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In acid drainage environments, biosulfidogenesis by sulfate-reducing bacteria (SRB) attenuates the extreme conditions by enabling the precipitation of metals as their sulfides, and the neutralization of acidity through proton consumption. So far, only a handful of moderately acidophilic SRB species have been described, most of which are merely acidotolerant. Here, a novel species within a novel genus of moderately acidophilic SRB is described, Acididesulfobacillus acetoxydans gen. nov. sp. nov. strain INE, able to grow at pH 3.8. Bioreactor studies with strain INE at optimum (5.0) and low (3.9) pH for growth showed that strain INE alkalinized its environment, and that this was more pronounced at lower pH. These studies also showed the capacity of strain INE to completely oxidize organic acids to CO2, which is uncommon among acidophilic SRB. Since organic acids are mainly in their protonated form at low pH, which increases their toxicity, their complete oxidation may be an acid stress resistance mechanism. Comparative proteogenomic and membrane lipid analysis further indicated that the presence of saturated ether-bound lipids in the membrane, and their relative increase at lower pH, was a protection mechanism against acid stress. Interestingly, other canonical acid stress resistance mechanisms, such as a Donnan potential and increased active charge transport, did not appear to be active.

Large amounts of water are purified by high pressure driven membrane filtration processes, i.e. nanofiltration (NF) and reverse osmosis (RO). In Chapter 1, we introduce the topics discussed in this Thesis. A common but major challenge in the operation of high pressure membrane filtration systems is the formation of deposits on the membrane surface. This is defined as fouling when it leads to a certain membrane performance decrease. The deposition of abiotic particles on the membrane can often be adequately prevented via pre-treatment of the feed water steps, or reduced by cleaning the membrane. Microbial presence on the membrane surface cannot be prevented, and the consequential growth and formation of recalcitrant biofilms on the membrane surface is difficult to control via membrane cleaning.Much scientific effort has been invested to understand which bacteria are involved in membrane fouling. This has shown that bacteria belonging to the Sphingomonadaceae family are detected frequently within membrane fouling layers. How bacteria function and interact on the membrane surface, and respond to cleaning agents is not well studied. This information might prove valuable to design alternative cleaning strategies. In this Thesis, we have physiologically characterized members of the Sphingomonadaceae family that were isolated from fouled membranes, and investigated the ability to remove membrane fouling layers using an alternative cleaning strategy.In Chapter 2, we provide on overview of the studies that have investigated the microorganisms involved in fouling of high-pressure membrane systems. Most of these studies focus on the bacterial community composition. The diversity of eukaryotic and archaeal species within membrane fouling layers is rarely studied. Comparison between the microbial diversity of the feed water and pre-treatment steps in relation to that of the membrane surface reveals large differences within the bacterial and archaeal diversity, but not within the

The microbial sulfur cycle plays an important role in acidic environments such as acid mine drainage (AMD) and acidic volcanic hot pools. The reduction of inorganic sulfur compounds such as sulfate (SO42-) and elemental sulfur (S80) results in the production of sulfide (H2S) (biosulfidogenesis). H2S reacts with dissolved metals to form metal sulfides, which precipitate out of solution and thereby lower metal concentrations in AMD environments. In addition, at low pH biosulfidogenesis from SO42- is proton-consuming, mitigating the acidity. (Bio)sulfidogenesis furthermore is an attractive technology for selective recovery of metals as metal sulfides from mining and metallurgy process waters. In industry this has been applied predominantly with neutrophilic sulfate-reducing bacteria (SRB). Process economics can be improved, however, by using S80 instead of SO42- as electron acceptor, as this enables a fourfold decrease in electron donor requirements. The use of S80 could also enable operation at more acidic pH and higher temperatures, as so far no extremely acidophilic (pHopt < 3.0) SRB are known, while multiple (thermo)acidophilic S80-reducing bacteria and archaea have been described. Biosulfidogenesis at (thermo)acidophilic conditions can further reduce process costs by enabling the combination of sulfidogenesis and metal precipitation from AMD or hot acidic metallurgy process waters in one reactor. In this thesis we investigated sulfidogenesis from S80 and SO42- at acidic pH and low to high temperature, focusing on laboratory-scale processes for application (chapter 2 and 3), and bioremediation of AMD-impacted environments (chapter 4 and 5)—acidic mine pit lakes and AMD sediments.In chapter 2 we describe a novel abiotic process for sulfidogenesis from S80 and H2, mediated by catalytic pyrite. The catalytic properties of pyrite likely are related to their size, since both pyrite formed in situ from the S80, H2S and Fe2+ present in the medium, and externally sourced

