a hypersaline alkaline lake. Extremophiles 8, 325-334. Barria, C., Malecki, M., and Arraiano, C.M. (2013). Bacterial adaptation to cold. Microbiology 159, 2437-2443. Blazewicz, S.J., Barnard, R.L., Daly, R.A., and Firestone, M.K. (2013). Evaluating rRNA as an indicator of microbial activity in environmental communities: limitations and uses. The ISME Journal 7, 2061-2068. Boden, R., Hutt, L.P., and Rae, A.W. (2017). Reclassification of Thiobacillus aquaesulis (Wood & Kelly, 1995) as Annwoodia aquaesulis gen. nov., comb. nov., transfer of Thiobacillus (Beijerinck, 1904) from the Hydrogenophilales to the Nitrosomonadales, proposal of

46 47 Hydrogenophilalia class. nov. within the ‘Proteobacteria’, and four Casanueva, A., Tuffin, M., Cary, C., and Cowan, D.A. (2010). Molecular new families within the orders Nitrosomonadales and Rhodocyclales. adaptations to psychrophily: the impact of 'omic' technologies. Trends International Journal of Systematic and Evolutionary Microbiology 67, in Microbiology 18, 374-381. 1191-1205. Caspi, R., Altman, T., Billington, R., Dreher, K., Foerster, H., Fulcher, C.A., Boisvert, S., Laviolette, F., and Corbeil, J. (2010). Ray: simultaneous assembly Holland, T.A., Keseler, I.M., Kothari, A., Kubo, A., Krummenacker, of reads from a mix of high-throughput sequencing technologies. M., Latendresse, M., Mueller, L.A., Ong, Q., Paley, S., Subhraveti, P., Journal of Computational Biology 17, 1519-1533. Weaver, D.S., Weerasinghe, D., Zhang, P., and Karp, P.D. (2014). The Bond, D.R., Holmes, D.E., Tender, L.M., and Lovley, D.R. (2002). Electrode- MetaCyc database of metabolic pathways and enzymes and the BioCyc reducing microorganisms that harvest energy from marine sediments. collection of Pathway/Genome Databases. Nucleic Acids Research 42, Science 295, 483-485. D459-471. Bretschger, O., Obraztsova, A., Sturm, C.A., Chang, I.S., Gorby, Y.A., Reed, Catal, T., Kavanagh, P., O'flaherty, V., and Leech, D. (2011). Generation of S.B., Culley, D.E., Reardon, C.L., Barua, S., and Romine, M.F. (2007a). electricity in microbial fuel cells at sub-ambient temperatures. Journal Current production and metal oxide reduction by Shewanella of Power Sources 196, 2676-2681. oneidensis MR-1 wild type and mutants. Applied and environmental Chao, J., Wang, W., Xiao, S.M., and Liu, X.D. (2008). Response of microbiology 73, 7003-7012. Acidithiobacillus ferrooxidans ATCC 23270 gene expression to acid Bretschger, O., Obraztsova, A., Sturm, C.A., Chang, I.S., Gorby, Y.A., Reed, stress. World Journal of Microbiology & Biotechnology 24, 2103-2109. S.B., Culley, D.E., Reardon, C.L., Barua, S., Romine, M.F., Zhou, J., Chintalapati, S., Kiran, M.D., and Shivaji, S. (2004). Role of membrane lipid Beliaev, A.S., Bouhenni, R., Saffarini, D., Mansfeld, F., Kim, B.H., fatty acids in cold adaptation. Cellular and Molecular Biology (Noisy- Fredrickson, J.K., and Nealson, K.H. (2007b). Current production and Le-Grand, France) 50, 631-642. metal oxide reduction by Shewanella oneidensis MR-1 wild type and Crick, F.H. (Year). "On protein synthesis", in: Symposia of the Society for mutants. Applied and Environmental Microbiology 73, 7003-7012. Experimental Biology), 8. Bridge, T.a.M., and Johnson, D.B. (1998). Reduction of soluble iron and D'amico, S., Collins, T., Marx, J.C., Feller, G., and Gerday, C. (2006). reductive dissolution of ferric iron-containing minerals by moderately Psychrophilic microorganisms: challenges for life. 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48 49 Dopson, M., Ni, G., and Sleutels, T.H. (2016). Possibilities for extremophilic and other microorganisms. Proceedings of the National Academy of microorganisms in microbial electrochemical systems. FEMS Sciences of the United States of America 103, 11358-11363. Microbiology Reviews 40, 164-181. Gould, W.D., King, M., Mohapatra, B.R., Cameron, R.A., Kapoor, A., and Dopson, M., Ossandon, F.J., Lövgren, L., and Holmes, D.S. (2014). Metal Koren, D.W. (2012). A critical review on destruction of thiocyanate in resistance or tolerance? Acidophiles confront high metal loads via both mining effluents. Minerals Engineering 34, 38-47. abiotic and biotic mechanisms. Frontiers in Microbiology 5, 157. Gregory, K.B., Bond, D.R., and Lovley, D.R. (2004). Graphite electrodes as Dutta, P.K., Rabaey, K., Yuan, Z., and Keller, J. (2008). Spontaneous electron donors for anaerobic respiration. Environmental Microbiology electrochemical removal of aqueous sulfide. 