Alcohol dehydrogenase system acts as the sole pathway for methanol oxidation in Desulfofundulus kuznetsovii strain TPOSR.

Desulfofundulus kuznetsovii is a thermophilic, spore-forming sulphate-reducing bacterium in the family Peptococcaceae. In this study, we describe a newly isolated strain of D. kuznetsovii, strain TPOSR, and compare its metabolism to the type strain D. kuznetsovii 17T. Both strains grow on a large variety of alcohols, such as methanol, ethanol and propane-diols, coupled to the reduction of sulphate. Strain 17T metabolizes methanol via two routes, one involving a cobalt-dependent methyl transferase and the other using a cobalt-independent alcohol dehydrogenase. However, strain TPOSR, which shares 97% average nucleotide identity with D. kuznetsovii strain 17T, lacks several genes from the methyl transferase operon found in strain 17T. The gene encoding the catalytically active methyl transferase subunit B is missing, indicating that strain TPOSR utilizes the alcohol dehydrogenase pathway exclusively. Both strains grew with methanol during cobalt starvation, but growth was impaired. Strain 17T was more sensitive to cobalt deficiency, due to the repression of its methyl transferase system. Our findings shed light on the metabolic diversity of D. kuznetsovii and their metabolic differences of encoding one or two routes for the conversion of methanol.

A Multifunctional Dehalobacter? Tandem Chloroform and Dichloromethane Degradation in a Mixed Microbial Culture.

Chloroform (CF) and dichloromethane (DCM) contaminate groundwater sites around the world but can be cleaned up through bioremediation. Although several strains of Dehalobacter restrictus can reduce CF to DCM and multiple Peptococcaceae can ferment DCM, these processes cannot typically happen simultaneously due to CF sensitivity in the known DCM-degraders or electron donor competition. Here, we present a mixed microbial culture that can simultaneously metabolize CF and DCM and create an additional enrichment culture fed only DCM. Through genus-specific quantitative polymerase chain reaction, we find that Dehalobacter grows while either CF alone or DCM alone is converted, indicating its involvement in both metabolic steps. Additionally, the culture was maintained for over 1400 days without the addition of an exogenous electron donor, and through electron balance calculations, we show that DCM metabolism would produce sufficient reducing equivalents (likely hydrogen) for CF respiration. Together, these results suggest intraspecies electron transfer could occur to continually reduce CF in the culture. Minimizing the addition of electron donor reduces the cost of bioremediation, and "self-feeding" could prolong bioremediation activity long after donor addition ends. Overall, understanding this mechanism informs strategies for culture maintenance and scale-up and benefits contaminated sites where the culture is employed for remediation worldwide.

Structure and functional capacity of a benzene-mineralizing, nitrate-reducing microbial community.

AIMS: How benzene is metabolized by microbes under anoxic conditions is not fully understood. Here, we studied the degradation pathways in a benzene-mineralizing, nitrate-reducing enrichment culture. METHODS AND RESULTS: Benzene mineralization was dependent on the presence of nitrate and correlated to the enrichment of a Peptococcaceae phylotype only distantly related to known anaerobic benzene degraders of this family. Its relative abundance decreased after benzene mineralization had terminated, while other abundant taxa-Ignavibacteriaceae, Rhodanobacteraceae and Brocadiaceae-slightly increased. Generally, the microbial community remained diverse despite the amendment of benzene as single organic carbon source, suggesting complex trophic interactions between different functional groups. A subunit of the putative anaerobic benzene carboxylase previously detected in Peptococcaceae was identified by metaproteomic analysis suggesting that benzene was activated by carboxylation. Detection of proteins involved in anaerobic ammonium oxidation (anammox) indicates that benzene mineralization was accompanied by anammox, facilitated by nitrite accumulation and the presence of ammonium in the growth medium. CONCLUSIONS: The results suggest that benzene was activated by carboxylation and further assimilated by a novel Peptococcaceae phylotype. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm the hypothesis that Peptococcaceae are important anaerobic benzene degraders.

Anaerobic Biodegradation of Chloroform and Dichloromethane with a Dehalobacter Enrichment Culture.

Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. Complete dechlorination of CF has been reported under anaerobic conditions by microbes that respire CF to DCM and others that biodegrade DCM. The objectives of this study were to ascertain if a commercially available bioaugmentation enrichment culture (KB-1 Plus CF) uses an oxidative or fermentative pathway for biodegradation of DCM and to determine if the products from DCM biodegradation can support organohalide respiration of CF to DCM in the absence of an exogenous electron donor. In various treatments with the KB-1 Plus CF culture to which 14C-CF was added, the predominant product was 14CO2, indicating that oxidation is the predominant pathway for DCM. Recovery of 14C-DCM when biodegradation was still in progress confirmed that CF first undergoes reductive dechlorination to DCM. 14C-labeled organic acids, including acetate and propionate, were also recovered, suggesting that synthesis of organic acids provides a sink for the electron equivalents from oxidation of DCM. When the biomass was washed to remove organic acids from prior additions of exogenous electron donor and only CF and DCM were added, the culture completely dechlorinated CF. The total amount of DCM added was not sufficient to provide the electron equivalents needed to reduce CF to DCM. Thus, the additional reducing power came via the DCM generated from CF reduction. Nevertheless, the rate of CF consumption was considerably lower compared to that of treatments that received an exogenous electron donor. IMPORTANCE Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. One way to address this problem is to add microbes to the subsurface that can biodegrade these compounds. While microbes are known that can accomplish this task, less is known about the pathways used under anaerobic conditions. Some use an oxidative pathway, resulting mainly in carbon dioxide. Others use a fermentative pathway, resulting in formation of organic acids. In this study, a commercially available bioaugmentation enrichment culture (KB-1 Plus CF) was evaluated using carbon-14 labeled chloroform. The main product formed was carbon dioxide, indicating the use of an oxidative pathway. The reducing power gained from oxidation was shown to support reductive dechlorination of CF to DCM. The results demonstrate the potential to achieve full dechlorination of CF and DCM to nonhazardous products that are difficult to identify in the field.

Stimulation of dissimilatory sulfate reduction in response to sulfate in microcosm incubations from two contrasting temperate peatlands near Ithaca, NY, USA.

Peatlands are responsible for over half of wetland methane emissions, yet major uncertainties remain regarding carbon flow, especially when increased availability of electron acceptors stimulates competing physiologies. We used microcosm incubations to study the effects of sulfate on microorganisms in two temperate peatlands, one bog and one fen. Three different electron donor treatments were used (13C-acetate, 13C-formate and a mixture of 12C short-chain fatty acids) to elucidate the responses of sulfate-reducing bacteria (SRB) and methanogens to sulfate stimulation. Methane production was measured and metagenomic sequencing was performed, with only the heavy DNA fraction sequenced from treatments receiving 13C electron donors. Our data demonstrate stimulation of dissimilatory sulfate reduction in both sites, with contrasting community responses. In McLean Bog (MB), hydrogenotrophic Deltaproteobacteria and acetotrophic Peptococcaceae lineages of SRB were stimulated, as were lineages with unclassified dissimilatory sulfite reductases. In Michigan Hollow Fen (MHF), there was little stimulation of Peptococcaceae populations, and a small stimulation of Deltaproteobacteria SRB populations only in the presence of formate as electron donor. Sulfate stimulated an increase in relative abundance of reads for both oxidative and reductive sulfite reductases, suggesting stimulation of an internal sulfur cycle. Together, these data indicate a stimulation of SRB activity in response to sulfate in both sites, with a stronger growth response in MB than MHF. This study provides valuable insights into microbial community responses to sulfate in temperate peatlands and is an important first step to understanding how SRB and methanogens compete to regulate carbon flow in these systems.

Phosphitispora fastidiosa gen. nov. sp. nov., a new dissimilatory phosphite-oxidizing anaerobic bacterium isolated from anaerobic sewage sludge.

