Microbiological versus Chemical Reductive Sulfidation: An Experimental and Theoretical Study.

Microbiological reductive sulfidation (RS) has rarely been documented, although it represents an efficient strategy for thiol formation. In this work, we reported on the sulfate-respiring bacterium Desulfovibrio sp.86 that has previously demonstrated RS activity toward the pesticide chlordecone. The purpose of this study was to assess its substrate versatility using a set of 28 carbonyls, to compare with chemical RS and to rationalize the observed trends using a dual experimental and theoretical approach. The chemical RS generally proceeds in two steps (S/O exchange using a sulfur donor like P4S10, reduction of the thione intermediate). Intriguingly, chlordecone was found to be converted into chlordecthiol following the first step. Hence, we designed a protocol and applied it to the 28 substrates to assess their propensity to be directly converted into thiols with the P4S10 treatment alone. Finally, we performed density functional theory calculations on these carbonyls and their thiocarbonyl derivatives to build a set of structural, electronic, and thermodynamic parameters. The results showed that chemical and microbiological RS probably involved two distinct mechanisms. Chemically, we observed that several carbonyls, possessing electron-withdrawing groups and/or aromatic rings, were directly transformed into thiols in the presence of P4S10. The correlation obtained with the electron affinity of the thiones led us to conclude that a probable single-electron reductive transfer occurred during the first step. We also found that Desulfovibrio sp.86 transformed a variety of aldehydes and ketones, without ever detecting thiones. No significant correlation was observed with the calculated parameters, but a relationship between aldehyde RS biotransformation and bacterial growth was observed. Differences in selectivity with chemical RS open the way for further applications in organic synthesis.

Genetic Analysis of Citrobacter sp.86 Reveals Involvement of Corrinoids in Chlordecone and Lindane Biotransformations.

Chlordecone (Kepone®) and γ-hexachlorocyclohexane (γ-HCH or lindane) have been used for decades in the French West Indies (FWI) resulting in long-term soil and water pollution. In a previous work, we have identified a new Citrobacter species (sp.86) that is able to transform chlordecone into numerous products under anaerobic conditions. No homologs to known reductive dehalogenases or other candidate genes were found in the genome sequence of Citrobacter sp.86. However, a complete anaerobic pathway for cobalamin biosynthesis was identified. In this study, we investigated whether cobalamin or intermediates of cobalamin biosynthesis was required for chlordecone microbiological transformation. For this purpose, we constructed a set of four Citrobacter sp.86 mutant strains defective in several genes belonging to the anaerobic cobalamin biosynthesis pathway. We monitored chlordecone and its transformation products (TPs) during long-term incubation in liquid cultures under anaerobic conditions. Chlordecone TPs were detected in the case of cobalamin-producing Citrobacter sp.86 wild-type strain but also in the case of mutants able to produce corrinoids devoid of lower ligand. In contrast, mutants unable to insert the cobalt atom in precorrin-2 did not induce any transformation of chlordecone. In addition, it was found that lindane, previously shown to be anaerobically transformed by Citrobacter freundii without evidence of a mechanism, was also degraded in the presence of the wild-type strain of Citrobacter sp.86. The lindane degradation abilities of the various Citrobacter sp.86 mutant strains paralleled chlordecone transformation. The present study shows the involvement of cobalt-containing corrinoids in the microbial degradation of chlorinated compounds with different chemical structures. Their increased production in contaminated environments could accelerate the decontamination processes.

Transformation of the recalcitrant pesticide chlordecone by Desulfovibrio sp.86 with a switch from ring-opening dechlorination to reductive sulfidation activity.

The insecticide chlordecone has been used in the French West Indies for decades, resulting in long term pollution, human health problems and social crisis. In addition to bacterial consortia and Citrobacter sp.86 previously described to transform chlordecone into three families of transformation products (A: hydrochlordecones, B: polychloroindenes and C: polychloroindenecarboxylic acids), another bacterium Desulfovibrio sp.86, showing the same abilities has been isolated and its genome was sequenced. Ring-opening dechlorination, leading to A, B and C families, was observed as previously described. Changing operating conditions in the presence of chlordecone gave rise to the formation of an unknown sulfur-containing transformation product instead of the aforementioned ones. Its structural elucidation enabled to conclude to a thiol derivative, which corresponds to an undocumented bacterial reductive sulfidation. Microbial experiments pointed out that the chlordecone thiol derivative was observed in anaerobiosis, and required the presence of an electron acceptor containing sulfur or hydrogen sulfide, in a confined atmosphere. It seems that this new reaction is also active on hydrochlordecones, as the 10-monohydrochlordecone A1 was transformed the same way. Moreover, the chlordecone thiol derivative called F1 was detected in several chlordecone contaminated mangrove bed sediments from Martinique Island, highlighting the environmental relevance of these results.

