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.

Sediment cooling triggers germination and sulfate reduction by heat-resistant thermophilic spore-forming bacteria.

Thermophilic endospores are widespread in cold marine sediments where the temperature is too low to support growth and activity of thermophiles in situ. These endospores are likely expelled from warm subsurface environments and subsequently dispersed by ocean currents. The endospore upper temperature limit for survival is 140°C, which can be tolerated in repeated short exposures, potentially enabling transit through hot crustal fluids. Longer-term thermal tolerance of endospores, and how long they could persist in an environment hotter than their maximum growth temperature, is less understood. To test whether thermophilic endospores can survive prolonged exposure to high temperatures, sediments were incubated at 80-90°C for 6, 12 or 463 days. Sediments were then cooled by 10-40°C, mimicking the cooling in subsurface oil reservoirs subjected to seawater injection. Cooling the sediments induced sulfate reduction, coinciding with an enrichment of endospore-forming Clostridia. Different Desulfofundulus, Desulfohalotomaculum, Desulfallas, Desulfotomaculum and Desulfofarcimen demonstrated different thermal tolerances, with some Desulfofundulus strains surviving for >1 year at 80°C. In an oil reservoir context, heat-resistant endospore-forming sulfate-reducing bacteria have a survival advantage if they are introduced to, or are resident in, an oil reservoir normally too hot for germination and growth, explaining observations of reservoir souring following cold seawater injection.

Desulfosporosinus spp. were the most predominant sulfate-reducing bacteria in pilot- and laboratory-scale passive bioreactors for acid mine drainage treatment.

Five types of sulfate-reducing passive bioreactors with rice bran as substrate were operated at three different mine sites under various operating conditions to investigate and compare the dominant sulfate-reducing bacteria (SRBs) involved in acid mine drainage (AMD) treatment. In all bioreactors, AMD was properly treated under the national effluent standard of Japan when 16 samples in total were taken from different depths of the bioreactors at different sampling times. Analysis of the microbiomes in the five bioreactors by Illumina sequencing showed that Desulfosporosinus spp. were dominant SRBs in all bioreactors (the relative abundances were ~ 26.0% of the total population) regardless of reactor configurations, sizes, and operating conditions. This genus is known to comprise spore-forming, acid-tolerant, and oxygen-resistant SRBs with versatile metabolic capabilities. Microbial populations of AMD water and soil samples (as inocula) from the respective mine sites were also analyzed to investigate the origin of the genus Desulfosporosinus. Desulfosporosinus spp. were detectable in most AMD water samples, even at low relative abundances (0.0025 to 0.0069% of total AMD population), suggesting that the genus Desulfosporosinus is present within the AMD water that flows into the bioreactor. These data strongly imply that the passive treatment system is a versatile and widely applicable process for AMD treatment.

Transcriptome analysis of a thermophilic and hydrogenogenic carboxydotroph Carboxydothermus pertinax.

A thermophilic and hydrogenogenic carboxydotroph, Carboxydothermus pertinax, performs hydrogenogenic CO metabolism in which CODH-II couples with distally encoded ECH. To enhance our knowledge of its hydrogenogenic CO metabolism, we performed whole transcriptome analysis of C. pertinax grown under 100% CO or 100% N2 using RNA sequencing. Of the 2577 genes, 36 and 64 genes were differentially expressed genes (DEGs) with false discovery rate adjusted P value < 0.05 when grown under 100% CO or 100% N2, respectively. Most of the DEGs were components of 23 gene clusters, suggesting switch between metabolisms via intensive expression changes in a relatively low number of gene clusters. Of the 9 significantly expressed gene clusters under 100% CO, CODH-II and ECH gene clusters were found. Only the ECH gene cluster was regulated by the CO-responsive transcriptional factor CooA, suggesting that others were separately regulated in the same transcriptional cascade as the ECH gene cluster. Of the 14 significantly expressed gene clusters under 100% N2, ferrous iron transport gene cluster involved in anaerobic respiration and prophage region were found. Considering that the expression of the temperate phage was strictly repressed under 100% CO, hydrogenogenic CO metabolism might be stable for C. pertinax.

Domestication of previously uncultivated Candidatus Desulforudis audaxviator from a deep aquifer in Siberia sheds light on its physiology and evolution.

