2,4-Dichlorophenoxyacetic acid degradation in methanogenic mixed cultures obtained from Brazilian Amazonian soil samples.

2,4-Dichlorophenoxyacetic acid (2,4-D) is the third most applied pesticide in Brazil to control broadleaf weeds in crop cultivation and pastures. Due to 2,4-D's high mobility and long half-life under anoxic conditions, this herbicide has high probability for groundwater contamination. Bioremediation is an attractive solution for 2,4-D contaminated anoxic environments, but there is limited understanding of anaerobic 2,4-D biodegradation. In this study, methanogenic enrichment cultures were obtained from Amazonian top soil (0-40 cm) and deep soil (50 -80 cm below ground) that biotransform 2,4-D (5 µM) to 4-chlorophenol and phenol. When these cultures were transferred (10% v/v) to fresh medium containing 40 µM or 160 µM 2,4-D, the rate of 2,4-D degradation decreased, and biotransformation did not proceed beyond 4-chlorophenol and 2,4-dichlorophenol in the top and deep soil cultures, respectively. 16S rRNA gene sequencing and qPCR of a selection of microbes revealed no significant enrichment of known organohalide-respiring bacteria. Furthermore, a member of the genus Cryptanaerobacter was identified as possibly responsible for phenol conversion to benzoate in the top soil inoculated culture. Overall, these results demonstrate the effect of 2,4-D concentration on biodegradation and microbial community composition, which are both important factors when developing pesticide bioremediation technologies.

Reductive dechlorination of 1,2-dichloroethane in the presence of chloroethenes and 1,2-dichloropropane as co-contaminants.

1,2-Dichloroethane (1,2-DCA) is one of the most abundant manmade chlorinated organic contaminants in the world. Reductive dechlorination of 1,2-DCA by organohalide-respiring bacteria (OHRB) can be impacted by other chlorinated contaminants such as chloroethenes and chloropropanes that can co-exist with 1,2-DCA at contaminated sites. The aim of this study was to evaluate the effect of chloroethenes and 1,2-dichloropropane (1,2-DCP) on 1,2-DCA dechlorination using sediment cultures enriched with 1,2-DCA as the sole chlorinated compound (EA culture) or with 1,2-DCA and tetrachloroethene (PCE) (EB culture), and to model dechlorination kinetics. Both cultures contained Dehalococcoides as most predominated OHRB, and Dehalogenimonas and Geobacter as other known OHRB. In sediment-free enrichments obtained from the EA and EB cultures, dechlorination of 1,2-DCA was inhibited in the presence of the same concentrations of either PCE, vinyl chloride (VC), or 1,2-DCP; however, concurrent dechlorination of dual chlorinated compounds was achieved. In contrast, 1,2-DCA dechlorination completely ceased in the presence of cis-dichloroethene (cDCE) and only occurred after cDCE was fully dechlorinated. In turn, 1,2-DCA did not affect dechlorination of PCE, cDCE, VC, and 1,2-DCP. In sediment-free enrichments obtained from the EA culture, Dehalogenimonas 16S rRNA gene copy numbers decreased 1-3 orders of magnitude likely due to an inhibitory effect of chloroethenes. Dechlorination with and without competitive inhibition fit Michaelis-Menten kinetics and confirmed the inhibitory effect of chloroethenes and 1,2-DCP on 1,2-DCA dechlorination. This study reinforces that the type of chlorinated substrate drives the selection of specific OHRB, and indicates that removal of chloroethenes and in particular cDCE might be necessary before effective removal of 1,2-DCA at sites contaminated with mixed chlorinated solvents.

Organohalide respiration in pristine environments: implications for the natural halogen cycle.

Halogenated organic compounds, also termed organohalogens, were initially considered to be of almost exclusively anthropogenic origin. However, over 5000 naturally synthesized organohalogens are known today. This has also fuelled the hypothesis that the natural and ancient origin of organohalogens could have primed development of metabolic machineries for their degradation, especially in microorganisms. Among these, a special group of anaerobic microorganisms was discovered that could conserve energy by reducing organohalogens as terminal electron acceptor in a process termed organohalide respiration. Originally discovered in a quest for biodegradation of anthropogenic organohalogens, these organohalide-respiring bacteria (OHRB) were soon found to reside in pristine environments, such as the deep subseafloor and Arctic tundra soil with limited/no connections to anthropogenic activities. As such, accumulating evidence suggests an important role of OHRB in local natural halogen cycles, presumably taking advantage of natural organohalogens. In this minireview, we integrate current knowledge regarding the natural origin and occurrence of industrially important organohalogens and the evolution and spread of OHRB, and describe potential implications for natural halogen and carbon cycles.