Natural Chlordecone Degradation Revealed by Numerous Transformation Products Characterized in Key French West Indies Environmental Compartments.

Production and use of the insecticide chlordecone has caused long-term environmental pollution in the James River area and the French West Indies (FWI) that has resulted in acute human-health problems and a social crisis. High levels of chlordecone in FWI soils, even after its ban decades ago, and the absence of detection of transformation products (TPs), have suggested that chlordecone is virtually nonbiodegradable in the environment. Here, we investigated laboratory biodegradation, consisting of bacterial liquid cultures and microcosms inoculated with FWI soils, using a dual nontargeted GC-MS and LC-HRMS approach. In addition to previously reported, partly characterized hydrochlordecones and polychloroindenes (families A and B), we discovered 14 new chlordecone TPs, assigned to four families (B, C, D, and E). Organic synthesis and NMR analyses allowed us to achieve the complete structural elucidation of 19 TPs. Members of TP families A, B, C, and E were detected in soil, sediment, and water samples from Martinique and include 17 TPs not initially found in commercial chlordecone formulations. 2,4,5,6,7-Pentachloroindene was the most prominent TP, with levels similar to those of chlordecone. Overall, our results clearly show that chlordecone pollution extends beyond the parent chlordecone molecule and includes a considerable number of previously undetected TPs. Structural diversity of the identified TPs illustrates the complexity of chlordecone degradation in the environment and raises the possibility of extensive worldwide pollution of soil and aquatic ecosystems by chlordecone TPs.

Microbial Degradation of a Recalcitrant Pesticide: Chlordecone.

Chlordecone (Kepone®) is a synthetic organochlorine insecticide (C10Cl10O) used worldwide mostly during the 1970 and 1980s. Its intensive application in the French West Indies to control the banana black weevil Cosmopolites sordidus led to a massive environmental pollution. Persistence of chlordecone in soils and water for numerous decades even centuries causes global public health and socio-economic concerns. In order to investigate the biodegradability of chlordecone, microbial enrichment cultures from soils contaminated by chlordecone or other organochlorines and from sludge of a wastewater treatment plant have been conducted. Different experimental procedures including original microcosms were carried out anaerobically over long periods of time. GC-MS monitoring resulted in the detection of chlorinated derivatives in several cultures, consistent with chlordecone biotransformation. More interestingly, disappearance of chlordecone (50 µg/mL) in two bacterial consortia was concomitant with the accumulation of a major metabolite of formula C9Cl5H3 (named B1) as well as two minor metabolites C10Cl9HO (named A1) and C9Cl4H4 (named B3). Finally, we report the isolation and the complete genomic sequences of two new Citrobacter isolates, closely related to Citrobacter amalonaticus, and that were capable of reproducing chlordecone transformation. Further characterization of these Citrobacter strains should yield deeper insights into the mechanisms involved in this transformation process.

Bacterial structure and spatiotemporal distribution in a horizontal subsurface flow constructed wetland.

In this study, bacterial community structure in a horizontal subsurface flow constructed wetland (HSF-CW) planted with Phragmites australis was investigated using the 16S rRNA cloning-sequencing technique. Two layer depths were considered: the rhizosphere zone (RH) and the deep-layer zone (DL) in different sampling periods. Bacteria-specific primers 008F and 1492R were used to amplify the 16S rRNA genes and construct six clone libraries. A total of 1,284 individual sequences were used to assess the HSF-CW diversity. Phylogenetic analysis of RH and DL clone libraries shows that 41.57 and 42.17 % of the 16S rRNA sequences are affiliated with the Proteobacteria in the RH and the DL, respectively. The remaining major phylogenetic groups are Bacteroidetes, Planctomycetes, and Chloroflexi with 11.78, 9.36, and 7.6 %, respectively, in the RH and 11.38, 6.48, and 7.65 % in the DL, respectively. Minor divisions such as Verrucomicrobia, TM7, Nitrospira, and Gemmatimonadetes represented <6 % of the total sequences, while 14.2 % were unidentified Bacteria. Among the Proteobacteria, the Alphaproteobacteria subclass is represented in both locations, while the Deltaproteobacteria and Gammaproteobacteria subclasses were predominant in the RH and the DL, respectively. Results suggest that Archaea and Bacteria in the HSF-CW are the essential actors in the nitrogen cycle and that the established microbial community is efficient in nitrogen removal from wastewater.

Characterization of rhizosphere prokaryotic diversity in a horizontal subsurface flow constructed wetland using a PCR cloning-sequencing based approach.

