Higher thermal remediation temperature facilitates the sequential bioaugmented reductive dechlorination.

The coupling of thermal remediation with microbial reductive dechlorination (MRD) has shown promising potential for the cleanup of chlorinated solvent contaminated sites. In this study, thermal treatment and bioaugmentation were applied in series, where prior higher thermal remediation temperature led to improved TCE dechlorination performance with both better organohalide-respiring bacteria (OHRB) colonization and electron donor availability. The 60 °C was found to be a key temperature point where the promotion effect became obvious. Amplicon sequencing and co-occurrence network analysis demonstrated that temperature was a more dominating factor than bioaugmentation that impacted microbial community structure. Higher temperature of prior thermal treatment resulted in the decrease of richness, diversity of indigenous microbial communities, and simplified the network structure, which benefited the build-up of newcoming microorganisms during bioaugmentation. Thus, the abundance of Desulfitobacterium increased from 0.11 % (25 °C) to 3.10 % (90 °C). Meanwhile, released volatile fatty acids (VFAs) during thermal remediation functioned as electron donors and boosted MRD. Our results provided temperature-specific information on synergistic effect of sequential thermal remediation and bioaugmentation, which contributed to better implementation of the coupled technologies in chloroethene-impacted sites.

Novel Soil Bacterium Strain Desulfitobacterium sp. PGC-3-9 Detoxifies Trichothecene Mycotoxins in Wheat via De-Epoxidation under Aerobic and Anaerobic Conditions.

Trichothecenes are the most common mycotoxins contaminating small grain cereals worldwide. The C12,13 epoxide group in the trichothecenes was identified as a toxic group posing harm to humans, farm animals, and plants. Aerobic biological de-epoxidation is considered the ideal method of controlling these types of mycotoxins. In this study, we isolated a novel trichothecene mycotoxin-de-epoxidating bacterium, Desulfitobacterium sp. PGC-3-9, from a consortium obtained from the soil of a wheat field known for the occurrence of frequent Fusarium head blight epidemics under aerobic conditions. Along with MMYPF media, a combination of two antibiotics (sulfadiazine and trimethoprim) substantially increased the relative abundance of Desulfitobacterium species from 1.55% (aerobic) to 29.11% (aerobic) and 28.63% (anaerobic). A single colony purified strain, PGC-3-9, was isolated and a 16S rRNA sequencing analysis determined that it was Desulfitobacterium. The PGC-3-9 strain completely de-epoxidated HT-2, deoxynivalenol (DON), nivalenol and 15-acetyl deoxynivalenol, and efficiently eliminated DON in wheat grains under aerobic and anaerobic conditions. The strain PGC-3-9 exhibited high DON de-epoxidation activity at a wide range of pH (6-10) and temperature (15-50 °C) values under both conditions. This strain may be used for the development of detoxification agents in the agriculture and feed industries and the isolation of de-epoxidation enzymes.

Flavodoxin hydroquinone provides electrons for the ATP-dependent reactivation of protein-bound corrinoid cofactors.

Corrinoid-dependent enzyme systems rely on the super-reduced state of the protein-bound corrinoid cofactor to be functional, for example, in methyl transfer reactions. Due to the low redox potential of the [CoII ]/[CoI ] couple, autoxidation of the corrinoid cofactor occurs and leads to the formation of the inactive [CoII ]-state. For the reactivation, which is an energy-demanding process, electrons have to be transferred from a physiological donor to the corrinoid cofactor by the help of a reductive activator protein. In this study, we identified reduced flavodoxin as electron donor for the ATP-dependent reduction of protein-bound corrinoid cofactors of bacterial O-demethylase enzyme systems. Reduced flavodoxin was generated enzymatically using pyruvate:ferredoxin/flavodoxin oxidoreductase rather than hydrogenase. Two of the four flavodoxins identified in Acetobacterium dehalogenans and Desulfitobacterium hafniense DCB-2 were functional in supplying electrons for corrinoid reduction. They exhibited a midpoint potential of about -400 mV (ESHE , pH 7.5) for the semiquinone/hydroquinone transition. Reduced flavodoxin could be replaced by reduced clostridial ferredoxin. It was shown that the low-potential electrons of reduced flavodoxin are first transferred to the iron-sulfur cluster of the reductive activator and finally to the protein-bound corrinoid cofactor. This study further highlights the importance of reduced flavodoxin, which allows maintaining a variety of enzymatic reaction cycles by delivering low-potential electrons.