All forms of life, from bacteria to humans, require sulfur as essential nutrient. Furthermore, many bacteria and archaea interact with sulfur compounds to obtain the energy they need for growth. Some of these sulfur microbes – for instance sulfate-reducing microorganisms – produce sulfide (H2S), which has the smell of rotten eggs and can create ‘dead zones’ in the ocean because of its toxicity. However, other sulfur microbes do the opposite and consume H2S, using it as electron donor for energy. Sulfur microbes thus have a big impact on the marine environment, and even on the global climate. As microbiologists, we want to find out who are these sulfur microbes, and what else do they do?To answer these questions, we focused on the Black Sea, the world’s largest anoxic basin. It is home to many different sulfur microbes which cannot tolerate oxygen and are yet to be studied. As tools, we used metagenomics and anaerobic cultivation. Metagenome-assembled-genomes (MAGs) were obtained from a cross-assembly of 15 metagenomes sampled at different water column depths. MAGs were then taxonomically classified, screened for functional marker genes to hypothesize their energy metabolism, and their relative abundance was profiled over depth to refine these hypotheses. We obtained medium-to-high-quality MAGs of various putative sulfur-oxidizing and sulfate-reducing bacteria, including known ones and novel ones. Specifically sulfate-reducing bacteria were surprisingly diverse and novel, meaning much is still to be learned about their role in the Black Sea and other marine waters with low oxygen levels.With anaerobic cultivation, we have brought new and exciting sulfur microbes from Black Sea samples into pure culture, allowing us to study their metabolism in detail. For instance, we isolated two strains of a novel bacterial family which cleave sulfate ester groups from polysaccharides to digest their food. Their genomes contain more genes for sulfate-cleaving enzymes (sulfatases) t

The conventional perception that the zone of sulfate reduction and methanogenesis are separated in high- and low-sulfate-containing marine sediments has recently been changed by studies demonstrating their co-occurrence in sediments. The presence of methanogens was linked to the presence of substrates that are not used by sulfate reducers. In the current study, we hypothesized that both groups can co-exist, consuming common substrates (H2 and/or acetate) in sediments. We enriched butyrate-degrading communities in sediment slurries originating from the sulfate, sulfate–methane transition, and methane zone of Aarhus Bay, Denmark. Sulfate was added at different concentrations (0, 3, 20 mM), and the slurries were incubated at 10 °C and 25 °C. During butyrate conversion, sulfate reduction and methanogenesis occurred simultaneously. The syntrophic butyrate degrader Syntrophomonas was enriched both in sulfate-amended and in sulfate-free slurries, indicating the occurrence of syntrophic conversions at both conditions. Archaeal community analysis revealed a dominance of Methanomicrobiaceae. The acetoclastic Methanosaetaceae reached high relative abundance in the absence of sulfate, while presence of acetoclastic Methanosarcinaceae was independent of the sulfate concentration, temperature, and the initial zone of the sediment. This study shows that there is no vertical separation of sulfate reducers, syntrophs, and methanogens in the sediment and that they all participate in the conversion of butyrate.