Geochemical and microbial community determinants of reductive dechlorination at a site biostimulated with glycerol.

Biostimulation is widely used to enhance reductive dechlorination of chlorinated ethenes in contaminated aquifers. However, the knowledge on corresponding biogeochemical responses is limited. In this study, glycerol was injected in an aquifer contaminated with cis-dichloroethene (cDCE), and geochemical and microbial shifts were followed for 265 days. Consistent with anoxic conditions and sulfate reduction after biostimulation, MiSeq 16S rRNA gene sequencing revealed temporarily increased relative abundance of Firmicutes, Bacteriodetes and sulfate reducing Deltaproteobacteria. In line with 13 C cDCE enrichment and increased Dehalococcoides mccartyi (Dcm) numbers, dechlorination was observed toward the end of the field experiment, albeit being incomplete with accumulation of vinyl chloride. This was concurrent with (i) decreased concentrations of dissolved organic carbon (DOC), reduced relative abundances of fermenting and sulfate reducing bacteria that have been suggested to promote Dcm growth by providing electron donor (H2 ) and essential corrinoid cofactors, (ii) increased sulfate concentration and increased relative abundance of Epsilonproteobacteria and Deferribacteres as putative oxidizers of reduced sulfur compounds. Strong correlations of DOC, relative abundance of fermenters and sulfate reducers, and dechlorination imply the importance of syntrophic interactions to sustain robust dechlorination. Tracking microbial and environmental parameters that promote/preclude enhanced reductive dechlorination should aid development of sustainable bioremediation strategies.

Systematics of haloarchaea and biotechnological potential of their hydrolytic enzymes.

Halophilic archaea, also referred to as haloarchaea, dominate hypersaline environments. To survive under such extreme conditions, haloarchaea and their enzymes have evolved to function optimally in environments with high salt concentrations and, sometimes, with extreme pH and temperatures. These features make haloarchaea attractive sources of a wide variety of biotechnological products, such as hydrolytic enzymes, with numerous potential applications in biotechnology. The unique trait of haloarchaeal enzymes, haloenzymes, to sustain activity under hypersaline conditions has extended the range of already-available biocatalysts and industrial processes in which high salt concentrations inhibit the activity of regular enzymes. In addition to their halostable properties, haloenzymes can also withstand other conditions such as extreme pH and temperature. In spite of these benefits, the industrial potential of these natural catalysts remains largely unexplored, with only a few characterized extracellular hydrolases. Because of the applied impact of haloarchaea and their specific ability to live in the presence of high salt concentrations, studies on their systematics have intensified in recent years, identifying many new genera and species. This review summarizes the current status of the haloarchaeal genera and species, and discusses the properties of haloenzymes and their potential industrial applications.

Concurrent Haloalkanoate Degradation and Chlorate Reduction by Pseudomonas chloritidismutans AW-1T.

Haloalkanoates are environmental pollutants that can be degraded aerobically by microorganisms producing hydrolytic dehalogenases. However, there is a lack of information about the anaerobic degradation of haloalkanoates. Genome analysis of Pseudomonas chloritidismutans AW-1T, a facultative anaerobic chlorate-reducing bacterium, showed the presence of two putative haloacid dehalogenase genes, the l-DEX gene and dehI, encoding an l-2-haloacid dehalogenase (l-DEX) and a halocarboxylic acid dehydrogenase (DehI), respectively. Hence, we studied the concurrent degradation of haloalkanoates and chlorate as a yet-unexplored trait of strain AW-1T The deduced amino acid sequences of l-DEX and DehI revealed 33 to 37% and 26 to 86% identities with biochemically/structurally characterized l-DEX and the d- and dl-2-haloacid dehalogenase enzymes, respectively. Physiological experiments confirmed that strain AW-1T can grow on chloroacetate, bromoacetate, and both l- and d-α-halogenated propionates with chlorate as an electron acceptor. Interestingly, growth and haloalkanoate degradation were generally faster with chlorate as an electron acceptor than with oxygen as an electron acceptor. In line with this, analyses of l-DEX and DehI dehalogenase activities using cell-free extract (CFE) of strain AW-1T grown on dl-2-chloropropionate under chlorate-reducing conditions showed up to 3.5-fold higher dehalogenase activity than the CFE obtained from AW-1T cells grown on dl-2-chloropropionate under aerobic conditions. Reverse transcription-quantitative PCR showed that the l-DEX gene was expressed constitutively independently of the electron donor (haloalkanoates or acetate) or acceptor (chlorate or oxygen), whereas the expression of dehI was induced by haloalkanoates. Concurrent degradation of organic and inorganic halogenated compounds by strain AW-1T represents a unique metabolic capacity in a single bacterium, providing a new piece of the puzzle of the microbial halogen cycle.IMPORTANCE Halogenated organic and inorganic compounds are important environmental pollutants that have carcinogenic and genotoxic effects on both animals and humans. Previous research studied the degradation of organic and inorganic halogenated compounds separately but not concurrently. This study shows concurrent degradation of halogenated alkanoates and chlorate as an electron donor and acceptor, respectively, coupled to growth in a single bacterium, Pseudomonas chloritidismutans AW-1T Hence, besides biogenesis of molecular oxygen from chlorate reduction enabling a distinctive placement of strain AW-1T between aerobic and anaerobic microorganisms, we can now add another unique metabolic potential of this bacterium to the roster. The degradation of different halogenated compounds under anoxic conditions by a single bacterium is also of interest for the natural halogen cycle in different aquatic and terrestrial ecosystems where ample natural production of halogenated compounds has been documented.