An enigmatic uncultured member of Firmicutes, Candidatus Desulforudis audaxviator (CDA), is known by its genome retrieved from the deep gold mine in South Africa, where it formed a single-species ecosystem fuelled by hydrogen from water radiolysis. It was believed that in situ conditions CDA relied on scarce energy supply and did not divide for hundreds to thousand years. We have isolated CDA strain BYF from a 2-km-deep aquifer in Western Siberia and obtained a laboratory culture growing with a doubling time of 28.5 h. BYF uses not only H2 but also various organic electron donors for sulfate respiration. Growth required elemental iron, and ferrous iron did not substitute for it. A complex intracellular organization included gas vesicles, internal membranes, and electron-dense structures enriched in phosphorus, iron, and calcium. Genome comparison of BYF with the South African CDA revealed minimal differences mostly related to mobile elements and prophage insertions. Two genomes harbored <800 single-nucleotide polymorphisms and had nearly identical CRISPR loci. We suggest that spores with the gas vesicles may facilitate global distribution of CDA followed by colonization of suitable subsurface environments. Alternatively, a slow evolution rate in the deep subsurface could result in high genetic similarity of CDA populations at two sites spatially separated for hundreds of millions of years.

Long-Term Transcriptional Activity at Zero Growth of a Cosmopolitan Rare Biosphere Member.

Microbial diversity in the environment is mainly concealed within the rare biosphere (all species with <0.1% relative abundance). While dormancy explains a low-abundance state very well, the mechanisms leading to rare but active microorganisms remain elusive. We used environmental systems biology to genomically and transcriptionally characterize "Candidatus Desulfosporosinus infrequens," a low-abundance sulfate-reducing microorganism cosmopolitan to freshwater wetlands, where it contributes to cryptic sulfur cycling. We obtained its near-complete genome by metagenomics of acidic peat soil. In addition, we analyzed anoxic peat soil incubated under in situ-like conditions for 50 days by Desulfosporosinus-targeted qPCR and metatranscriptomics. The Desulfosporosinus population stayed at a constant low abundance under all incubation conditions, averaging 1.2 × 106 16S rRNA gene copies per cm³ soil. In contrast, transcriptional activity of "Ca. Desulfosporosinus infrequens" increased at day 36 by 56- to 188-fold when minor amendments of acetate, propionate, lactate, or butyrate were provided with sulfate, compared to the no-substrate-control. Overall transcriptional activity was driven by expression of genes encoding ribosomal proteins, energy metabolism, and stress response but not by expression of genes encoding cell growth-associated processes. Since our results did not support growth of these highly active microorganisms in terms of biomass increase or cell division, they had to invest their sole energy for maintenance, most likely counterbalancing acidic pH conditions. This finding explains how a rare biosphere member can contribute to a biogeochemically relevant process while remaining in a zero-growth state over a period of 50 days.IMPORTANCE The microbial rare biosphere represents the largest pool of biodiversity on Earth and constitutes, in sum of all its members, a considerable part of a habitat's biomass. Dormancy or starvation is typically used to explain the persistence of low-abundance microorganisms in the environment. We show that a low-abundance microorganism can be highly transcriptionally active while remaining in a zero-growth state for at least 7 weeks. Our results provide evidence that this zero growth at a high cellular activity state is driven by maintenance requirements. We show that this is true for a microbial keystone species, in particular a cosmopolitan but permanently low-abundance sulfate-reducing microorganism in wetlands that is involved in counterbalancing greenhouse gas emissions. In summary, our results provide an important step forward in understanding time-resolved activities of rare biosphere members relevant for ecosystem functions.

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism.

Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway.IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and "Candidatus Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

Co-occurrence network analysis reveals thermodynamics-driven microbial interactions in methanogenic bioreactors.