Recently, we isolated two marine strains, F1T and F21T, which together with Kiritimatiella glycovorans L21-Fru-ABT are the only pure cultures of the class Kiritimatiellae within the phylum Verrucomicrobiota. Here, we present an in-depth genome-guided characterization of both isolates with emphasis on their exopolysaccharide synthesis. The strains only grew fermentatively on simple carbohydrates and sulfated polysaccharides. Strains F1T, F21T and K. glycovorans reduced elemental sulfur, ferric citrate and anthraquinone-2,6-disulfonate during anaerobic growth on sugars. Both strains produced exopolysaccharides during stationary phase, probably with intracellularly stored glycogen as energy and carbon source. Exopolysaccharides included N-sulfated polysaccharides probably containing hexosamines and thus resembling glycosaminoglycans. This implies that the isolates can both degrade and produce sulfated polysaccharides. Both strains encoded an unprecedently high number of glycoside hydrolase genes (422 and 388, respectively), including prevalent alpha-L-fucosidase genes, which may be necessary for degrading complex sulfated polysaccharides such as fucoidan. Strain F21T encoded three putative glycosaminoglycan sulfotransferases and a putative sulfate glycosaminoglycan biosynthesis gene cluster. Based on phylogenetic and chemotaxonomic analyses, we propose the taxa Pontiella desulfatans F1T gen. nov., sp. nov. and Pontiella sulfatireligans F21T sp. nov. as representatives of the Pontiellaceae fam. nov. within the class Kiritimatiellae.

The genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1T and D. butyratoxydans MSL71T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20 mM sulfate or 20 mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.

The marine environment contains a large diversity of sulfated polysaccharides and other glycopolymers. Saccharolytic microorganisms degrade these compounds through hydrolysis, which includes the hydrolysis of sulfate groups from sugars by sulfatases. Various marine bacteria of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum have exceptionally high numbers of sulfatase genes associated with the degradation of sulfated polysaccharides. However, thus far no sulfatase-rich marine anaerobes are known. In this study, we aimed to isolate marine anaerobes using sulfated polysaccharides as substrate. Anoxic enrichment cultures were set up with a mineral brackish marine medium, inoculated with anoxic Black Sea sediment sampled at 2,100 m water depth water and incubated at 15∘C (in situ T = 8∘C) for several weeks. Community analysis by 16S rRNA gene amplicon sequencing revealed the enrichment of Kiritimatiellaeota clade R76-B128 bacteria in the enrichments with the sulfated polysaccharides fucoidan and iota-carrageenan as substrate. We isolated two strains, F1 and F21, which represent a novel family within the order of the Kiritimatiellales. They were capable of growth on various mono-, di-, and polysaccharides, including fucoidan. The desulfation of iota-carrageenan by strain F21 was confirmed quantitatively by an increase in free sulfate concentration. Strains F1 and F21 represent the first marine sulfatase-rich anaerobes, encoding more sulfatases (521 and 480, 8.0 and 8.4% of all coding sequences, respectively) than any other microorganism currently known. Specific encoded sulfatase subfamilies could be involved in desulfating fucoidan (S1_15, S1_17 and S1_25) and iota-carrageenan (S1_19). Strains F1 and F21 had a sulfatase gene classification profile more similar to aerobic than anaerobic sulfatase-rich PVC bacteria, including Kiritimatiella glycovorans, the only other cultured representative within the Kiritimatiellaeot

Since 1928 with the discovery of penicillin, the value of microbes in our society significantly was reconsidered. Nowadays, 60% of commercial drugs and products mimic or derive from microbialmetabolites. After almost a century, can we find new compounds and where? For addressing thisquestion, we need a large-scale screening of the microbial capabilities. Trichococcus species have multiple genes for producing 1,3-propanediol (1,3-PDO), which synthesizes the partially biodegradable plastic PTT. Based on this, we developed a strategy for analyzing 90,000 bacterial genomes that eventually generated information for every microbial characteristic. The outstanding factor is that all this information is stored in a database that can be easily mined for everything. This collective andunbiased strategy resulted in identifying the key genes for efficient production of 1,3-PDO. We discovered 187 novel candidates that can produce 1,3-PDO and some were in the lab confirmed. Another result of the screening was about Trichococcus patagoniensis. This bacterium grows in minus 5 degrees without oxygen and was discovered by NASA scientists to simulate life in other planets. When it is cold and without oxygen, T. patagoniensis “extra-terrestrial” properties allow it to create its own ”blanket” by producing exopolymer saccharides. We characterized this cryoprotectant compound as inulin, which prevents crystallization of water and many plants use it for preserving their roots in subzero temperatures. Furthermore, inulin is a commercial prebiotic and is connected with gut health. Considering the bacterial kingdom, there are limited members producing inulin and none of them wereidentified as prychrotolerant species. T. patagoniensisis produces plenty of inulin and due to its robustness, easily can be the next biofactory for the compound.The applied methods in this PhD thesis is a platform for mining every bacterial or metabolic information. All the knowledge is there and we need to dive int