Geochemical Parameters and Reductive Dechlorination Determine Aerobic Cometabolic vs Aerobic Metabolic Vinyl Chloride Biodegradation at Oxic/Anoxic Interface of Hyporheic Zones.

Hyporheic zones mediate vinyl chloride (VC) biodegradation in groundwater discharging into surface waters. At the oxic/anoxic interface (OAI) of hyporheic zones subjected to redox oscillations, VC is degraded via coexisting aerobic ethenotrophic and anaerobic reductive dechlorination pathways. However, the identity of aerobic VC degradation pathways (cometabolic vs metabolic) and their interactions with reductive dechlorination in relation to riverbed sediment geochemistry remain ill-defined. We addressed this using microcosms containing OAI sediments incubated under fluctuating oxic/anoxic atmosphere. Under oxic atmosphere, aerobic metabolic VC oxidation was absent in sediments with high total organic carbon (TOC) and VC was reductively dechlorinated to ethene. Ethene was oxidized by ethenotrophs that can degrade VC cometabolically. Contrastingly, VC was metabolically oxidized by ethenotrophs in low-TOC sediments with low reductive dechlorination potential. Accordingly, enrichment and isolation of metabolic VC-oxidizing ethenotrophs was successful only from the low-TOC sediment. Sequence analysis of etnE genes from the microcosms as well phylogenetic typing of the isolates showed that ethenotrophs in the sediments were facultative anaerobic Proteobacteria capable of coping with OAI-associated redox fluctuations. Our results suggest that local sediment heterogeneity supports/selects divergent VC degradation processes at the OAI and that high reductive dechlorination potential suppresses development of aerobic metabolic VC oxidation potential.

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.

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.

Evaluation of solid polymeric organic materials for use in bioreactive sediment capping to stimulate the degradation of chlorinated aliphatic hydrocarbons.

In situ bioreactive capping is a promising technology for mitigation of surface water contamination by discharging polluted groundwater. Organohalide respiration (OHR) of chlorinated ethenes in bioreactive caps can be stimulated through incorporation of solid polymeric organic materials (SPOMs) that provide a sustainable electron source for organohalide respiring bacteria. In this study, wood chips, hay, straw, tree bark and shrimp waste, were assessed for their long term applicability as an electron donor for OHR of cis-dichloroethene (cDCE) and vinyl chloride (VC) in sediment microcosms. The initial release of fermentation products, such as acetate, propionate and butyrate led to the onset of extensive methane production especially in microcosms amended with shrimp waste, straw and hay, while no considerable stimulation of VC dechlorination was obtained in any of the SPOM amended microcosms. However, in the longer term, short chain fatty acids accumulation decreased as well as methanogenesis, whereas high dechlorination rates of VC and cDCE were established with concomitant increase of Dehalococcoides mccartyi and vcrA and bvcA gene numbers both in the sediment and on the SPOMs. A numeric simulation indicated that a capping layer of 40 cm with hay, straw, tree bark or shrimp waste is suffice to reduce the groundwater VC concentration below the threshold level of 5 µg/l before discharging into the Zenne River, Belgium. Of all SPOMs, the persistent colonization of tree bark by D. mccartyi combined with the lowest stimulation of methanogenesis singled out tree bark as a long-term electron donor for OHR of cDCE/VC in bioreactive caps.