Methanogenic bioreactors have been applied to treat purified terephthalic acid (PTA) wastewater containing complex aromatic compounds, such as terephthalic acid, para-toluic acid and benzoic acid. This study characterized the interaction of microbial populations in 42 samples obtained from 10 PTA-degrading methanogenic bioreactors. Approximately, 54 dominant populations (11 methanogens, 8 syntrophs and 35 functionally unknown clades) that represented 73.9% of total 16S rRNA gene iTag sequence reads were identified. Co-occurrence analysis based on the abundance of dominant OTUs showed two non-overlapping networks centred around aromatic compound- (group AR: Syntrophorhabdaceae, Syntrophus and Pelotomaculum) and fatty acid- (group FA: Smithella and Syntrophobacter) degrading syntrophs. Group AR syntrophs have no direct correlation with hydrogenotrophic methanogens, while those from group FA do. As degradation of aromatic compounds has a wider thermodynamic window than fatty acids, Group AR syntrophs may be less influenced by fluctuations in hydrogenotrophic methanogen abundance or may non-specifically interact with diverse methanogens. In both groups, network analysis reveals full-scale- and lab-scale-specific uncultivated taxa that may mediate interactions between syntrophs and methanogens, suggesting that those uncultivated taxa may support the degradation of aromatic compounds through uncharted ecophysiological traits. These observations suggest that organisms from multiple niches orchestrate their metabolic capacity in multiple interaction networks to effectively degrade PTA wastewater.

Recoding of the selenocysteine UGA codon by cysteine in the presence of a non-canonical tRNACys and elongation factor SelB.

In many organisms, the UGA stop codon is recoded to insert selenocysteine (Sec) into proteins. Sec incorporation in bacteria is directed by an mRNA element, known as the Sec-insertion sequence (SECIS), located downstream of the Sec codon. Unlike other aminoacyl-tRNAs, Sec-tRNASec is delivered to the ribosome by a dedicated elongation factor, SelB. We recently identified a series of tRNASec-like tRNA genes distributed across Bacteria that also encode a canonical tRNASec. These tRNAs contain sequence elements generally recognized by cysteinyl-tRNA synthetase (CysRS). While some of these tRNAs contain a UCA Sec anticodon, most have a GCA Cys anticodon. tRNASec with GCA anticodons are known to recode UGA codons. Here we investigate the clostridial Desulfotomaculum nigrificans tRNASec-like tRNACys, and show that this tRNA is acylated by CysRS, recognized by SelB, and capable of UGA recoding with Cys in Escherichia coli. We named this non-canonical group of tRNACys as 'tRNAReC' (Recoding with Cys). We performed a comprehensive survey of tRNAReC genes to establish their phylogenetic distribution, and found that, in a particular lineage of clostridial Pelotomaculum, the Cys identity elements of tRNAReC had mutated. This novel tRNA, which contains a UCA anticodon, is capable of Sec incorporation in E. coli, albeit with lower efficiency relative to Pelotomaculum tRNASec. We renamed this unusual tRNASec derived from tRNAReC as 'tRNAReU' (Recoding with Sec). Together, our results suggest that tRNAReC and tRNAReU may serve as safeguards in the production of selenoproteins and - to our knowledge - they provide the first example of programmed codon-anticodon mispairing in bacteria.

A benzene-degrading nitrate-reducing microbial consortium displays aerobic and anaerobic benzene degradation pathways.

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.

A switch of chlorinated substrate causes emergence of a previously undetected native Dehalobacter population in an established Dehalococcoides-dominated chloroethene-dechlorinating enrichment culture.

Chlorobenzenes are soil and groundwater pollutants of concern that can be reductively dehalogenated by organohalide-respiring bacteria from the genera Dehalococcoides and Dehalobacter. The bioaugmentation culture KB-1® harbours Dehalococcoides mccartyi spp. that reductively dehalogenate trichloroethene to ethene. It contains more than 30 reductive dehalogenase genes; some of them are highly similar to genes found in the chlorobenzene-respiring Dehalococcoides mccartyi strain CBDB1. We explored the chlorobenzene dehalogenation capability of the KB-1 enrichment culture using 1,2,4-trichlorobenzene (1,2,4-TCB). We achieved adaptation of KB-1 to 1,2,4-TCB that is dehalogenated to a mixture of dichlorobenzenes, and subsequently to monochlorobenzene and benzene. Surprisingly, a native Dehalobacter population, and not a Dehalococcoides population, couples the dechlorination of 1,2,4-TCB to growth achieving an average yield of 1.1 ± 0.6 × 1013 cells per mole of Cl- released. Interestingly, the dechlorination of 1,2,4-TCB occurs alongside the complete dechlorination of trichloroethene to ethene in cultures fed both electron acceptors. Dehalobacter was not previously identified as a major player in KB-1, but its ecological niche was favoured by the introduction of 1,2,4-TCB. Based on 16S rRNA phylogeny, Dehalobacter populations seem to cluster into specialised clades, and are likely undergoing substrate specialisation as a strategy to reduce competition for electron acceptors.