High pressure membrane filtration processes such as nanofiltration (NF) and reverse osmosis (RO) are capable to produce high quality water, for industrial applications and human consumption, from virtually any feed water source. Biofouling, the unwanted biofilm formation causing negative effects on membrane performance, contributes significantly to the operational costs and downtime of membrane filtration installations. In this thesis, the microbiological aspects and process technological aspects of NF and RO membrane biofouling have been extensively investigated in full-scale industrial installations and in simplified laboratory systems. An operational definition of NF and RO biofouling is given and generally applied preventative and corrective measures to manage membrane biofouling are reviewed. Anoxic groundwater treating NF installations (including their microbial communities), have been studied and compared to oxic NF systems and RO systems. The anoxic groundwater treating NF installations were shown to be much more resistant towards biofouling development, when compared to their oxic feed water treating counterparts. Membrane cleaning, the first-choice remedy against biofouling development, was studied in full-scale installations and in a laboratory setup. It was shown that aged foulants could not be removed by chemical cleaning without compromising membrane integrity. It was concluded that the prevention of biofilm formation, rather than the control of biofilm formation (cleaning), should be the main focus of a successful anti-biofouling approach. Biofouling microorganisms from the Sphingomondaceae family, which have been isolated from the fouled membranes obtained from the full-scale studies, were then physiologically characterized. It was shown that the Sphingomonadaceae membrane isolates share many features that are uncommon for other members of the Sphingomonadaceae family. Those ‘uncommon’ features were then linked to the specific physiological traits th

Synthesis gas (syngas) fermentation is a process capable of processing a gaseous substrate via fermentation into commodity chemicals and fuels. Gas (mainly consisting of hydrogen, carbon monoxide and carbon dioxide) fed to the fermentation process can be obtained from a wide variety of sources, including off-gases from industry, gasification of solid carbon wastes (e.g. municipal waste, lignocellulosic biomass) or gas derived from electrochemical reduction/physicochemical reduction processes. Current limitations of the fermentation process are the relatively poorly understood physiology and genetics of the biocatalysts involved. Therefore the work described in this thesis aimed at unravelling of the syngas metabolism of acetogenic and methanogenic strains, with main focus on carbon monoxide metabolism. In addition, the application of synthetic co-cultures for syngas fermentation was explored in order to assess if such cultivation approach could lead to broadening of the syngas fermentation product spectrum. In addition to co-cultivation proof-of-concept studies for application, new fundamental insights on the metabolism of the involved biocatalysts were obtained.

Degradation of long-chain fatty acids (LCFAs) in methanogenic environments is a syntrophic process involving the activity of LCFA-degrading bacteria and hydrogen-utilizing methanogens. If methanogens are inhibited, other hydrogen scavengers are needed to achieve complete LCFA degradation. In this work, we developed two different oleate (C18:1 LCFA)-degrading anaerobic enrichment cultures, one methanogenic (ME) and another in which methanogenesis was inhibited (IE). Inhibition of methanogens was attained by adding a solution of 2-bromoethanesulfonate (BrES), which turned out to consist of a mixture of BrES and isethionate. Approximately 5 times faster oleate degradation was accomplished by the IE culture compared with the ME culture. A bacterium closely related to Syntrophomonas zehnderi (99% 16S rRNA gene identity) was the main oleate degrader in both enrichments, in syntrophic relationship with hydrogenotrophic methanogens from the genera Methanobacterium and Methanoculleus (in ME culture) or with a bacterium closely related to Desulfovibrio aminophilus (in IE culture). A Desulfovibrio species was isolated, and its ability to utilize hydrogen was confirmed. This bacterium converted isethionate to acetate and sulfide, with or without hydrogen as electron donor. This bacterium also utilized BrES but only after 3 months of incubation. Our study shows that syntrophic oleate degradation can be coupled to desulfonation.IMPORTANCE In anaerobic treatment of complex wastewater containing fat, oils, and grease, high long-chain fatty acid (LCFA) concentrations may inhibit microbial communities, particularly those of methanogens. Here, we investigated if anaerobic degradation of LCFAs can proceed when methanogens are inhibited and in the absence of typical external electron acceptors, such as nitrate, iron, or sulfate. Inhibition studies were performed with the methanogenic inhibitor 2-bromoethanesulfonate (BrES). We noticed that, after autoclaving, BrES underwent partial hydr