Mobilization and transformation of arsenic from ternary complex OM-Fe(III)-As(V) in the presence of As(V)-reducing bacteria.

Organic matter (OM) was proved to have a high affinity for arsenic (As) in the presence of ferric iron (Fe(III)), the formed ternary complex OM-Fe(III)-As(V) were frequently studied before; however, the mobilization and transformation of As from OM-Fe(III)-As(V) in the presence of As(V)-reducing bacteria remains unclear. Two different strains (Desulfitobacterium sp. DJ-3, Exiguobacterium sp. DJ-4) were incubated with OM-Fe(III)-As(V) to assess the biotransformation of As and Fe. Results showed that Desulfitobacterium sp. DJ-3 could substantially stimulate the reduction and release of OM-Fe complexed As(V) and resulted in notable As(III) release (30 mg/L). The linear combination fitting result of k3-weighted As K-edge EXAFS spectra showed that 56% of OM-Fe-As(V) was transformed to OM-Fe-As(III) after 144 h. Besides, strain DJ-3 could also reduce OM complexed Fe(III), which lead to the decomposition of ternary complex and the release of 11.8 mg/g Fe(II), this microbial Fe(III) reduction process has resulted in 11% more As liberation from OM-Fe(III)-As(V) than without bacteria. In contrast, Exiguobacterium sp. DJ-4 could only reduce free As(V) but cannot stimulate As release from the complex. Our study provides the first evidence for microbial As reduction and release from ternary complex OM-Fe(III)-As(V), which could be of great importance in As geochemical circulation.

Structures in Tetrahydrofolate Methylation in Desulfitobacterial Glycine Betaine Metabolism at Atomic Resolution.

Enzymes orchestrating methylation between tetrahydrofolate (THF) and cobalamin (Cbl) are abundant among all domains of life. During energy production in Desulfitobacterium hafniense, MtgA catalyzes the methyl transfer from methylcobalamin (Cbl-CH3 ) to THF in the catabolism of glycine betaine (GB). Despite its lack of sequence identity with known structures, we could show that MtgA forms a homodimeric complex of two TIM barrels. Atomic crystallographic insights into the interplay of MtgA with THF as well as analysis of a trapped reaction intermediate (THF-CH3 )+ reveal conformational rearrangements during the transfer reaction. Whereas residues for THF methylation are conserved, the binding mode for the THF glutamyl-p-aminobenzoate moiety (THF tail) is unique. Apart from snapshots of individual reaction steps of MtgA, structure-based mutagenesis combined with enzymatic activity assays allowed a mechanistic description of the methyl transfer between Cbl-CH3 and THF. Altogether, the THF-tail-binding motion observed in MtgA is unique compared to other THF methyltransferases and therefore contributes to the general understanding of THF-mediated methyl transfer.

Engineered in situ biogeochemical transformation as a secondary treatment following ISCO - A field test.

ISCO using activated sodium persulphate is a widely used technology for treating chlorinated solvent source zones. In sensitive areas, however, high groundwater sulphate concentrations following treatment may be a drawback. In situ biogeochemical transformation, a technology that degrades contaminants via reduced iron minerals formed by microbial activity, offers a potential solution for such sites, the bioreduction of sulphate and production of iron sulphides that abiotically degrade chlorinated ethenes acting as a secondary technology following ISCO. This study assesses this approach in the field using hydrochemical and molecular tools, solid phase analysis and geochemical modelling. Following a neutralisation and bioaugmentation, favourable conditions for iron- and sulphate-reducers were created, resulting in a remarkable increase in their relative abundance. The abundance of dechlorinating bacteria (Dehalococcoides mccartyi, Dehalobacter sp. and Desulfitobacterium spp.) remained low throughout this process. The activity of iron- and sulphate-reducers was further stimulated through application of magnetite plus starch and microiron plus starch, resulting in an increase in ferrous iron concentration (from

Isolation, characterization and bioaugmentation of an acidotolerant 1,2-dichloroethane respiring Desulfitobacterium species from a low pH aquifer.