Organohalide respiration potential in marine sediments from Aarhus Bay.

Organohalide respiration (OHR), catalysed by reductive dehalogenases (RDases), plays an important role in halogen cycling. Natural organohalides and putative RDase-encoding genes have been reported in Aarhus Bay sediments, however, OHR has not been experimentally verified. Here we show that sediments of Aarhus Bay can dehalogenate a range of organohalides, and different organohalides differentially affected microbial community compositions. PCE-dechlorinating cultures were further examined by 16S rRNA gene-targeted quantitative PCR and amplicon sequencing. Known organohalide-respiring bacteria (OHRB) including Dehalococcoides, Dehalobacter and Desulfitobacterium decreased in abundance during transfers and serial dilutions, suggesting the importance of yet uncharacterized OHRB in these cultures. Switching from PCE to 2,6-DBP led to its complete debromination to phenol in cultures with and without sulfate. 2,6-DBP debrominating cultures differed in microbial composition from PCE-dechlorinating cultures. Desulfobacterota genera recently verified to include OHRB, including Desulfovibrio and Desulfuromusa, were enriched in all microcosms, whereas Halodesulfovibrio was only enriched in cultures without sulfate. Hydrogen and methane were detected in cultures without sulfate. Hydrogen likely served as electron donor for OHR and methanogenesis. This study shows that OHR can occur in marine environments mediated by yet unknown OHRB, suggesting their role in natural halogen cycling.

Inter-species Metabolic Interactions in an In-vitro Minimal Human Gut Microbiome of Core Bacteria.

Knowledge of the functional roles and interspecies interactions are crucial for improving our understanding of the human intestinal microbiome in health and disease. However, the complexity of the human intestinal microbiome and technical challenges in investigating it pose major challenges. In this proof-of-concept study, we rationally designed, assembled and experimentally tested a synthetic Diet-based Minimal Microbiome (Db-MM) consisting of ten core intestinal bacterial species that together are capable of efficiently converting dietary fibres into short chain fatty acids (SCFAs). Despite their genomic potential for metabolic competition, all ten bacteria coexisted during growth on a mixture of dietary fibres, including pectin, inulin, xylan, cellobiose and starch. By integrated analyses of metabolite production, community composition and metatranscriptomics-based gene expression data, we identified interspecies metabolic interactions leading to production of key SCFAs such as butyrate and propionate. While public goods, such as sugars liberated from colonic fibres, are harvested by non-degraders, some species thrive by cross-feeding on energetically challenging substrates, including the butyrogenic conversion of acetate and lactate. Using a reductionist approach in an in-vitro system combined with functional measurements, our study provides key insights into the complex interspecies metabolic interactions between core intestinal bacterial species.

Selective pressure on microbial communities in a drinking water aquifer - Geochemical parameters vs. micropollutants.

Groundwater quality is crucial for drinking water production, but groundwater resources are increasingly threatened by contamination with pesticides. As pesticides often occur at micropollutant concentrations, they are unattractive carbon sources for microorganisms and typically remain recalcitrant. Exploring microbial communities in aquifers used for drinking water production is an essential first step towards understanding the fate of micropollutants in groundwater. In this study, we investigated the interaction between groundwater geochemistry, pesticide presence, and microbial communities in an aquifer used for drinking water production. Two groundwater monitoring wells in The Netherlands were sampled in 2014, 2015, and 2016. In both wells, water was sampled from five discrete depths ranging from 13 to 54 m and was analyzed for geochemical parameters, pesticide concentrations and microbial community composition using 16S rRNA gene sequencing and qPCR. Groundwater geochemistry was stable throughout the study period and pesticides were heterogeneously distributed at low concentrations (µg L-1 range). Microbial community composition was also stable throughout the sampling period. Integration of a unique dataset of chemical and microbial data showed that geochemical parameters and to a lesser extent pesticides exerted selective pressure on microbial communities. Microbial communities in both wells showed similar composition in the deeper aquifer, where pumping results in horizontal flow. This study provides insight into groundwater parameters that shape microbial community composition. This information can contribute to the future implementation of remediation technologies to guarantee safe drinking water production.

Proteogenomic analysis of Georgfuchsia toluolica revealed unexpected concurrent aerobic and anaerobic toluene degradation.