Anaerobic syntrophic acetate oxidation (SAO) is a thermodynamically unfavorable process involving a syntrophic acetate oxidizing bacterium (SAOB) that forms interspecies electron carriers (IECs). These IECs are consumed by syntrophic partners, typically hydrogenotrophic methanogenic archaea or sulfate reducing bacteria. In this work, the metabolism and occurrence of SAOB at extremely haloalkaline conditions were investigated, using highly enriched methanogenic (M-SAO) and sulfate-reducing (S-SAO) cultures from south-western Siberian hypersaline soda lakes. Activity tests with the M-SAO and S-SAO cultures and thermodynamic calculations indicated that H2 and formate are important IECs in both SAO cultures. Metagenomic analysis of the M-SAO cultures showed that the dominant SAOB was ‘Candidatus Syntrophonatronum acetioxidans,’ and a near-complete draft genome of this SAOB was reconstructed. ‘Ca. S. acetioxidans’ has all genes necessary for operating the Wood–Ljungdahl pathway, which is likely employed for acetate oxidation. It also encodes several genes essential to thrive at haloalkaline conditions; including a Na+-dependent ATP synthase and marker genes for ‘salt-out‘ strategies for osmotic homeostasis at high soda conditions. Membrane lipid analysis of the M-SAO culture showed the presence of unusual bacterial diether membrane lipids which are presumably beneficial at extreme haloalkaline conditions. To determine the importance of SAO in haloalkaline environments, previously obtained 16S rRNA gene sequencing data and metagenomic data of five different hypersaline soda lake sediment samples were investigated, including the soda lakes where the enrichment cultures originated from. The draft genome of ‘Ca. S. acetioxidans’ showed highest identity with two metagenome-assembled genomes (MAGs) of putative SAOBs that belonged to the highly abundant and diverse Syntrophomonadaceae family present in the soda lake sediments. The 16S rRNA ge

Prokaryotes in natural environments respond rapidly to high concentrations of chemicals and physical stresses. Exposure to anthropogenic toxic substances—such as oil, chlorinated solvents, or antibiotics—favors the evolution of resistant phenotypes, some of which can use contaminants as an exclusive carbon source or as electron donors and acceptors. Microorganisms similarly adapt to extreme pH, metal, or osmotic stress. The metabolic plasticity of prokaryotes can thus be harnessed for bioremediation and can be exploited in a variety of ways, ranging from stimulated natural attenuation to bioaugmentation and from wastewater treatment to habitat restoration.

Anaerobic digestion relies on complex microbial communities that closely interact in the anaerobic degradation of biomass and organic waste material to methane and carbon dioxide. The adoption of high-throughput molecular methods and the holistic “omics” approach in applied microbial ecology has greatly extended our view on the manifold metabolic diversity and trophic networks in the microbiomes thriving in anaerobic bioreactors. In this chapter, current concepts in metagenomics and microbial ecology of anaerobic digestion are described. Recent advances in gene-centric and genome-centric approaches and their application on lab-scale and production-scale biogas reactors have paved the way to a knowledge-based microbial resource management in anaerobic bioreactors. The adoption of systems biology principles in systems ecology of reactor microbiomes will open up new perspectives in process control and optimization of biotechnological processes relying on complex open mixed cultures.