A Desulfitobacterium sp. strain AusDCA of the Peptococcaceae family capable of respiring 1,2-dichloroethane (1,2-DCA) to ethene anaerobically with ethanol or hydrogen as electron donor at pH 5.0 with optimal range between pH 6.5-7.5 was isolated from an acidic aquifer near Sydney, Australia. Strain AusDCA is distant (94% nucleotide identity) from its nearest phylogenetic neighbor, D. metallireducens, and could represent a new species. Reference gene-based quantification of growth indicated a doubling time of 2 days in cultures buffered at pH 7.2, and a yield of 7.66 (± 4.0) × 106 cells µmol-1 of 1,2-DCA. A putative 1,2-DCA reductive dehalogenase was translated from a dcaAB locus and had high amino acid identity (97.3% for DcaA and 100% for DcaB) to RdhA1B1 of the 1,2-DCA respiring Dehalobacter strain WL. Proteomic analysis confirmed DcaA expression in the pure culture. Dehalogenation of 1,2-DCA (1.6 mM) was observed in batch cultures established from groundwater at pH 5.5 collected 38 days after in situ bioaugmentation but not in cultures established with groundwater collected at the same time from wells not receiving bioaugmentation. Overall, strain AusDCA can tolerate lower pH than previously characterized organohalide respiring bacteria and remained viable in groundwater at pH 5.5.

Desulfitobacterium contributes to the microbial transformation of 2,4,5-T by methanogenic enrichment cultures from a Vietnamese active landfill.

The herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was a major component of Agent Orange, which was used as a defoliant in the Vietnam War. Little is known about its degradation under anoxic conditions. Established enrichment cultures using soil from an Agent Orange bioremediation plant in southern Vietnam with pyruvate as potential electron donor and carbon source were shown to degrade 2,4,5-T via ether cleavage to 2,4,5-trichlorophenol (2,4,5-TCP), which was further dechlorinated to 3,4-dichlorophenol. Pyruvate was initially fermented to hydrogen, acetate and propionate. Hydrogen was then used as the direct electron donor for ether cleavage of 2,4,5-T and subsequent dechlorination of 2,4,5-TCP. 16S rRNA gene amplicon sequencing indicated the presence of bacteria and archaea mainly belonging to the Firmicutes, Bacteroidetes, Spirochaetes, Chloroflexi and Euryarchaeota. Desulfitobacterium hafniense was identified as the dechlorinating bacterium. Metaproteomics of the enrichment culture indicated higher protein abundances of 60 protein groups in the presence of 2,4,5-T. A reductive dehalogenase related to RdhA3 of D. hafniense showed the highest fold change, supporting its function in reductive dehalogenation of 2,4,5-TCP. Despite an ether-cleaving enzyme not being detected, the inhibition of ether cleavage but not of dechlorination, by 2-bromoethane sulphonate, suggested that the two reactions are catalysed by different organisms.

Metabolic flexibility of a prospective bioremediator: Desulfitobacterium hafniense Y51 challenged in chemostats.

Desulfitobacterium hafniense Y51 has been widely used in investigations of perchloroethylene (PCE) biodegradation, but limited information exists on its other physiological capabilities. We investigated how D. hafniense Y51 confronts the debilitating limitations of not having enough electron donor (lactate), or electron acceptor (fumarate) during cultivation in chemostats. The residual concentrations of the substrates supplied in excess were much lower than expected. Transcriptomics, proteomics and fluxomics were integrated to investigate how this phenomenon was regulated. Through diverse regulation at both transcriptional and translational levels, strain Y51 turned to fermenting the excess lactate and disproportionating the excess fumarate under fumarate- and lactate-limiting conditions respectively. Genes and proteins related to the utilization of a variety of alternative electron donors and acceptors absent from the medium were induced, apparently involving the Wood-Ljungdahl pathway. Through this metabolic flexibility, D. hafniense Y51 may be able to switch between different metabolic capabilities under limiting conditions.