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50 51 Dopson, M., Ni, G., and Sleutels, T.H. (2016). Possibilities for extremophilic and other microorganisms. Proceedings of the National Academy of microorganisms in microbial electrochemical systems. FEMS Sciences of the United States of America 103, 11358-11363. Microbiology Reviews 40, 164-181. Gould, W.D., King, M., Mohapatra, B.R., Cameron, R.A., Kapoor, A., and Dopson, M., Ossandon, F.J., Lövgren, L., and Holmes, D.S. (2014). Metal Koren, D.W. (2012). A critical review on destruction of thiocyanate in resistance or tolerance? Acidophiles confront high metal loads via both mining effluents. Minerals Engineering 34, 38-47. abiotic and biotic mechanisms. Frontiers in Microbiology 5, 157. Gregory, K.B., Bond, D.R., and Lovley, D.R. (2004). Graphite electrodes as Dutta, P.K., Rabaey, K., Yuan, Z., and Keller, J. (2008). Spontaneous electron donors for anaerobic respiration. Environmental Microbiology electrochemical removal of aqueous sulfide. 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Characterization of Psychrotrophic Strains of Thiobacillus- Microarray and genetic analysis of electron transfer to electrodes in Ferrooxidans from the Environment of a Uranium-Mine. Journal of Geobacter sulfurreducens. Environmental Microbiology 8, 1805-1815. General and Applied Microbiology 32, 169-175. Horn, G., Hofweber, R., Kremer, W., and Kalbitzer, H.R. (2007). Structure and Franks, A.E. (2010). Transcriptional analysis in microbial fuel cells: common function of bacterial cold shock proteins. Cellular and Molecular Life pitfalls in global gene expression studies of microbial biofilms. FEMS Sciences 64, 1457-1470. Microbiology Letters 307, 111-112. Hugerth, L.W., Wefer, H.A., Lundin, S., Jakobsson, H.E., Lindberg, M., Rodin, Gauthier, M.J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M., Bonin, S., Engstrand, L., and Andersson, A.F. (2014). DegePrime, a program P., and Bertrand, J.C. (1992). Marinobacter hydrocarbonoclasticus gen. for degenerate primer design for broad-taxonomic-range PCR in nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading microbial ecology studies. Applied and Environmental Microbiology marine bacterium. International Journal of Systematic and 80, 5116-5123. Evolutionary Microbiology 42, 568-576. Janssen, A.J., Meijer, S., Bontsema, J., and Lettinga, G. (1998). Application of Geppert, F., Liu, D., Van Eerten-Jansen, M., Weidner, E., Buisman, C., and Ter the redox potential for controling a sulfide oxidizing bioreactor. Heijne, A. (2016). Bioelectrochemical Power-to-Gas: State of the Art Biotechnology and Bioengineering 60, 147-155. and Future Perspectives. Trends in Biotechnology 34, 879-894. Jensen, A.B., and Webb, C. (1995). Treatment of H2s-Containing Gases - a Gorby, Y.A., Yanina, S., Mclean, J.S., Rosso, K.M., Moyles, D., Dohnalkova, Review of Microbiological Alternatives. 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52 53 Joshi, N., and Fass, J. (2011). "Sickle: A sliding-window, adaptive, quality- Li, W.W., Yu, H.Q., and He, Z. (2014). Towards sustainable wastewater based trimming tool for FastQ files (Version 1.33)[Software]".). treatment by using microbial fuel cells-centered technologies. Energy Jourdin, L., Grieger, T., Monetti, J., Flexer, V., Freguia, S., Lu, Y., Chen, J., & Environmental Science 7, 911-924. Romano, M., Wallace, G.G., and Keller, J. (2015). High Acetic Acid Liljeqvist, M., Ossandon, F.J., Gonzalez, C., Rajan, S., Stell, A., Valdes, J., Production Rate Obtained by Microbial Electrosynthesis from Carbon Holmes, D.S., and Dopson, M. (2015). Metagenomic analysis reveals Dioxide. Environmental Science & Technology 49, 13566-13574. adaptations to a cold-adapted lifestyle in a low-temperature acid mine Kanehisa, M., and Goto, S. (2000). KEGG: kyoto encyclopedia of genes and drainage stream. FEMS Microbiology Ecology 91. genomes. Nucleic Acids Research 28, 27-30. 