Denitrifying Betaproteobacteria play a key role in the anaerobic degradation of monoaromatic hydrocarbons. We performed a multi-omics study to better understand the metabolism of the representative organism Georgfuchsia toluolica strain G5G6 known as a strict anaerobe coupling toluene oxidation with dissimilatory nitrate and Fe(III) reduction. Despite the genomic potential for degradation of different carbon sources, we did not find sugar or organic acid transporters, in line with the inability of strain G5G6 to use these substrates. Using a proteomics analysis, we detected proteins of fumarate-dependent toluene activation, membrane-bound nitrate reductase, and key components of the metal-reducing (Mtr) pathway under both nitrate- and Fe(III)-reducing conditions. High abundance of the multiheme cytochrome MtrC implied that a porin-cytochrome complex was used for respiratory Fe(III) reduction. Remarkably, strain G5G6 contains a full set of genes for aerobic toluene degradation, and we detected enzymes of aerobic toluene degradation under both nitrate- and Fe(III)-reducing conditions. We further detected an ATP-dependent benzoyl-CoA reductase, reactive oxygen species detoxification proteins, and cytochrome c oxidase indicating a facultative anaerobic lifestyle of strain G5G6. Correspondingly, we found diffusion through the septa a substantial source of oxygen in the cultures enabling concurrent aerobic and anaerobic toluene degradation by strain G5G6.

High biodiversity in a benzene-degrading nitrate-reducing culture is sustained by a few primary consumers.

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and energy source and nitrate as the electron acceptor. Using genome-centric metagenomics plus metatranscriptomics, we demonstrate that most of the community members likely feed on metabolic left-overs or on necromass while only a few of them, from families Rhodocyclaceae and Peptococcaceae, are candidates to degrade benzene. We verify with an additional succession experiment using metabolomics and metabarcoding that these few community members are the actual drivers of benzene degradation. As such, we hypothesize that high species richness is maintained and the complexity of a natural community is stabilized in a controlled environment by the interdependencies between the few benzene degraders and the rest of the community members, ultimately resulting in a food web with different trophic levels.

Metagenomic- and Cultivation-Based Exploration of Anaerobic Chloroform Biotransformation in Hypersaline Sediments as Natural Source of Chloromethanes.

Chloroform (CF) is an environmental contaminant that can be naturally formed in various environments ranging from forest soils to salt lakes. Here we investigated CF removal potential in sediments obtained from hypersaline lakes in Western Australia. Reductive dechlorination of CF to dichloromethane (DCM) was observed in enrichment cultures derived from sediments of Lake Strawbridge, which has been reported as a natural source of CF. No CF removal was observed in abiotic control cultures without artificial electron donors, indicating biotic CF dechlorination in the enrichment cultures. Increasing vitamin B12 concentration from 0.04 to 4 µM in enrichment cultures enhanced CF removal and reduced DCM formation. In cultures amended with 4 µM vitamin B12 and 13C labelled CF, formation of 13CO2 was detected. Known organohalide-respiring bacteria and reductive dehalogenase genes were neither detected using quantitative PCR nor metagenomic analysis of the enrichment cultures. Rather, members of the order Clostridiales, known to co-metabolically transform CF to DCM and CO2, were detected. Accordingly, metagenome-assembled genomes of Clostridiales encoded enzymatic repertoires for the Wood-Ljungdahl pathway and cobalamin biosynthesis, which are known to be involved in fortuitous and nonspecific CF transformation. This study indicates that hypersaline lake microbiomes may act as a filter to reduce CF emission to the atmosphere.

Long-Chain Fatty Acids Degradation by Desulfomonile Species and Proposal of "Candidatus Desulfomonile Palmitatoxidans".