In this study, we report transcription of genes involved in aerobic and anaerobic benzene degradation pathways in a benzene-degrading denitrifying continuous culture. Transcripts associated with the family Peptococcaceae dominated all samples (21–36% relative abundance) indicating their key role in the community. We found a highly transcribed gene cluster encoding a presumed anaerobic benzene carboxylase (AbcA and AbcD) and a benzoate-coenzyme A ligase (BzlA). Predicted gene products showed >96% amino acid identity and similar gene order to the corresponding benzene degradation gene cluster described previously, providing further evidence for anaerobic benzene activation via carboxylation. For subsequent benzoyl-CoA dearomatization, bam-like genes analogous to the ones found in other strict anaerobes were transcribed, whereas gene transcripts involved in downstream benzoyl-CoA degradation were mostly analogous to the ones described in facultative anaerobes. The concurrent transcription of genes encoding enzymes involved in oxygenase-mediated aerobic benzene degradation suggested oxygen presence in the culture, possibly formed via a recently identified nitric oxide dismutase (Nod). Although we were unable to detect transcription of Nod-encoding genes, addition of nitrite and formate to the continuous culture showed indication for oxygen production. Such an oxygen production would enable aerobic microbes to thrive in oxygen-depleted and nitrate-containing subsurface environments contaminated with hydrocarbons.

Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17T, isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment

There is a worldwide increasing energy demand while fossil resources are getting depleted. Using organic waste to produce valuable and renewable products such as organic acids including lactate and succinate is a promising strategy. In this study, starch waste was selected as the main substrate as it is abundant in many places and has low-value. Gut microorganisms from cow rumen fluids and guinea pig fecal samples were used as inocula to catalyze organic acid production. Microbial diversity involved in organic acid production was investigated. Succinate yields were as high as 4.52 mmol SA / gram Starch. Starch waste and rumen fluid are suitable for lactate and succinate production. In addition, three novel organic acid producing bacteria were obtained and some may have potential for future applications.

Syntrophic methanogenic associations between acetogenic bacteria and methanogenic archaea are essential for the complete mineralization of organic compounds to methane and CO2. Propionate and butyrate are important intermediates in anaerobic digestion. In the absence of inorganic electron acceptors these short chain fatty acids can only be degraded if the products acetate, hydrogen and formate, are kept low by methanogens. However, when sulfate is available the conditions change, and propionate and butyrate can be oxidized coupled to sulfate reduction. Several sulfate-reducing bacteria are able to grow in syntrophic associations with methanogens, but others not. In this thesis, a functional analysis of protein domains was performed on a selected group of bacteria with the ability to grow on short chain fatty acids alone, or in syntrophy with methanogens. Genome analysis revealed that the presence of periplasmic formate dehydrogenases, most probably involved in interspecies electron transfer, differentiated syntrophic from non-syntrophic butyrate and propionate degraders. Moreover, the metabolic flexibility of the propionate-degrading bacterium Syntrophobacter fumaroxidans was investigated. S. fumaroxidans can couple propionate oxidation to sulfate reduction or can degrade propionate in syntrophic lifestyle with H2 and formate scavenging microorganisms. Propionate-grown cultures of S. fumaroxidans with sulfate as electron acceptor, or in syntrophy with Methanospirillum hungatei or Desulfovibrio desulfuricans were studied. We found that S. fumaroxidans is prone to oxidize propionate in syntrophy despite the availability of sulfate to grow on its own. A comparative proteomic analysis of propionate degradation by S. fumaroxidans in five growth conditions, including axenic and cocultures, was performed. This analysis gave a thorough overview of the propionate metabolism of S. fumaroxidans. Details on the energy conservation mechanisms and electron transfer to syntrophic

1-Hexadecene-contaminated wastewater is produced in oil refineries and can be treated in methanogenic bioreactors, although generally at low conversion rates. In this study, a microbial culture able to degrade 1-hexadecene was enriched, and different stimulation strategies were tested for enhancing 1-hexadecene conversion to methane. Seven and three times faster methane production was obtained in cultures stimulated with yeast extract or lactate, respectively, while cultures amended with crotonate lost the ability to degrade 1-hexadecene. Methane production from 1-hexadecene was not enhanced by the addition of extra hydrogenotrophic methanogens. Bacteria closely related to Syntrophus and Smithella were detected in 1-hexadecene-degrading cultures, but not in the ones amended with crotonate, which suggests the involvement of these bacteria in 1-hexadecene degradation. Genes coding for alkylsuccinate synthase alpha-subunit were detected in cultures degrading 1-hexadecene, indicating that hydrocarbon activation may occur by fumarate addition. These findings are novel and show that methane production from 1-hexadecene is improved by the addition of yeast extract or lactate. These extra electron donors may be considered as a potential bioremediation strategy of oil-contaminated sites with bioenergy generation through methane production.