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52 53 Begley, R.F., and Rothberg, J.M. (2005). Genome sequencing in Orell, A., Navarro, C.A., Rivero, M., Aguilar, J.S., and Jerez, C.A. (2012). microfabricated high-density picolitre reactors. Nature 437, 376-380. Inorganic polyphosphates in extremophiles and their possible functions. Marsili, E., Baron, D.B., Shikhare, I.D., Coursolle, D., Gralnick, J.A., and Bond, Extremophiles 16, 573-583. D.R. (2008). Shewanella Secretes flavins that mediate extracellular Oren, A. (2008). Microbial life at high salt concentrations: phylogenetic and electron transfer. Proceedings of the National Academy of Sciences of metabolic diversity. Saline Systems 4, 2. the United States of America 105, 3968-3973. Orr, W.L., and Sinninghe Damsté, J.S. (1990). "Geochemistry of sulfur in Martin, S., Marquez, M.C., Sanchez-Porro, C., Mellado, E., Arahal, D.R., and petroleum systems." ACS Publications). Ventosa, A. (2003). 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Applied and Environmental Microbiology Parks, D.H., Imelfort, M., Skennerton, C.T., Hugenholtz, P., and Tyson, G.W. 65, 4715-4724. (2015). CheckM: assessing the quality of microbial genomes recovered Muller, H., Bosch, J., Griebler, C., Damgaard, L.R., Nielsen, L.P., Lueders, T., from isolates, single cells, and metagenomes. Genome Research 25, and Meckenstock, R.U. (2016). Long-distance electron transfer by 1043-1055. cable bacteria in aquifer sediments. The ISME Journal 10, 2010-2019. Peter Jurtshuk, J. (1996). "Bacterial Metabolism," in Medical Microbiology, ed. Myers, C.R., and Myers, J.M. (2004). Shewanella oneidensis MR-1 restores S. Baron.). menaquinone synthesis to a menaquinone-negative mutant. Applied Phadtare, S., and Inouye, M. (2004). Genome-wide transcriptional analysis of and Environmental Microbiology 70, 5415-5425. the cold shock response in wild-type and cold-sensitive, quadruple-csp- Nealson, K.H., Belz, A., and Mckee, B. (2002). Breathing metals as a way of deletion strains of Escherichia coli. Journal of Bacteriology 186, 7007- life: geobiology in action. Antonie van Leeuwenhoek 81, 215-222. 7014. Nevin, K.P., Woodard, T.L., Franks, A.E., Summers, Z.M., and Lovley, D.R. Pop, M., and Salzberg, S.L. (2008). Bioinformatics challenges of new (2010). Microbial electrosynthesis: feeding microbes electricity to sequencing technology. Trends in Genetics 24, 142-149. convert carbon dioxide and water to multicarbon extracellular organic Poser, A., Lohmayer, R., Vogt, C., Knoeller, K., Planer-Friedrich, B., Sorokin, compounds. MBio 1, e00103-00110. D., Richnow, H.H., and Finster, K. (2013). Disproportionation of Newman, D.K., and Kolter, R. (2000). A role for excreted quinones in elemental sulfur by haloalkaliphilic bacteria from soda lakes. extracellular electron transfer. Nature 405, 94-97. Extremophiles 17, 1003-1012. Ni, G., Christel, S., Roman, P., Wong, Z.L., Bijmans, M.F., and Dopson, M. Potter, M.C. (1911). Electrical effects accompanying the decomposition of (2016). Electricity generation from an inorganic sulfur compound organic compounds. Proceedings of the Royal Society of London Series containing mining wastewater by acidophilic microorganisms. B-Containing Papers of a Biological Character 84, 260-276. Research in Microbiology 167, 568-575. Qiu, X., Wu, L., Huang, H., Mcdonel, P.E., Palumbo, A.V., Tiedje, J.M., and Zhou, J. (2001). Evaluation of PCR-generated chimeras, mutations,

54 55 Begley, R.F., and Rothberg, J.M. (2005). Genome sequencing in Orell, A., Navarro, C.A., Rivero, M., Aguilar, J.S., and Jerez, C.A. (2012). microfabricated high-density picolitre reactors. Nature 437, 376-380. Inorganic polyphosphates in extremophiles and their possible functions. Marsili, E., Baron, D.B., Shikhare, I.D., Coursolle, D., Gralnick, J.A., and Bond, Extremophiles 16, 573-583. D.R. (2008). Shewanella Secretes flavins that mediate extracellular Oren, A. (2008). Microbial life at high salt concentrations: phylogenetic and electron transfer. Proceedings of the National Academy of Sciences of metabolic diversity. Saline Systems 4, 2. the United States of America 105, 3968-3973. Orr, W.L., and Sinninghe Damsté, J.S. (1990). "Geochemistry of sulfur in Martin, S., Marquez, M.C., Sanchez-Porro, C., Mellado, E., Arahal, D.R., and petroleum systems." ACS Publications). Ventosa, A. (2003). Marinobacter lipolyticus sp. nov., a novel Padan, E., Bibi, E., Ito, M., and Krulwich, T.A. (2005). Alkaline pH moderate halophile with lipolytic activity. International Journal of homeostasis in bacteria: new insights. Biochimica et Biophysica Acta Systematic and Evolutionary Microbiology 53, 1383-1387. 