Performance of biological wastewater treatment systems may be related to the composition and activity of microbial populations they contain. However, little information is known regarding microbial community inhabiting these ecosystems. The purpose of this study was to investigate archaeal and bacterial diversity, using cultivation-independent molecular techniques, in a constructed wetland receiving domestic wastewater. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by PCR using primers specific for archaeal and bacterial domains. A high microbial diversity was detected. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 31.3 % of the OTUs, followed by the Bacteroidetes (14.8 %), Planctomycetales (13.8 %), Actinobacteria (12 %), and Chloroflexi (8.2 %). Sequences affiliated with minor phylogenetic divisions such as the TM7, Nitrospira, OP10, and BRC1 are represented by <6 % of total OTUs. The Archaea domain was represented by the Thaumarchaeota phylum dominated by the Candidatus Nitrososphaera genus.

The structure and spatio-temporal distribution of the Archaea in a horizontal subsurface flow constructed wetland.

In this study, archaeal community structure and temporal dynamics were monitored, using 16S rRNA clone libraries construction from a horizontal subsurface flow constructed wetland. Phylogenetic assignation of 1026 16S rRNA gene sequences shows that 96.2% of the total operational taxonomic units (OTUs) were affiliated with Thaumarchaeota, a newly proposed archaeal phylum and 3.7% with unclassified Archaea. Among the total sequences, 42% and 40.2% were affiliated with Candidatus Nitrososphaera and unclassified Nitrosopumilus respectively with more than 99% similarity. Results suggest that several dominant and active nitrifiers may benefit from the micro-aerobic conditions around the reed roots to perform ammonia oxidation. The archaeal diversity detected in the rhizosphere zone is clearly different from that detected in the bottom basin. This engineered habitat revealed the reed root and the water composition effects on the archaeal diversity.

Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence.

BACKGROUND: Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed. RESULTS: C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms. CONCLUSIONS: Analysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes.

Towards the definition of a core of microorganisms involved in anaerobic digestion of sludge.

The microbial consortium involved in anaerobic digestion has not yet been precisely characterized and this process remains a 'black box' with limited efficiency. In this study, seven anaerobic sludge digesters were selected based on technology, type of sludge, process and water quality. The prokaryotic community of these digesters was examined by constructing and analysing a total of 9890 16S rRNA gene clones. Libraries were constructed using primers specific for the Bacteria and Archaea domains for each digester, respectively. After phylogenetic affiliation, the libraries were compared using statistical tools to determine the similarities or differences among the seven digesters. Results show that the prokaryotic community of an anaerobic digester is composed of phylotypes commonly found in all anaerobic digesters sampled and also of specific phylotypes. The Archaea community is represented by an equilibrium among a restricted number of operational taxonomic units (OTUs). These OTUs are affiliated with Methanosarcinales, Methanomicrobiales and Arc I phylogenetic groups. Statistical analysis revealed that the Bacteria community can be described as a three component model: one-third making up a core group of phylotypes common to most of the digesters, one-third are phylotypes shared among a few digesters and another one-third are specific phylotypes. The core group is composed of only six OTUs affiliated with Chloroflexi, Betaproteobacteria, Bacteroidetes and Synergistetes. Its role in anaerobic degradation appears critical to investigate. This comparison of anaerobic digester populations is a first step towards a future understanding of the relationship among biodiversity, operating conditions and digester efficiency.

Cloacibacillus evryensis gen. nov., sp. nov., a novel asaccharolytic, mesophilic, amino-acid-degrading bacterium within the phylum 'Synergistetes', isolated from an anaerobic sludge digester.

A novel anaerobic, mesophilic, amino-acid-utilizing bacterium, strain 158T, was isolated from an anaerobic digester of a wastewater treatment plant. Cells of strain 158T were non-motile, rod-shaped (2.0-3.0 x 0.8-1.0 microm) and stained Gram-negative. Optimal growth occurred at 37 degrees C and pH 7.0 in an anaerobic basal medium containing 1 % Casamino acids. Strain 158T fermented arginine, histidine, lysine and serine and showed growth on yeast extract, brain-heart infusion (BHI) medium and tryptone, but not on carbohydrates, organic acids or alcohols. The end products of degradation were: acetate, butyrate, H2 and CO2 from arginine; acetate, propionate, butyrate, H2 and CO2 from lysine; and acetate, propionate, butyrate, valerate, H2 and CO2 from histidine, serine, BHI medium, Casamino acids and tryptone. The DNA G+C content was 55.8 mol%. The 16S rRNA gene sequence of strain 158T showed only 92.6 % sequence similarity with that of Synergistes jonesii, the only described species of the 'Synergistes' group. The major cellular fatty acids were iso-C(15:0) (16.63 %), iso-C(15:0) 3-OH (12.41 %) and C(17:1)omega6c (9.46 %) and the polar fatty acids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylamine; these fatty acid profiles did not resemble those of any recognized bacterial species. Due to the considerable differences in genotypic, phenotypic and phylogenetic characteristics between strain 158T and those of its nearest relative, it is proposed that strain 158T represents a novel species in a new genus, Cloacibacillus evryensis gen. nov., sp. nov., in the phylum 'Synergistetes'. The type strain is 158T (=DSM 19522T=JCM 14828T).