Purinyl-cobamide is a native prosthetic group of reductive dehalogenases.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon-halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

Variable dual carbon-bromine stable isotope fractionation during enzyme-catalyzed reductive dehalogenation of brominated ethenes.

The potential of compound-specific stable isotope analysis (CSIA) to characterize biotransformation of brominated organic compounds (BOCs) was assessed and compared to chlorinated analogues. Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S catalyzed the dehalogenation of tribromoethene (TBE) to either vinyl bromide (VB) or ethene, respectively. Significantly lower isotope fractionation was observed for TBE dehalogenation by S. multivorans (εC = -1.3 ± 0.2‰) compared to D. hafniense (εC = -7.7 ± 1.5‰). However, higher fractionation was observed for dibromoethene (DBE) dehalogenation by S. multivorans (εC = -16.8 ± 1.8‰ and -21.2 ± 1.6‰ for trans- and cis-1,2- (DBE) respectively), compared to D. hafniense PCE-S (εC = -9.5 ± 1.2‰ and -14.5 ± 0.7‰ for trans-1,2-DBE and cis-1,2-DBE, respectively). Significant, but similar, bromine fractionation was observed for for S. multivorans (εBr = -0.53 ± 0.15‰, -1.03 ± 0.26‰, and -1.18 ± 0.13‰ for trans-1,2-DBE, cis-1,2-DBE and TBE, respectively) and D. hafniense PCE-S (εBr = -0.97 ± 0.28‰, -1.16 ± 0.36‰, and -1.34 ± 0.32‰ for cis-1,2-DBE, TBE and trans-1,2-DBE, respectively). Variable CBr dual-element slopes were estimated at Λ (εC/εBr) = 1.03 ± 0.2, 17.9 ± 5.8, and 29.9 ± 11.0 for S. multivorans debrominating TBE, cis-1,2-DBE and trans-1,2-DBE, respectively, and at 7.14 ± 1.6, 8.27 ± 3.7, and 8.92 ± 2.4 for D. hafniense PCE-S debrominating trans-1,2-DBE, TBE and cis-1,2-DBE, respectively. A high variability in isotope fractionation, which was substrate property related, was observed for S. multivorans but not D. hafniense, similar as observed for chlorinated ethenes, and may be due to rate-limiting steps preceding the bond-cleavage or differences in the reaction mechanism. Overall, significant isotope fractionation was observed and, therefore, CSIA can be applied to monitor the fate of brominated ethenes in the environment. Isotope effects differences, however, are not systematically comparable to chlorinated ethenes.

Use of Bacteria To Stabilize Archaeological Iron.

Iron artifacts are common among the findings of archaeological excavations. The corrosion layer formed on these objects requires stabilization after their recovery, without which the destruction of the item due to physicochemical damage is likely. Current technologies for stabilizing the corrosion layer are lengthy and generate hazardous waste products. Therefore, there is a pressing need for an alternative method for stabilizing the corrosion layer on iron objects. The aim of this study was to evaluate an alternative conservation-restoration method using bacteria. For this, anaerobic iron reduction leading to the formation of stable iron minerals in the presence of chlorine was investigated for two strains of Desulfitobacterium hafniense (strains TCE1 and LBE). Iron reduction was observed for soluble Fe(III) phases as well as for akaganeite, the most troublesome iron compound in the corrosion layer of archaeological iron objects. In terms of biogenic mineral production, differential efficiencies were observed in assays performed on corroded iron coupons. Strain TCE1 produced a homogeneous layer of vivianite covering 80% of the corroded surface, while on the coupons treated with strain LBE, only 10% of the surface was covered by the same mineral. Finally, an attempt to reduce iron on archaeological objects was performed with strain TCE1, which led to the formation of both biogenic vivianite and magnetite on the surface of the artifacts. These results demonstrate the potential of this biological treatment for stabilizing archaeological iron as a promising alternative to traditional conservation-restoration methods.IMPORTANCE Since the Iron Age, iron has been a fundamental material for the building of objects used in everyday life. However, due to its reactivity, iron can be easily corroded, and the physical stability of the object built is at risk. This is particularly true for archaeological objects on which a potentially unstable corrosion layer is formed during the time the object is buried. After excavation, changes in environmental conditions (e.g., higher oxygen concentration or lower humidity) alter the stability of the corrosion layer and can lead to the total destruction of the object. In this study, we demonstrate the feasibility of an innovative treatment based on bacterial iron reduction and biogenic mineral formation to stabilize the corrosion layer and protect these objects.