1717, 67-88. Martin, W., Baross, J., Kelley, D., and Russell, M.J. (2008). Hydrothermal vents Pant, D., Van Bogaert, G., Diels, L., and Vanbroekhoven, K. (2010). A review and the origin of life. Nature Reviews Microbiology 6, 805-814. of the substrates used in microbial fuel cells (MFCs) for sustainable Meulenberg, R., Scheer, E.J., Pronk, J.T., Hazeu, W., Bos, P., and Kuenen, J.G. energy production. Bioresource Technology 101, 1533-1543. (1993). Metabolism of Tetrathionate in Thiobacillus-Acidophilus. Parada, A.E., Needham, D.M., and Fuhrman, J.A. (2016). Every base matters: FEMS Microbiology Letters 112, 167-172. assessing small subunit rRNA primers for marine microbiomes with Michels, M., and Bakker, E.P. (1985). Generation of a large, protonophore- mock communities, time series and global field samples. sensitive proton motive force and pH difference in the acidophilic Environmental Microbiology 18, 1403-1414. bacteria Thermoplasma acidophilum and Bacillus acidocaldarius. Park, H.S., Kim, B.H., Kim, H.S., Kim, H.J., Kim, G.T., Kim, M., Chang, I.S., Journal of Bacteriology 161, 231-237. Park, Y.K., and Chang, H.I. (2001). A novel electrochemically active Miller, D.N., Bryant, J.E., Madsen, E.L., and Ghiorse, W.C. (1999). Evaluation and Fe(III)-reducing bacterium phylogenetically related to Clostridium and optimization of DNA extraction and purification procedures for butyricum isolated from a microbial fuel cell. Anaerobe 7, 297-306. soil and sediment samples. Applied and Environmental Microbiology Parks, D.H., Imelfort, M., Skennerton, C.T., Hugenholtz, P., and Tyson, G.W. 65, 4715-4724. (2015). CheckM: assessing the quality of microbial genomes recovered Muller, H., Bosch, J., Griebler, C., Damgaard, L.R., Nielsen, L.P., Lueders, T., from isolates, single cells, and metagenomes. Genome Research 25, and Meckenstock, R.U. (2016). Long-distance electron transfer by 1043-1055. cable bacteria in aquifer sediments. The ISME Journal 10, 2010-2019. Peter Jurtshuk, J. (1996). "Bacterial Metabolism," in Medical Microbiology, ed. Myers, C.R., and Myers, J.M. (2004). Shewanella oneidensis MR-1 restores S. Baron.). menaquinone synthesis to a menaquinone-negative mutant. Applied Phadtare, S., and Inouye, M. (2004). Genome-wide transcriptional analysis of and Environmental Microbiology 70, 5415-5425. the cold shock response in wild-type and cold-sensitive, quadruple-csp- Nealson, K.H., Belz, A., and Mckee, B. (2002). Breathing metals as a way of deletion strains of Escherichia coli. Journal of Bacteriology 186, 7007- life: geobiology in action. Antonie van Leeuwenhoek 81, 215-222. 7014. Nevin, K.P., Woodard, T.L., Franks, A.E., Summers, Z.M., and Lovley, D.R. Pop, M., and Salzberg, S.L. (2008). Bioinformatics challenges of new (2010). Microbial electrosynthesis: feeding microbes electricity to sequencing technology. Trends in Genetics 24, 142-149. convert carbon dioxide and water to multicarbon extracellular organic Poser, A., Lohmayer, R., Vogt, C., Knoeller, K., Planer-Friedrich, B., Sorokin, compounds. MBio 1, e00103-00110. D., Richnow, H.H., and Finster, K. (2013). Disproportionation of Newman, D.K., and Kolter, R. (2000). A role for excreted quinones in elemental sulfur by haloalkaliphilic bacteria from soda lakes. extracellular electron transfer. Nature 405, 94-97. Extremophiles 17, 1003-1012. Ni, G., Christel, S., Roman, P., Wong, Z.L., Bijmans, M.F., and Dopson, M. Potter, M.C. (1911). Electrical effects accompanying the decomposition of (2016). Electricity generation from an inorganic sulfur compound organic compounds. Proceedings of the Royal Society of London Series containing mining wastewater by acidophilic microorganisms. B-Containing Papers of a Biological Character 84, 260-276. Research in Microbiology 167, 568-575. Qiu, X., Wu, L., Huang, H., Mcdonel, P.E., Palumbo, A.V., Tiedje, J.M., and Zhou, J. (2001). Evaluation of PCR-generated chimeras, mutations,

54 55 and heteroduplexes with 16S rRNA gene-based cloning. Applied and Reimers, C.E., Girguis, P., Stecher, H.A., Tender, L.M., Ryckelynck, N., and Environmental Microbiology 67, 880-887. Whaling, P. (2006). Microbial fuel cell energy from an ocean cold seep. Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, Geobiology 4, 123-136. J., and Glockner, F.O. (2013). The SILVA ribosomal RNA gene Ren, C.Q., Liu, D.X., Bai, Z.Q., and Li, T.H. (2005). Corrosion behavior of oil database project: improved data processing and web-based tools. tube steel in simulant solution with hydrogen sulfide and carbon Nucleic Acids Research 41, D590-596. dioxide. Materials Chemistry and Physics 93, 305-309. Quatrini, R., Appia-Ayme, C., Denis, Y., Jedlicki, E., Holmes, D.S., and Richter, H., Nevin, K.P., Jia, H.F., Lowy, D.A., Lovley, D.R., and Tender, L.M. Bonnefoy, V. (2009). Extending the models for iron and sulfur (2009). Cyclic voltammetry of biofilms of wild type and mutant oxidation in the extreme acidophile Acidithiobacillus ferrooxidans. Geobacter sulfurreducens on fuel cell anodes indicates possible roles BMC Genomics 10, 394. of OmcB, OmcZ, type IV pili, and protons in extracellular electron Rabaey, K. (2010). "Bioelectrochemical systems: a new approach towards transfer. Energy & Environmental Science 2, 506-516. environmental and industrial biotechnology," in Bioelectrochemical Rivkina, E.M., Friedmann, E.I., Mckay, C.P., and Gilichinsky, D.A. (2000). systems : from extracellular electron transfer to biotechnological Metabolic activity of permafrost bacteria below the freezing point. application, eds. K. Rabaey, L. Angenent, U. Schröder & J. Keller. Applied and Environmental Microbiology 66, 3230-3233. (London, UK: IWA Publishing), 1-16. Robertson, C.E., Harris, J.K., Wagner, B.D., Granger, D., Browne, K., Tatem, Rabaey, K., Angenent, L., Schroder, U., and Keller, J. (2009). B., Feazel, L.M., Park, K., Pace, N.R., and Frank, D.N. (2013). Explicet: Bioelectrochemical systems. IWA publishing. graphical user interface software for metadata-driven management, Rabaey, K., Boon, N., Siciliano, S.D., Verhaege, M., and Verstraete, W. (2004). analysis and visualization of microbiome data. Bioinformatics 29, Biofuel cells select for microbial consortia that self-mediate electron 3100-3101. transfer. Applied and Environmental Microbiology 70, 5373-5382. Rocca, J.D., Hall, E.K., Lennon, J.T., Evans, S.E., Waldrop, M.P., Cotner, J.B., Rabaey, K., Read, S.T., Clauwaert, P., Freguia, S., Bond, P.L., Blackall, L.L., Nemergut, D.R., Graham, E.B., and Wallenstein, M.D. (2015). and Keller, J. (2008). Cathodic oxygen reduction catalyzed by bacteria Relationships between protein-encoding gene abundance and in microbial fuel cells. The ISME Journal 2, 519-527. corresponding process are commonly assumed yet rarely observed. The Rabaey, K., Rodriguez, J., Blackall, L.L., Keller, J., Gross, P., Batstone, D., ISME Journal 9, 1693-1699. Verstraete, W., and Nealson, K.H. (2007). Microbial ecology meets Roman, P., Lipinska, J., Bijmans, M.F.M., Sorokin, D.Y., Keesman, K.J., and electrochemistry: electricity-driven and driving communities. The Janssen, A.J.H. (2016). Inhibition of a biological sulfide oxidation ISME Journal 1, 9-18. under haloalkaline conditions by thiols and diorgano polysulfanes. Rabaey, K., Van De Sompel, K., Maignien, L., Boon, N., Aelterman, P., Water Research 101, 448-456. Clauwaert, P., De Schamphelaire, L., Pham, H.T., Vermeulen, J., Rousseau, R., Dominguez-Benetton, X., Delia, M.L., and Bergel, A. (2013). Verhaege, M., Lens, P., and Verstraete, W. (2006). Microbial fuel cells Microbial bioanodes with high salinity tolerance for microbial fuel for sulfide removal. Environmental Science & Technology 40, 5218- cells and microbial electrolysis cells. Electrochemistry 5224. Communications 33, 1-4. Raes, S.M., Jourdin, L., Buisman, C.J., and Strik, D.P. (2017). Continuous Russell, M.J., Hall, A.J., and Gize, A.P. (1990). Pyrite and the Origin of Life. Long Term Bioelectrochemical Chain Elongation to Butyrate. Nature 344, 387-387. ChemElectroChem 4, 386-395. Samartzidou, H., Mehrazin, M., Xu, Z.H., Benedik, M.J., and Delcour, A.H. Rawlings, D.E.,- Tributsch, H., and Hansford, G.S. (1999). Reasons why (2003). Cadaverine inhibition of porin plays a role in cell survival at 'Leptospirillum'-like species rather than Thiobacillus ferrooxidans are acidic pH. Journal of Bacteriology 185, 13-19. the dominant iron-oxidizing bacteria in many commercial processes for Sanger, F., Nicklen, S., and Coulson, A.R. (1977). DNA sequencing with chain- the biooxidation of pyrite and related ores. Microbiology-Reading 145, terminating inhibitors. Proceedings of the National Academy of 5-13. Sciences of the United States of America 74, 5463-5467. Reguera, G., Mccarthy, K.D., Mehta, T., Nicoll, J.S., Tuominen, M.T., and Schelert, J., Dixit, V., Hoang, V., Simbahan, J., Drozda, M., and Blum, P. Lovley, D.R. (2005). Extracellular electron transfer via microbial (2004). Occurrence and characterization of mercury resistance in the nanowires. Nature 435, 1098-1101.