A new strictly anaerobic bacterium, strain DYL19T, was enriched and isolated with phosphite as the sole electron donor and CO2 as a single carbon source and electron acceptor from anaerobic sewage sludge sampled at a sewage treatment plant in Constance, Germany. It is a Gram-positive, spore-forming, slightly curved, rod-shaped bacterium which oxidizes phosphite to phosphate while reducing CO2 to biomass and small amounts of acetate. Optimal growth is observed at 30 °C, pH 7.2, with a doubling time of 3 days. Beyond phosphite, no further inorganic or organic electron donor can be used, and no other electron acceptor than CO2 is reduced. Sulphate inhibits growth with phosphite and CO2. The G+C content is 45.95 mol%, and dimethylmenaquinone-7 is the only quinone detectable in the cells. On the basis of 16S rRNA gene sequence analysis and other chemotaxonomic properties, strain DYL19T is described as the type strain of a new genus and species, Phosphitispora fastidiosa gen. nov., sp. nov.

A gap-filling algorithm for prediction of metabolic interactions in microbial communities.

The study of microbial communities and their interactions has attracted the interest of the scientific community, because of their potential for applications in biotechnology, ecology and medicine. The complexity of interspecies interactions, which are key for the macroscopic behavior of microbial communities, cannot be studied easily experimentally. For this reason, the modeling of microbial communities has begun to leverage the knowledge of established constraint-based methods, which have long been used for studying and analyzing the microbial metabolism of individual species based on genome-scale metabolic reconstructions of microorganisms. A main problem of genome-scale metabolic reconstructions is that they usually contain metabolic gaps due to genome misannotations and unknown enzyme functions. This problem is traditionally solved by using gap-filling algorithms that add biochemical reactions from external databases to the metabolic reconstruction, in order to restore model growth. However, gap-filling algorithms could evolve by taking into account metabolic interactions among species that coexist in microbial communities. In this work, a gap-filling method that resolves metabolic gaps at the community level was developed. The efficacy of the algorithm was tested by analyzing its ability to resolve metabolic gaps on a synthetic community of auxotrophic Escherichia coli strains. Subsequently, the algorithm was applied to resolve metabolic gaps and predict metabolic interactions in a community of Bifidobacterium adolescentis and Faecalibacterium prausnitzii, two species present in the human gut microbiota, and in an experimentally studied community of Dehalobacter and Bacteroidales species of the ACT-3 community. The community gap-filling method can facilitate the improvement of metabolic models and the identification of metabolic interactions that are difficult to identify experimentally in microbial communities.

Microbial analysis and enrichment of anaerobic phenol and p-cresol degrading consortia with addition of AQDS.

Quinones and humus are ubiquitous in the biosphere and play an important role in the anaerobic biodegradation and biotransformation of organic acids, poisonous compounds as well as inorganic compounds. The impact of humic model compound, anthraquinone-2, 6-disulfonate (AQDS) on anaerobic phenol and p-cresol degradation were studied. Four methanogenic AQDS-free phenol and p-cresol enrichments and two phenol-AQDS enrichments were obtained using two sludges with potential biodegradability of phenol and cresol isomers as inoculum. 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that syntrophic aromatic compound degrading bacterium Syntrophorhabdus aromaticivorans was dominant in four AQDS-free enrichments, whereas phenol degrading Cryptanaerobacter phenolicus was dominant in two phenol-AQDS enrichments. Neither co-culture of S. aromaticivorans with Methanospirillum hungatei nor two phenol-AQDS enrichments could metabolize phenol using AQDS as the terminal electron acceptor. Further degradation experiments suggested that C. phenolicus related microbes in two phenol-AQDS enrichments were responsible for the conversion of phenol to benzoate, and benzoate was further degraded by benzoate degraders of Syntrophus aciditrophicus or Sporotomaculum syntrophicum to acetate.

Desulfosporosinus metallidurans sp. nov., an acidophilic, metal-resistant sulfate-reducing bacterium from acid mine drainage.