Mutualistic interaction between dichloromethane- and chloromethane-degrading bacteria in an anaerobic mixed culture.

The microbial mixed culture RM grows with dichloromethane (DCM) as the sole energy source generating acetate, methane, chloride and biomass as products. Chloromethane (CM) was not an intermediate during DCM utilization consistent with the observation that CM could not replace DCM as a growth substrate. Interestingly, cultures that received DCM and CM together degraded both compounds concomitantly. Transient hydrogen (H2 ) formation reaching a maximum concentration of 205 ± 13 ppmv was observed in cultures growing with DCM, and the addition of exogenous H2 at concentrations exceeding 3000 ppmv impeded DCM degradation. In contrast, CM degradation in culture RM had a strict requirement for H2 . Following five consecutive transfers on CM and H2 , Acetobacterium 16S rRNA gene sequences dominated the culture and the DCM-degrader Candidatus Dichloromethanomonas elyunquensis was eliminated, consistent with the observation that the culture lost the ability to degrade DCM. These findings demonstrate that culture RM harbours different populations responsible for anaerobic DCM and CM metabolism, and further imply that the DCM and CM degradation pathways are mechanistically distinct. H2 generated during DCM degradation is consumed by the hydrogenotrophic CM degrader, or may fuel other hydrogenotrophic processes, including organohalide respiration, methanogenesis and H2 /CO2 reductive acetogenesis.

Marinisporobacter balticus gen. nov., sp. nov., Desulfosporosinus nitroreducens sp. nov. and Desulfosporosinus fructosivorans sp. nov., new spore-forming bacteria isolated from subsurface sediments of the Baltic Sea.

Four novel Gram-stain-positive, endospore-forming bacteria of the order Clostridiales were isolated from subsurface sediments sampled during International Ocean Discovery Program Expedition 347 to the Baltic Sea. One strain (59.4MT) grew as an obligate heterotroph by aerobic respiration and anaerobically by fermentation. Optimum growth was observed with 0.5 % NaCl at 25 °C and pH 7.0-7.3. Analysis of 16S rRNA gene sequences of 59.4MT revealed Alkaliphilus transvaalensis (92.3 % identity), Candidatus Geosporobacter ferrireducens (92.2 %), Geosporobacter subterraneus (91.9 %) and Alkaliphilus peptidifermentans (91.7 %) to be the closest relatives. On the basis of the results of phenotypic and genotypic analyses, we propose that strain 59.4MT represents a novel species within a novel genus, Marinisporobacter balticus gen. nov., sp. nov., with the type strain 59.4MT (=DSM 102940T=JCM 31103T). Three other strains, 59.4F, 59.4BT and 63.6FT, were affiliated with the genus Desulfosporosinus and grew as strictly anaerobic sulfate reducers. These strains additionally used thiosulfate, elemental sulfur, sulfite and DMSO as electron acceptors and hydrogen as an electron donor. Strains 59.4F and 59.4BT had identical 16S rRNA gene sequences, which were most similar to those of Desulfosporosinus lacus (97.8 %), Desulfosporosinus hippei (97.3 %) and Desulfosporosinus orientis (97.3 %). Strain 63.6FT was closely related to D. lacus (97.7 %), Desulfosporosinus meridiei (96.6 %) and D. hippei (96.5 %). The similarity of 16S rRNA gene sequences of strains 59.4BT and 63.6FT was 96.6 %. We propose the new names Desulfosporosinus nitroreducens sp. nov., incorporating strain 59.4F (=DSM 101562=JCM 31104) and the type strain 59.4BT (=DSM 101608T=JCM 31105T), and Desulfosporosinus fructosivorans sp. nov., with the type strain 63.6FT (=DSM 101609T=JCM 31106T).