56 57 and heteroduplexes with 16S rRNA gene-based cloning. Applied and Reimers, C.E., Girguis, P., Stecher, H.A., Tender, L.M., Ryckelynck, N., and Environmental Microbiology 67, 880-887. Whaling, P. (2006). Microbial fuel cell energy from an ocean cold seep. Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, Geobiology 4, 123-136. J., and Glockner, F.O. (2013). The SILVA ribosomal RNA gene Ren, C.Q., Liu, D.X., Bai, Z.Q., and Li, T.H. (2005). Corrosion behavior of oil database project: improved data processing and web-based tools. tube steel in simulant solution with hydrogen sulfide and carbon Nucleic Acids Research 41, D590-596. dioxide. Materials Chemistry and Physics 93, 305-309. Quatrini, R., Appia-Ayme, C., Denis, Y., Jedlicki, E., Holmes, D.S., and Richter, H., Nevin, K.P., Jia, H.F., Lowy, D.A., Lovley, D.R., and Tender, L.M. Bonnefoy, V. (2009). Extending the models for iron and sulfur (2009). Cyclic voltammetry of biofilms of wild type and mutant oxidation in the extreme acidophile Acidithiobacillus ferrooxidans. Geobacter sulfurreducens on fuel cell anodes indicates possible roles BMC Genomics 10, 394. of OmcB, OmcZ, type IV pili, and protons in extracellular electron Rabaey, K. (2010). "Bioelectrochemical systems: a new approach towards transfer. Energy & Environmental Science 2, 506-516. environmental and industrial biotechnology," in Bioelectrochemical Rivkina, E.M., Friedmann, E.I., Mckay, C.P., and Gilichinsky, D.A. (2000). systems : from extracellular electron transfer to biotechnological Metabolic activity of permafrost bacteria below the freezing point. application, eds. K. Rabaey, L. Angenent, U. Schröder & J. Keller. Applied and Environmental Microbiology 66, 3230-3233. (London, UK: IWA Publishing), 1-16. Robertson, C.E., Harris, J.K., Wagner, B.D., Granger, D., Browne, K., Tatem, Rabaey, K., Angenent, L., Schroder, U., and Keller, J. (2009). B., Feazel, L.M., Park, K., Pace, N.R., and Frank, D.N. (2013). Explicet: Bioelectrochemical systems. IWA publishing. graphical user interface software for metadata-driven management, Rabaey, K., Boon, N., Siciliano, S.D., Verhaege, M., and Verstraete, W. (2004). analysis and visualization of microbiome data. Bioinformatics 29, Biofuel cells select for microbial consortia that self-mediate electron 3100-3101. transfer. Applied and Environmental Microbiology 70, 5373-5382. Rocca, J.D., Hall, E.K., Lennon, J.T., Evans, S.E., Waldrop, M.P., Cotner, J.B., Rabaey, K., Read, S.T., Clauwaert, P., Freguia, S., Bond, P.L., Blackall, L.L., Nemergut, D.R., Graham, E.B., and Wallenstein, M.D. (2015). and Keller, J. (2008). Cathodic oxygen reduction catalyzed by bacteria Relationships between protein-encoding gene abundance and in microbial fuel cells. The ISME Journal 2, 519-527. corresponding process are commonly assumed yet rarely observed. The Rabaey, K., Rodriguez, J., Blackall, L.L., Keller, J., Gross, P., Batstone, D., ISME Journal 9, 1693-1699. Verstraete, W., and Nealson, K.H. (2007). Microbial ecology meets Roman, P., Lipinska, J., Bijmans, M.F.M., Sorokin, D.Y., Keesman, K.J., and electrochemistry: electricity-driven and driving communities. The Janssen, A.J.H. (2016). Inhibition of a biological sulfide oxidation ISME Journal 1, 9-18. under haloalkaline conditions by thiols and diorgano polysulfanes. Rabaey, K., Van De Sompel, K., Maignien, L., Boon, N., Aelterman, P., Water Research 101, 448-456. Clauwaert, P., De Schamphelaire, L., Pham, H.T., Vermeulen, J., Rousseau, R., Dominguez-Benetton, X., Delia, M.L., and Bergel, A. (2013). Verhaege, M., Lens, P., and Verstraete, W. (2006). Microbial fuel cells Microbial bioanodes with high salinity tolerance for microbial fuel for sulfide removal. Environmental Science & Technology 40, 5218- cells and microbial electrolysis cells. Electrochemistry 5224. Communications 33, 1-4. Raes, S.M., Jourdin, L., Buisman, C.J., and Strik, D.P. (2017). Continuous Russell, M.J., Hall, A.J., and Gize, A.P. (1990). Pyrite and the Origin of Life. Long Term Bioelectrochemical Chain Elongation to Butyrate. Nature 344, 387-387. ChemElectroChem 4, 386-395. Samartzidou, H., Mehrazin, M., Xu, Z.H., Benedik, M.J., and Delcour, A.H. Rawlings, D.E.,- Tributsch, H., and Hansford, G.S. (1999). Reasons why (2003). Cadaverine inhibition of porin plays a role in cell survival at 'Leptospirillum'-like species rather than Thiobacillus ferrooxidans are acidic pH. Journal of Bacteriology 185, 13-19. the dominant iron-oxidizing bacteria in many commercial processes for Sanger, F., Nicklen, S., and Coulson, A.R. (1977). DNA sequencing with chain- the biooxidation of pyrite and related ores. Microbiology-Reading 145, terminating inhibitors. Proceedings of the National Academy of 5-13. Sciences of the United States of America 74, 5463-5467. Reguera, G., Mccarthy, K.D., Mehta, T., Nicoll, J.S., Tuominen, M.T., and Schelert, J., Dixit, V., Hoang, V., Simbahan, J., Drozda, M., and Blum, P. Lovley, D.R. (2005). Extracellular electron transfer via microbial (2004). Occurrence and characterization of mercury resistance in the nanowires. Nature 435, 1098-1101.