A novel, spore-forming, acidophilic and metal-resistant sulfate-reducing bacterium, strain OLT, was isolated from a microbial mat in a tailing dam at a gold ore mining site. Cells were slightly curved immotile rods, 0.5 µm in diameter and 2.0-3.0 µm long. Cells were stained Gram-negative, despite the Gram-positive cell structure revealed by electron microscopy of ultrathin layers. OLT grew at pH 4.0-7.0 with an optimum at 5.5. OLT utilised H2, lactate, pyruvate, malate, formate, propionate, ethanol, glycerol, glucose, fructose, sucrose, peptone and tryptone as electron donors for sulfate reduction. Sulfate, sulfite, thiosulfate, nitrate and fumarate were used as electron acceptors in the presence of lactate. Elemental sulfur, iron (III), and arsenate did not serve as electron acceptors. The major cellular fatty acids were C16:1ω7c (39.0 %) and C16 : 0 (12.1 %). The draft genome of OLT was 5.29 Mb in size and contained 4909 protein-coding genes. The 16S rRNA gene sequence placed OLT within the phylum Firmicutes, class Clostridia, family Peptococcaceae, genus Desulfosporosinus. Desulfosporosinus nitroreducens 59.4BT was the closest relative with 97.6 % sequence similarity. On the basis of phenotypic and phylogenetic characteristics, strain OLT represents a novel species within the genus Desulfosporosinus, for which we propose the name Desulfosporosinus metallidurans sp. nov. with the type strain OLT (=DSM 104464T=VKM В-3021T).

Anaerobic Microbial Metabolism of Dichloroacetate.

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising "Candidatus Dichloromethanomonas elyunquensis" strain RM utilizes DCA as an energy source, and the transient formation of formate, H2, and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated "Candidatus Dichloromethanomonas elyunquensis" strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg-1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.IMPORTANCE Dichloroacetate (DCA) is ubiquitous in the environment due to natural formation via biological and abiotic chlorination processes and the turnover of chlorinated organic materials (e.g., humic substances). Additional sources include DCA usage as a chemical feedstock and cancer drug and its unintentional formation during drinking water disinfection by chlorination. Despite the ubiquitous presence of DCA, its fate under anoxic conditions has remained obscure. We discovered an anaerobic bacterium capable of metabolizing DCA, identified the enzyme responsible for DCA dehalogenation, and elucidated a novel DCA fermentation pathway. The findings have implications for the turnover of DCA and the carbon and electron flow in electron acceptor-depleted environments and the human gastrointestinal tract.

Evolutionary stasis of a deep subsurface microbial lineage.

Sulfate-reducing bacteria Candidatus Desulforudis audaxviator (CDA) were originally discovered in deep fracture fluids accessed via South African gold mines and have since been found in geographically widespread deep subsurface locations. In order to constrain models for subsurface microbial evolution, we compared CDA genomes from Africa, North America and Eurasia using single cell genomics. Unexpectedly, 126 partial single amplified genomes from the three continents, a complete genome from of an isolate from Eurasia, and metagenome-assembled genomes from Africa and Eurasia shared >99.2% average nucleotide identity, low frequency of SNP's, and near-perfectly conserved prophages and CRISPRs. Our analyses reject sample cross-contamination, recent natural dispersal, and unusually strong purifying selection as likely explanations for these unexpected results. We therefore conclude that the analyzed CDA populations underwent only minimal evolution since their physical separation, potentially as far back as the breakup of Pangea between 165 and 55 Ma ago. High-fidelity DNA replication and repair mechanisms are the most plausible explanation for the highly conserved genome of CDA. CDA presents a stark contrast to the current model organisms in microbial evolutionary studies, which often develop adaptive traits over far shorter periods of time.

Two Marine Desulfotomaculum spp. of Different Origin are Capable of Utilizing Acetone and Higher Ketones.

Degradation of acetone and higher ketones has been described in detail for aerobic and nitrate-reducing bacteria. Among sulfate-reducing bacteria, degradation of acetone and other ketones is still an uncommon ability and has not been understood completely yet. In the present work, we show that Desulfotomaculum arcticum and Desulfotomaculum geothermicum are able to degrade acetone and butanone. Total proteomics of cell-free extracts of both organisms indicated an involvement of a thiamine diphosphate-dependent enzyme, a B12-dependent mutase, and a specific dehydrogenase during acetone degradation. Similar enzymes were recently described to be involved in acetone degradation by Desulfococcus biacutus. As there are so far only two described sulfate reducers able to degrade acetone, D. arcticum and D. geothermicum represent two further species with this capacity. All these bacteria appear to degrade acetone via the same set of enzymes and therefore via the same pathway.