The marine environment contains a large diversity of sulfated polysaccharides and other glycopolymers. Saccharolytic microorganisms degrade these compounds through hydrolysis, including the hydrolysis of sulfate groups from sugars by sulfatases. Various marine bacteria of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum have exceptionally high numbers of sulfatase genes associated with the degradation of sulfated polysaccharides. However, thus far no sulfatase-rich marine anaerobes are known. In this study, we aimed to isolate marine anaerobes using sulfated polysaccharide as substrate. Anoxic enrichment cultures were set up with a mineral brackish marine medium, which was inoculated with anoxic Black Sea sediment from 2100 m depth and incubated at 15°C (in situ T = 8°C) for several weeks. Community analysis by 16S rRNA gene amplicon sequencing revealed the enrichment of Kiritimatiellaeota clade R76-B128 bacteria with the sulfated polysaccharides fucoidan and iota-carrageenan as substrate. We isolated two strains, F1 and F21, which represent a novel family within the order of the Kiritimatiellales. They were capable of growth on various mono-, di- and polysaccharides, including fucoidan. The desulfation of iota-carrageenan by strain F21 was confirmed quantitatively by an increase in free sulfate concentration. Strains F1 and F21 represent the first marine sulfatase-rich anaerobes, encoding more sulfatases (521 and 480, 8.0% and 8.4% of all coding sequences, respectively) than any other microorganism currently known. Specific encoded sulfatase subfamilies could be involved in desulfating fucoidan (S1_15, S1_17 and S1_25) and iota-carrageenan (S1_19). Strains F1 and F21 had a sulfatase gene classification profile more similar to aerobic than anaerobic sulfatase-rich PVC bacteria, including Kiritimatiella glycovorans, the only other cultured representative within the Kiritimatiellaeota. Both strains encoded a single anaerobic sulfatase-maturating enzyme w, we set up anoxic enrichment cultures in duplicate with a mineral brackish marine medium and one of several polysaccharides as substrate. The cultures were inoculated with anoxic Black Sea sediment from 2100 m depth and incubated at 15°C (in situ Ta = 8°C) for several weeks. Community analysis by 16S rRNA gene amplicon sequencing revealed that various microbial clades without cultured representatives were enriched, indicating a potential hydrolytic and/or fermentative metabolism. We observed an enrichment of Kiritimatiellaeota (formerly Verrucomicrobia subdivision five) clade R76-B128 bacteria with the sulfated polysaccharide fucoidan as substrate.We isolated six strains of Kiritimatiellaeota R76-B128 which form two novel genera and a novel family within the order of the Kiritimatiellales, based on analysis of their 16S rRNA genes. Strikingly, the genomes of strains F1 and F21 contain the highest numbers (521, 480 resp.) and percentages (8.0% and 8.4% of all coding sequences, resp.) of sulfatase genes reported for bacterial genomes to date. Moreover, the sulfatase classification of strains F1, F21, K. glycovorans and nine other sulfatase-rich PVC bacteria showed that strains F1 and F21 harbored the highest numbers of sulfatase subfamilies (39, 37 resp.).