56 57 hyperthermophilic archaeon Sulfolobus solfataricus by use of gene Sun, M., Mu, Z.X., Chen, Y.P., Sheng, G.P., Liu, X.W., Chen, Y.Z., Zhao, Y., disruption. Journal of Bacteriology 186, 427-437. Wang, H.L., Yu, H.Q., Wei, L., and Ma, F. (2009). Microbe-assisted Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. sulfide oxidation in the anode of a microbial fuel cell. Environmental Bioinformatics 30, 2068-2069. Science & Technology 43, 3372-3377. Segata, N., Bornigen, D., Morgan, X.C., and Huttenhower, C. (2013). Suzuki, M.T., and Giovannoni, S.J. (1996). Bias caused by template annealing PhyloPhlAn is a new method for improved phylogenetic and in the amplification of mixtures of 16S rRNA genes by PCR. Applied taxonomic placement of microbes. Nature Communications 4, 2304. and Environmental Microbiology 62, 625-630. Sharon, I., and Banfield, J.F. (2013). Microbiology. Genomes from Thorup, C., Schramm, A., Findlay, A.J., Finster, K.W., and Schreiber, L. (2017). metagenomics. Science 342, 1057-1058. Disguised as a Sulfate Reducer: Growth of the Deltaproteobacterium Sheu, S.H., and Weng, H.S. (2001). Treatment of olefin plant spent caustic by Desulfurivibrio alkaliphilus by Sulfide Oxidation with Nitrate. Mbio 8, combination of neutralization and Fenton reaction. Water Research 35, e00671-00617. 2017-2021. Tyson, G.W., Chapman, J., Hugenholtz, P., Allen, E.E., Ram, R.J., Richardson, Shi, L., Dong, H.L., Reguera, G., Beyenal, H., Lu, A.H., Liu, J., Yu, H.Q., and P.M., Solovyev, V.V., Rubin, E.M., Rokhsar, D.S., and Banfield, J.F. Fredrickson, J.K. (2016). Extracellular electron transfer mechanisms (2004). Community structure and metabolism through reconstruction between microorganisms and minerals. Nature Reviews Microbiology of microbial genomes from the environment. Nature 428, 37-43. 14, 651-662. Vaiopoulou, E., Provijn, T., Prevoteau, A., Pikaar, I., and Rabaey, K. (2016). Shi, L., Squier, T.C., Zachara, J.M., and Fredrickson, J.K. 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Thioalkalivibrio jannaschii sp. nov., novel species of haloalkaliphilic, Vargas, M., Malvankar, N.S., Tremblay, P.L., Leang, C., Smith, J.A., Patel, P., obligately chemolithoautotrophic sulfur-oxidizing bacteria from Snoeyenbos-West, O., Nevin, K.P., and Lovley, D.R. (2013). Aromatic hypersaline alkaline Mono Lake (California). International Journal of amino acids required for pili conductivity and long-range extracellular Systematic and Evolutionary Microbiology 52, 913-920. electron transport in Geobacter sulfurreducens. MBio 4, e00105-00113. Sorokin, D.Y., Lysenko, A.M., Mityushina, L.L., Tourova, T.P., Jones, B.E., Větrovský, T., & Baldrian, P. (2013). The variability of the 16S rRNA gene in Rainey, F.A., Robertson, L.A., and Kuenen, G.J. (2001). bacterial genomes and its consequences for bacterial community Thioalkalimicrobium aerophilum gen. nov., sp. nov. and analyses. PloS one, 8(2), e57923. Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus Vincke, E., Van Wanseele, E., Monteny, J., Beeldens, A., De Belie, N., Taerwe, gen. nov., sp. nov., Thioalkalivibrio nitratis sp.nov., novel and L., Van Gemert, D., and Verstraete, W. (2002). Influence of polymer Thioalkalivibrio denitrificancs sp. nov., novel obligately alkaliphilic addition on biogenic sulfuric acid attack of concrete. International and obligately chemolithoautotrophic sulfur-oxidizing bacteria from Biodeterioration & Biodegradation 49, 283-292. soda lakes. International Journal of Systematic and Evolutionary Wachtershauser, G. (1990). Evolution of the first metabolic cycles. Proceedings Microbiology 51, 565-580. of the National Academy of Sciences of the United States of America Soto, H., Nava, F., Leal, J., and Jara, J. (1995). Regeneration of cyanide by 87, 200-204. ozone oxidation of thiocyanate in cyanidation tailings. Minerals Wächtershäuser, G. (1988). 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58 59 hyperthermophilic archaeon Sulfolobus solfataricus by use of gene Sun, M., Mu, Z.X., Chen, Y.P., Sheng, G.P., Liu, X.W., Chen, Y.Z., Zhao, Y., disruption. Journal of Bacteriology 186, 427-437. Wang, H.L., Yu, H.Q., Wei, L., and Ma, F. (2009). Microbe-assisted Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. sulfide oxidation in the anode of a microbial fuel cell. Environmental Bioinformatics 30, 2068-2069. Science & Technology 43, 3372-3377. Segata, N., Bornigen, D., Morgan, X.C., and Huttenhower, C. (2013). Suzuki, M.T., and Giovannoni, S.J. (1996). Bias caused by template annealing PhyloPhlAn is a new method for improved phylogenetic and in the amplification of mixtures of 16S rRNA genes by PCR. Applied taxonomic placement of microbes. Nature Communications 4, 2304. and Environmental Microbiology 62, 625-630. Sharon, I., and Banfield, J.F. (2013). Microbiology. Genomes from Thorup, C., Schramm, A., Findlay, A.J., Finster, K.W., and Schreiber, L. (2017). metagenomics. Science 342, 1057-1058. Disguised as a Sulfate Reducer: Growth of the Deltaproteobacterium Sheu, S.H., and Weng, H.S. (2001). Treatment of olefin plant spent caustic by Desulfurivibrio alkaliphilus by Sulfide Oxidation with Nitrate. Mbio 8, combination of neutralization and Fenton reaction. Water Research 35, e00671-00617. 