Novel dichloromethane-fermenting bacteria in the Peptococcaceae family.

Dichloromethane (DCM; CH2Cl2) is a toxic groundwater pollutant that also has a detrimental effect on atmospheric ozone levels. As a dense non-aqueous phase liquid, DCM migrates vertically through groundwater to low redox zones, yet information on anaerobic microbial DCM transformation remains scarce due to a lack of cultured organisms. We report here the characterisation of DCMF, the dominant organism in an anaerobic enrichment culture (DFE) capable of fermenting DCM to the environmentally benign product acetate. Stable carbon isotope experiments demonstrated that the organism assimilated carbon from DCM and bicarbonate via the Wood-Ljungdahl pathway. DCMF is the first anaerobic DCM-degrading population also shown to metabolise non-chlorinated substrates. It appears to be a methylotroph utilising the Wood-Ljungdahl pathway for metabolism of methyl groups from methanol, choline, and glycine betaine. The flux of these substrates from subsurface environments may either directly (DCM, methanol) or indirectly (choline, glycine betaine) affect the climate. Community profiling and cultivation of cohabiting taxa in culture DFE without DCMF suggest that DCMF is the sole organism in this culture responsible for substrate metabolism, while the cohabitants persist via necromass recycling. Genomic and physiological evidence support placement of DCMF in a novel genus within the Peptococcaceae family, 'Candidatus Formimonas warabiya'.

Microbial reduction of As(V)-loaded Schwertmannite by Desulfosporosinus meridiei.

Arsenic-rich schwertmannite may cause arsenic (As) release during phase transition. In this study, microbial sulfidogenesis on As(V)-loaded schwertmannite (As-Sch) and associated As mobility at different SO42- concentrations were investigated under anaerobic conditions by Desulfosporosinus meridiei (D. meridiei). For biotic treatments, the more SO42- was added, the more Fe3+ was reduced to Fe2+, and the more As(V) was released during the reductive dissolution of As-Sch. The reduction of As(V) to As(III) by D. meridiei resulted in a higher concentration, toxicity, solubility and mobility of As than the corresponding abiotic treatments. However, compared with the abiotic treatments, a variety of new minerals (such as mackinawite, vivianite, sulfur, As2S3, and parasymplesite) were generated in the biotic treatments, and the As concentration in aqueous solution was less than 1 µM at the end of the incubation period regardless of the presence of SO42-. The results of continuous extraction of different species of As from secondary minerals showed that the effect of microorganisms decreased As content of amorphous iron oxide-bound phase, while increasing that bound on the surface of iron oxide surface-bound phase, thus increasing As fluidity. Our findings indicated that under anaerobic conditions, D. meridiei sulfidogenesis can trigger significant As mobilization in the early stage and remove As from the aqueous solutions when new minerals are formed at a later stage.

Effective Se reduction by lactate-stimulated indigenous microbial communities in excavated waste rocks.

Waste rocks generated from tunnel excavation contain the metalloid selenium (Se) and its concentration sometimes exceeds the environmental standards. The possibility and effectiveness of dissolved Se removal by the indigenous microorganisms are unknown. Chemical analyses and high-throughput 16S rRNA gene sequencing were implemented to investigate the functional and structural responses of the rock microbial communities to the Se and lactate amendment. During anaerobic incubation of the amended rock slurries from two distinct sites, dissolved Se concentrations decreased significantly, which coincided with lactate degradation to acetate and/or propionate. Sequencing indicated that relative abundances of Desulfosporosinus burensis increased drastically from 0.025 % and 0.022% to 67.584% and 63.716 %, respectively, in the sites. In addition, various Desulfosporosinus spp., Symbiobacterium-related species and Brevibacillus ginsengisoli, as well as the Se(VI)-reducing Desulfitobacterium hafniense, proliferated remarkably. They are capable of incomplete lactate oxidation to acetate as only organic metabolite, strongly suggesting their involvement in dissimilatory Se reduction. Furthermore, predominance of Pelosinus fermentans that ferments lactate to propionate and acetate implied that Se served as the electron sink for its fermentative lactate degradation. These results demonstrated that the indigenous microorganisms played vital roles in the lactate-stimulated Se reduction, leading to the biological Se immobilization treatment of waste rocks.