A psychrotolerant anaerobe, strain ART1T, was isolated from a psychrophilic anaerobic digester treating . 16S rRNA gene sequence of strain ART1T was highly similar to those of other Trichococcus species (> 99%), but digital DNA-DNA hybridization (dDDH) values were lower than 70% indicating that strain ART1 is a new species of the genus Trichococcus. Cells of strain ART1T were immotile cocci and stained Gram-positive. Growth was optimal at pH 7.5 and cells could grow in a temperature range of 0 to 37°C (optimum 30°C). Strain ART1T could degrade several carbohydrates, and the main products from glucose fermentation are lactate, acetate, formate, and ethanol. ., A psychrotolerant anaerobe, strain ART1T, was isolated from a psychrophilic anaerobic digester treating . 16S rRNA gene sequence of strain ART1T was highly similar to those of other Trichococcus species (> 99%), but digital DNA-DNA hybridization (dDDH) values were lower than 70% indicating that strain ART1 is a new species of the genus Trichococcus. Cells of strain ART1T were immotile cocci and stained Gram-positive. Growth was optimal at pH 7.5 and cells could grow in a temperature range of 0 to 37°C (optimum 30°C). Strain ART1T could degrade several carbohydrates, and the main products from glucose fermentation are lactate, acetate, formate, and ethanol. .

Propionate, butyrate, acetate, hydrogen and formate are the major intermediates of organic matter degradation. Sulfate-reducing bacteria (SRB) contribute significantly to the consumption of these substrates in sulfate-rich marine sediments. In sulfate-depleted sediments, however, complete degradation of propionate or butyrate is only possible via syntrophic cooperation of acetogenic bacteria and methanogenic archaea. Despite that the predominance of SRB in sulfate-rich and methanogens in sulfate-depleted sediments was reported, recent studies showed that both types of microorganism could be present in upper and lower parts of marine sediments. In this thesis, propionate and butyrate conversions and the involved microbial community in sulfate, sulfate-methane transition and methane zone sediment of Aarhus Bay, Denmark were studied using sediment slurry incubations. Interspecies hydrogen transfer and coexistence during acetate degradation were investigated in mixed pure cultures. In Chapter 2, interspecies hydrogen transfer between aceticlastic Methanosaeta concilii and hydrogenotrophic microorganisms, Desulfovibrio vulgaris or Methanococcus maripaludis, was investigated. Additionally, coexistence of M. concilii and Desulfobacter latus growing on acetate under sulfidogenic conditions was studied. The results of Chapter 2 showed that D. vulgaris could reduce sulfate and grow on leaked hydrogen from M. concilii. Hydrogen leakage from M. concilii provides an explanation for biogeochemical zonation both for competitive (e.g. acetate) and non-competitive substrates (methyl compounds), and this indicates the possible coexistence of SRB and methanogens in sulfate-rich environments. In chapter 3 and 4, long term incubations were examined focusing on butyrate and propionate conversion and the microbial community dynamics in sediment slurry enrichments at different sulfate (o, 3 and 20 mM) concentrations and incubation temperatures (10°C and 25°C). Sulfate reduction is the domi

Anaerobic microbial processes are commonly applied in the treatment of domestic and industrial wastewaters. Anaerobic digestion (AD) of wastewater has received a great deal of attention, but many aspects related to the complex interactions between microorganism, and how that is affected by the presence of certain toxic, are not yet fully understood. A particular case of this is the effect of heavy metals or chlorinated compounds. These compounds are known to have a strong impact in methanogens, a phylogenetic diverse group responsible for the last step of the AD process. The negative effect of sulphate towards methanogenesis is mainly related to outcompetition of methanogens by sulphate-reducing bacteria (SRB), or to toxicity caused by the sulphide generated from sulphate reduction. Heavy metals are part of many enzymes and cofactors and, in low concentrations, may beneficiate microbial activity. However, high concentrations of metals may disrupt enzyme function and structure. In cases where metal concentration is high, the presence of sulphate or sulphide might be favourable because sulphide precipitate with metals and detoxify the environment. In Chapter 2 we provide a review on the current knowledge on the effects of heavy metals and sulphate on AD, with special focus on methanogenesis. From this literature study, it came out that the influence of some metals, such as Co, is not extensively studied and that the potential of biologically produced sulphide as metal detoxification method in AD is still quite unexplored. In Chapter 3 we explored different strategies to improve methane production. Low concentrations of Ni and Co were supplemented to anaerobic sludge and the impact on methane production was evaluated. Although in contrast with other studies, no beneficial effect of metal supplementation was observed. Further on, the impact of high concentrations of Ni and Co added to anaerobic sludge was evaluated, as well as the use of sulphide as a detoxification str