2017-2021. Tyson, G.W., Chapman, J., Hugenholtz, P., Allen, E.E., Ram, R.J., Richardson, Shi, L., Dong, H.L., Reguera, G., Beyenal, H., Lu, A.H., Liu, J., Yu, H.Q., and P.M., Solovyev, V.V., Rubin, E.M., Rokhsar, D.S., and Banfield, J.F. Fredrickson, J.K. (2016). Extracellular electron transfer mechanisms (2004). Community structure and metabolism through reconstruction between microorganisms and minerals. Nature Reviews Microbiology of microbial genomes from the environment. Nature 428, 37-43. 14, 651-662. Vaiopoulou, E., Provijn, T., Prevoteau, A., Pikaar, I., and Rabaey, K. (2016). Shi, L., Squier, T.C., Zachara, J.M., and Fredrickson, J.K. (2007). Respiration Electrochemical sulfide removal and caustic recovery from spent of metal (hydr)oxides by Shewanella and Geobacter: a key role for caustic streams. Water Research 92, 38-43. multihaem c-type cytochromes. Molecular Microbiology 65, 12-20. Van Dijk, E.L., Jaszczyszyn, Y., and Thermes, C. (2014). Library preparation Slonczewski, J.L., Fujisawa, M., Dopson, M., and Krulwich, T.A. (2009). methods for next-generation sequencing: tone down the bias. Cytoplasmic pH measurement and homeostasis in bacteria and archaea. Experimental Cell Research 322, 12-20. Advances in Microbial Physiology 55, 1-79, 317. Van Zyl, A.W., Harrison, S.T., and Van Hille, R.P. (2011). "Biodegradation of Sorokin, D.Y., Gorlenko, V.M., Tourova, T.P., Tsapin, A.I., Nealson, K.H., and thiocyanate by a mixed microbial population", in: Mine Water– Kuenen, G.J. (2002). Thioalkalimicrobium cyclicum sp. nov. and Managing the Challenges (IMWA). (Aachen, Germany). Thioalkalivibrio jannaschii sp. nov., novel species of haloalkaliphilic, Vargas, M., Malvankar, N.S., Tremblay, P.L., Leang, C., Smith, J.A., Patel, P., obligately chemolithoautotrophic sulfur-oxidizing bacteria from Snoeyenbos-West, O., Nevin, K.P., and Lovley, D.R. (2013). Aromatic hypersaline alkaline Mono Lake (California). International Journal of amino acids required for pili conductivity and long-range extracellular Systematic and Evolutionary Microbiology 52, 913-920. electron transport in Geobacter sulfurreducens. MBio 4, e00105-00113. Sorokin, D.Y., Lysenko, A.M., Mityushina, L.L., Tourova, T.P., Jones, B.E., Větrovský, T., & Baldrian, P. (2013). The variability of the 16S rRNA gene in Rainey, F.A., Robertson, L.A., and Kuenen, G.J. (2001). bacterial genomes and its consequences for bacterial community Thioalkalimicrobium aerophilum gen. nov., sp. nov. and analyses. PloS one, 8(2), e57923. Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus Vincke, E., Van Wanseele, E., Monteny, J., Beeldens, A., De Belie, N., Taerwe, gen. nov., sp. nov., Thioalkalivibrio nitratis sp.nov., novel and L., Van Gemert, D., and Verstraete, W. (2002). Influence of polymer Thioalkalivibrio denitrificancs sp. nov., novel obligately alkaliphilic addition on biogenic sulfuric acid attack of concrete. International and obligately chemolithoautotrophic sulfur-oxidizing bacteria from Biodeterioration & Biodegradation 49, 283-292. soda lakes. International Journal of Systematic and Evolutionary Wachtershauser, G. (1990). Evolution of the first metabolic cycles. Proceedings Microbiology 51, 565-580. of the National Academy of Sciences of the United States of America Soto, H., Nava, F., Leal, J., and Jara, J. (1995). Regeneration of cyanide by 87, 200-204. ozone oxidation of thiocyanate in cyanidation tailings. Minerals Wächtershäuser, G. (1988). Pyrite formation, the first energy source for life: a Engineering 8, 273-281. hypothesis. Systematic and Applied Microbiology 10, 207-210. Sulonen, M.L., Kokko, M.E., Lakaniemi, A.M., and Puhakka, J.A. (2015). Wickham, H. (2017). Tidyverse: Easily Install and Load’Tidyverse’packages. Electricity generation from tetrathionate in microbial fuel cells by URL https://CRAN. R-project. org/package= tidyverse. R package acidophiles. Journal of Hazardous Materials 284, 182-189. version 1.1: 51.

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