'Candidatus Dichloromethanomonas elyunquensis' gen. nov., sp. nov., a dichloromethane-degrading anaerobe of the Peptococcaceae family.

Taxonomic assignments of anaerobic dichloromethane (DCM)-degrading bacteria remain poorly constrained but are important for understanding the microbial diversity of organisms contributing to DCM turnover in environmental systems. We describe the taxonomic classification of a novel DCM degrader in consortium RM obtained from pristine Rio Mameyes sediment. Phylogenetic analysis of full-length 16S rRNA gene sequences demonstrated that the DCM degrader was most closely related to members of the genera Dehalobacter and Syntrophobotulus, but sequence similarities did not exceed 94% and 93%, respectively. Genome-aggregate average amino acid identities against Peptococcaceae members did not exceed 66%, suggesting that the DCM degrader does not affiliate with any described genus. Phylogenetic analysis of conserved single-copy functional genes supported that the DCM degrader represents a novel clade. Growth strictly depended on the presence of DCM, which was consumed at a rate of 160±3µmolL-1 d-1. The DCM degrader attained 5.25×107±1.0×107 cells per µmol DCM consumed. Fluorescence in situ hybridization revealed rod-shaped cells 4±0.8µm long and 0.4±0.1µm wide. Based on the unique phylogenetic, genomic, and physiological characteristics, we propose that the DCM degrader represents a new genus and species, 'Candidatus Dichloromethanomonas elyunquensis'.

Benzene degradation in a denitrifying biofilm reactor: activity and microbial community composition.

Benzene is an aromatic compound and harmful for the environment. Biodegradation of benzene can reduce the toxicological risk after accidental or controlled release of this chemical in the environment. In this study, we further characterized an anaerobic continuous biofilm culture grown for more than 14 years on benzene with nitrate as electron acceptor. We determined steady state degradation rates, microbial community composition dynamics in the biofilm, and the initial anaerobic benzene degradation reactions. Benzene was degraded at a rate of 0.15 µmol/mg protein/day and a first-order rate constant of 3.04/day which was fourfold higher than rates reported previously. Bacteria belonging to the Peptococcaceae were found to play an important role in this anaerobic benzene-degrading biofilm culture, but also members of the Anaerolineaceae were predicted to be involved in benzene degradation or benzene metabolite degradation based on Illumina MiSeq analysis of 16S ribosomal RNA genes. Biomass retention in the reactor using a filtration finger resulted in reduction of benzene degradation capacity. Detection of the benzene carboxylase encoding gene, abcA, and benzoic acid in the culture vessel indicated that benzene degradation proceeds through an initial carboxylation step.

Dechlorination of three tetrachlorobenzene isomers by contaminated harbor sludge-derived enrichment cultures follows thermodynamically favorable reactions.

Dechlorination patterns of three tetrachlorobenzene isomers, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-TeCB, were studied in anoxic microcosms derived from contaminated harbor sludge. The removal of doubly, singly, and un-flanked chlorine atoms was noted in 1,2,3,4- and 1,2,3,5-TeCB fed microcosms, whereas only singly flanked chlorine was removed in 1,2,4,5-TeCB microcosms. The thermodynamically more favorable reactions were selectively followed by the enriched cultures with di- and/or mono-chlorobenzene as the main end products of the reductive dechlorination of all three isomers. Based on quantitative PCR analysis targeting 16S rRNA genes of known organohalide-respiring bacteria, the growth of Dehalococcoides was found to be associated with the reductive dechlorination of all three isomers, while growth of Dehalobacter, another known TeCB dechlorinator, was only observed in one 1,2,3,5-TeCB enriched microcosm among biological triplicates. Numbers of Desulfitobacterium and Geobacter as facultative dechlorinators were rather stable suggesting that they were not (directly) involved in the observed TeCB dechlorination. Bacterial community profiling suggested bacteria belonging to the phylum Bacteroidetes and the order Clostridiales as well as sulfate-reducing members of the class Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to fastidious dechlorinators. Our results provide a better understanding of thermodynamically preferred TeCB dechlorinating pathways in harbor environments and microbial guilds enriched and active in anoxic TeCB dechlorinating microcosms.

Desulfonatronospira sulfatiphila sp. nov., and Desulfitispora elongata sp. nov., two novel haloalkaliphilic sulfidogenic bacteria from soda lakes.