Assessment of the tolerance to Fe, Cu and Zn of a sulfidogenic sludge generated from hydrothermal vents sediments as a basis for its application on metals precipitation.

A paramour factor limiting metal-microorganism interaction is the metal ion concentration, and the metal precipitation efficiency driven by microorganisms is sensitive to metal ion concentration. The aim of the work was to determine the tolerance of the sulfidogenic sludge generated from hydrothermal vent sediments at microcosms level to different concentrations of Fe, Cu and Zn and the effect on the microbial community. In this study the chemical oxygen demand (COD) removal, sulfate-reducing activity (SRA) determination, inhibition effect through the determination of IC50, and the characterization of the bacterial community´s diversity were conducted. The IC50 on SRA was 34 and 81 mg/L for Zn and Cu, respectively. The highest sulfide concentration (H2S mg/L) and % of sulfate reduction obtained were: 511.30 ± 0.75 and 35.34 ± 0.51 for 50 mg/L of Fe, 482.48 ± 6.40 and 33.35 ± 0.44 for 10 mg/L of Cu, 442.26 ± 17.1 and 30.57 ± 1.18 for 10 mg/L of Zn, respectively. The COD removal rates were of 71.81 ± 7.6, 53.92 ± 1.07 and 57.68 ± 10.2 mg COD/ L d for Fe (50 mg/L), Cu (40 mg/L) and Zn (20 mg/L), respectively. Proteobacteria, Firmicutes, Chloroflexi and Actinobacteria were common phyla to four microcosms (stabilized sulfidogenic and added with Fe, Cu or Zn). The dsrA genes of Desulfotomaculum acetoxidans, Desulfotomaculum gibsoniae and Desulfovibrio desulfuricans were expressed in the microcosms supporting the SRA results. The consortia could be explored for ex-situ bioremediation purposes in the presence of the metals tested in this work.

Design, application, and microbiome of sulfate-reducing bioreactors for treatment of mining-influenced water.

Sulfate-reducing bioreactors, also called biochemical reactors, represent a promising option for passive treatment of mining-influenced water (MIW) based on similar technology to aerobic/anaerobic-constructed wetlands and vertical-flow wetlands. MIW from each mine site has a variety of site-specific properties related to its treatment; therefore, design factors, including the organic substrates and inorganic materials packed into the bioreactor, must be tested and evaluated before installation of full-scale sulfate-reducing bioreactors. Several full-scale sulfate-reducing bioreactors operating at mine sites provide examples, but holistic understanding of the complex treatment processes occurring inside the bioreactors is lacking. With the recent introduction of high-throughput DNA sequencing technologies, microbial processes within bioreactors may be clarified based on the relationships between operational parameters and key microorganisms identified using high-resolution microbiome data. In this review, the test design procedures and precedents of full-scale bioreactor application for MIW treatment are briefly summarized, and recent knowledge on the sulfate-reducing microbial communities of field-based bioreactors from fine-scale monitoring is presented.Key points• Sulfate-reducing bioreactors are promising for treatment of mining-influenced water.• Various design factors should be tested for application of full-scale bioreactors.• Introduction of several full-scale passive bioreactor systems at mine sites.• Desulfosporosinus spp. can be one of the key bacteria within field-based bioreactors.

Response of Isovalerate-Degrading Methanogenic Microbial Community to Inhibitors.