Microbial communities with the ability to convert long-chain fatty acids (LCFA) coupled to sulfate reduction can be important in the removal of these compounds from wastewater. In this work, an enrichment culture, able to oxidize the long-chain fatty acid palmitate (C16 : 0) coupled to sulfate reduction, was obtained from anaerobic granular sludge. Microscopic analysis of this culture, designated HP culture, revealed that it was mainly composed of one morphotype with a typical collar-like cell wall invagination, a distinct morphological feature of the Desulfomonile genus. 16S rRNA gene amplicon and metagenome-assembled genome (MAG) indeed confirmed that the abundant phylotype in HP culture belong to Desulfomonile genus [ca. 92% 16S rRNA gene sequences closely related to Desulfomonile spp.; and ca. 82% whole genome shotgun (WGS)]. Based on similar cell morphology and average nucleotide identity (ANI) (77%) between the Desulfomonile sp. in HP culture and the type strain Desulfomonile tiedjei strain DCB-1T, we propose a novel species designated as "Candidatus Desulfomonile palmitatoxidans." This bacterium shares 94.3 and 93.6% 16S rRNA gene identity with Desulfomonile limimaris strain DCB-MT and D. tiedjei strain DCB-1T, respectively. Based on sequence abundance of Desulfomonile-morphotype in HP culture, its predominance in the microscopic observations, and presence of several genes coding for enzymes involved in LCFA degradation, the proposed species "Ca. Desulfomonile palmitatoxidans" most probably plays an important role in palmitate degradation in HP culture. Analysis of the growth of HP culture and D. tiedjei strain DCB-1T with short- (butyrate), medium- (caprylate) and long-chain fatty acids (palmitate, stearate, and oleate) showed that both cultures degraded all fatty acids coupled to sulfate reduction, except oleate that was only utilized by HP culture. In the absence of sulfate, neither HP culture, nor D. tiedjei strain DCB-1T degraded palmitate when incubated with Methanobacterium formicicum as a possible methanogenic syntrophic partner. Unlike D. tiedjei strain DCB-1T, "Ca. Desulfomonile palmitatoxidans" lacks reductive dehalogenase genes in its genome, and HP culture was not able to grow by organohalide respiration. An emended description of the genus Desulfomonile is proposed. Our study reveals an unrecognized LCFA degradation feature of the Desulfomonile genus.

Organohalide-respiring Desulfoluna species isolated from marine environments.

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.

Discovered by genomics: putative reductive dehalogenases with N-terminus transmembrane helixes.

Attempts for bioremediation of toxic organohalogens resulted in the identification of organohalide-respiring bacteria harbouring reductive dehalogenases (RDases) enzymes. RDases consist of the catalytic subunit (RdhA, encoded by rdhA) that does not have membrane-integral domains, and a small putative membrane anchor (RdhB, encoded by rdhB) that (presumably) locates the A subunit to the outside of the cytoplasmic membrane. Recent genomic studies identified a putative rdh gene in an uncultured deltaproteobacterial genome that was not accompanied by an rdhB gene, but contained transmembrane helixes in N-terminus. Therefore, rather than having a separate membrane anchor protein, this putative RDase is likely a hybrid of RdhA and RdhB, and directly connected to the membrane with transmembrane helixes. However, functionality of the hybrid putative RDase remains unknown. Further analysis showed that the hybrid putative rdh genes are present in the genomes of pure cultures and uncultured members of Bacteriodetes and Deltaproteobacteria, but also in the genomes of the candidate divisions. The encoded hybrid putative RDases have cytoplasmic or exoplasmic C-terminus localization, and cluster phylogenetically separately from the existing RDase groups. With increasing availability of (meta)genomes, more diverse and likely novel rdh genes are expected, but questions regarding their functionality and ecological roles remain open.

Influence of different redox conditions and dissolved organic matter on pesticide biodegradation in simulated groundwater systems.

Insights into the influence of redox conditions, that is the availability of electron acceptors, and dissolved organic matter (DOM) on pesticide biodegradation in groundwater are key to understanding the environmental fate of pesticides in natural groundwater systems. Here, the influence of redox conditions and supplemental DOM addition on biodegradation of pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,6-dichlorobenzamide (BAM), mecoprop-p (MCPP) and bentazone, was tested in microcosm and subsequent column experiments. Pesticide degradation, functional genes and changes in specific fractions and quantity of DOM were systematically quantified. In aerobic microcosm experiments, the highest 2,4-D degradation rate was obtained with the presence of more assimilable DOM. In column experiments, minimal pesticide degradation (≤33.77%) in any anaerobic redox conditions was observed in the absence of DOM. However, in the presence of DOM, 2,4-D biodegradation was considerably enhanced under nitrate-reducing conditions (from 23.5 ±â€¯10.2% to 82.3 ±â€¯11.6%) and in a column without external electron acceptor amendment (from -6.3 ±â€¯12.6% to 31.1 ±â€¯36.3%). Observed preferential depletion of the fulvic acid fraction of DOM provides indications for specific functional DOM properties. The qPCR results show an increase in microbial biomass and functional genes (tfdA) in liquid phase after DOM addition. The results of this work provide insights into the interplays among DOM, redox geochemistry, and pesticide biodegradation, and show the potential of a novel approach - DOM addition to groundwater systems - for in situ biostimulation technology to remove pesticides from groundwater systems.