Two novel haloalkaliphilic bacteria with dissimilatory sulfidogenic metabolism were recovered from syntrophic associations obtained from anaerobic sediments of hypersaline soda lakes in Kulunda Steppe (Altai, Russia). Strain ASO3-2T was a member of a sulfidogenic syntrophic association oxidizing acetate at extremely haloalkaline conditions, and was isolated in pure culture using formate as electron donor and sulfate as electron acceptor. It was identified as representing a novel member of the genus Desulfonatronospira within the Deltaproteobacteria. In contrast to the two known species of this genus, the novel isolate was able to grow with formate as electron donor and sulfate, as well as with sulfite, as electron acceptor. Strain Acr1T was a minor component in a soda lake syntrophic association converting benzoate to methane and acetate. It became dominant in a subculture fed with crotonate. While growing on crotonate, strain Acr1T formed unusually long cells filled with polyhydroxyalkanoate-like granules. Its metabolism was limited to fermentation of crotonate and pyruvate and the ability to utilize thiosulfate and sulfur/polysulfide as electron acceptor. Strain Acr1T was identified as representing a novel member of the genus Desulfitispora in the class Clostridia. Both isolates were obligately haloalkaliphilic with extreme salt tolerance. On the basis of phenotypic and phylogenetic analyses, the novel sulfidogenic isolates from soda lakes are proposed to represent two novel species: Desulfonatronospira sulfatiphila sp. nov. (ASO3-2T=DSM 100427=UNIQEM U993T) and Desulfitispora elongata sp. nov. (Acr1T=DSM 29990=UNIQEM U994T).

The sequence capture by hybridization: a new approach for revealing the potential of mono-aromatic hydrocarbons bioattenuation in a deep oligotrophic aquifer.

The formation water of a deep aquifer (853 m of depth) used for geological storage of natural gas was sampled to assess the mono-aromatic hydrocarbons attenuation potential of the indigenous microbiota. The study of bacterial diversity suggests that Firmicutes and, in particular, sulphate-reducing bacteria (Peptococcaceae) predominate in this microbial community. The capacity of the microbial community to biodegrade toluene and m- and p-xylenes was demonstrated using a culture-based approach after several hundred days of incubation. In order to reveal the potential for biodegradation of these compounds within a shorter time frame, an innovative approach named the solution hybrid selection method, which combines sequence capture by hybridization and next-generation sequencing, was applied to the same original water sample. The bssA and bssA-like genes were investigated as they are considered good biomarkers for the potential of toluene and xylene biodegradation. Unlike a PCR approach which failed to detect these genes directly from formation water, this innovative strategy demonstrated the presence of the bssA and bssA-like genes in this oligotrophic ecosystem, probably harboured by Peptococcaceae. The sequence capture by hybridization shows significant potential to reveal the presence of genes of functional interest which have low-level representation in the biosphere.

Isolation of a sulfide-producing bacterial consortium from cooling-tower water: Evaluation of corrosive effects on galvanized steel.

Sulfidogenic Clostridia and sulfate reducing bacteria (SRB) often cohabit in nature. The presence of these microorganisms can cause microbially influenced corrosion (MIC) of materials in different ways. To investigate this aspect, bacteria were isolated from cooling tower water and used in corrosion tests of galvanized steel. The identity of the isolates was determined by comparative sequence analysis of PCR-amplified 16S rDNA gene fragments, separated by denaturing gradient gel electrophoresis (DGGE). This analysis showed that, in spite of the isolation process, colonies were not pure and consisted of a mixture of bacteria affiliated with Desulfosporosinus meridiei and Clostridium sp. To evaluate the corrosive effect, galvanized steel coupons were incubated with a mixed culture for 4, 8, 24, 72, 96, 168, 360 and 744 h, along with a control set in sterile culture medium only. The corrosion rate was determined by weight loss, and biofilm formation and corroded surfaces were observed by scanning electron microscopy (SEM). Although the sulfide-producing bacterial consortium led to a slight increase in the corrosion of galvanized steel coupons, when compared to the previous studies it can be said that Clostridium sp. can reduce the corrosive effect of the Desulfosporosinus sp. strain.