Isovalerate is one of the key intermediates during anaerobic digestion treating protein-containing waste/wastewater. Investigating the effect of different kinds of inhibitors on isovalerate-degrading microbial community is necessary to develop measures for improving the effectiveness of the treatment plants. In the present study, dynamic changes in the isovalerate-degrading microbial community in presence of inhibitors (ammonium, sulfide, mixed ammonium and sulfide, and chlortetracycline (CTC)) were investigated using high-throughput sequencing of 16S rRNA gene. Our observations showed that the isovalerate-degrading microbial community responded differently to different inhibitors and that the isovalerate degradation and gas production were strongly repressed by each inhibitor. We found that sulfide inhibited both isovalerate oxidation followed by methanogenesis, while ammonium, mixed ammonium and sulfide, and CTC mainly inhibited isovalerate oxidation. Genera classified into Proteobacteria and Chloroflexi were less sensitive to inhibitors. The two dominant genera, which are potential syntrophic isovalerate oxidizers, exhibited different responses to inhibitors that the unclassified_Peptococcaceae_3 was more sensitive to inhibitors than the unclassified_Syntrophaceae. Upon comparison to acetoclastic methanogen Methanosaeta, hydrogenotrophic methanogens Methanoculleus and Methanobacterium were less sensitive to inhibitors.

Mineralization versus fermentation: evidence for two distinct anaerobic bacterial degradation pathways for dichloromethane.

Dichloromethane (DCM) is an anthropogenic pollutant with ozone destruction potential that is also formed naturally. Under anoxic conditions, fermentation of DCM to acetate and formate has been reported in axenic culture Dehalobacterium formicoaceticum, and to acetate, H2 and CO2 in mixed culture RM, which harbors the DCM degrader 'Candidatus Dichloromethanomonas elyunquensis'. RM cultures produced 28.1 ± 2.3 µmol of acetate from 155.6 ± 9.3 µmol DCM, far less than the one third (i.e., about 51.9 µmol) predicted based on the assumed fermentation model, and observed in cultures of Dehalobacterium formicoaceticum. Temporal metabolite analyses using gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy revealed that no 13C-labeled acetate was formed in 13C-DCM-grown RM cultures, indicating acetate was not a direct product of DCM metabolism. The data were reconciled with DCM mineralization and H2 consumption via CO2 reduction to acetate and methane by homoacetogenic and methanogenic partner populations, respectively. In contrast, Dehalobacterium formicoaceticum produced 13C-labeled acetate and formate from 13C-DCM, consistent with a fermentation pathway. Free energy change calculations predicted that organisms with the mineralization pathway are the dominant DCM consumers in environments with H2 <100 ppmv. These findings have implications for carbon and electron flow in environments where DCM is introduced through natural production processes or anthropogenic activities.

Methanogenic biodegradation of iso-alkanes and cycloalkanes during long-term incubation with oil sands tailings.

Microbes indigenous to oil sands tailings ponds methanogenically biodegrade certain hydrocarbons, including n-alkanes and monoaromatics, whereas other hydrocarbons such as iso- and cycloalkanes are more recalcitrant. We tested the susceptibility of iso- and cycloalkanes to methanogenic biodegradation by incubating them with mature fine tailings (MFT) collected from two depths (6 and 31 m below surface) of a tailings pond, representing different lengths of exposure to hydrocarbons. A mixture of five iso-alkanes and three cycloalkanes was incubated with MFT for 1700 d. Iso-alkanes were completely biodegraded in the order 3-methylhexane > 4-methylheptane > 2-methyloctane > 2-methylheptane, whereas 3-ethylhexane and ethylcyclopentane were only partially depleted and methylcyclohexane and ethylcyclohexane were not degraded during incubation. Pyrosequencing of 16S rRNA genes showed enrichment of Peptococcaceae (Desulfotomaculum) and Smithella in amended cultures with acetoclastic (Methanosaeta) and hydrogenotrophic methanogens (Methanoregula and Methanoculleus). Bioaugmentation of MFT by inoculation with MFT-derived enrichment cultures reduced the lag phase before onset of iso-alkane and cycloalkane degradation. However, the same enrichment culture incubated without MFT exhibited slower biodegradation kinetics and less CH4 production, implying that the MFT solid phase (clay minerals) enhanced methanogenesis. These results help explain and predict continued emissions of CH4 from oil sands tailings repositories in situ.