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.

Enrichment of Desulfitobacterium spp. from forest and grassland soil using the O-demethylation of phenyl methyl ethers as a growth-selective process.

The O-demethylation of phenyl methyl ethers under anaerobic conditions is a metabolic feature of acetogens and Desulfitobacterium spp. Desulfitobacteria as well as most acetogens are Gram-positive bacteria with a low GC content and belong to the phylum Firmicutes. The consumption of the phenyl methyl ether syringate was studied in enrichment cultures originating from five different topsoils. Desulfitobacterium spp. were detected in all topsoils via quantitative PCR. Desulfitobacteria could be enriched using the O-demethylation of syringate as a growth-selective process. The enrichment was significantly favoured by an external electron acceptor such as 3-chloro-4-hydroxyphenylacetate or thiosulfate. Upon cultivation in the presence of syringate and thiosulfate, which naturally occur in soil, a maximum number of 16S rRNA gene copies of Desulfitobacterium spp. was reached within the first three subcultivation steps and accounted for 3-10% of the total microbial community depending on the soil type. Afterwards, a loss of Desulfitobacterium gene copies was observed. Community analyses revealed that Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were the main phyla in the initial soil samples. Upon addition of syringate and thiosulfate as growth substrates, these phyla were rapidly outcompeted by Firmicutes, which were under-represented in soil. The main Firmicutes genera identified were Alkalibaculum, Clostridium, Sporobacterium, Sporomusa and Tissierella, which might be responsible for outcompeting the desulfitobacteria. Most of these organisms belong to the acetogens, which have previously been described to demethylate phenyl methyl ethers. The shift of the native community structure to almost exclusively Firmicutes supports the participation of members of this phylum in environmental demethylation processes.

Resiliency of Stable Isotope Fractionation (δ(13)C and δ(37)Cl) of Trichloroethene to Bacterial Growth Physiology and Expression of Key Enzymes.

Quantification of in situ (bio)degradation using compound-specific isotope analysis requires a known and constant isotope enrichment factor (ε). Because reported isotope enrichment factors for microbial dehalogenation of chlorinated ethenes vary considerably we studied the potential effects of metabolic adaptation to TCE respiration on isotope fractionation (δ(13)C and δ(37)Cl) using a model organism (Desulfitobacterium hafniesne Y51), which only has one reductive dehalogenase (PceA). Cells grown on TCE for the first time showed exponential growth until 10(9) cells/mL. During exponential growth, the cell-normalized amount of PceA enzyme increased steadily in the presence of TCE (up to 21 pceA transcripts per cell) but not with alternative substrates (<1 pceA transcript per cell). Cultures initially transferred or subcultivated on TCE showed very similar isotope fractionation, both for carbon (εcarbon: -8.6‰ ± 0.3‰ or -8.8‰ ± 0.2‰) and chlorine (εchlorine: -2.7‰ ± 0.3‰) with little variation (0.7‰) for the different experimental conditions. Thus, TCE isotope fractionation by D. hafniense strain Y51 was affected by neither growth phase, pceA transcription, or translation, nor by PceA content per cell, suggesting that transport limitations did not affect isotope fractionation. Previously reported variable ε values for other organohalide-respiring bacteria might thus be attributed to different expression levels of their multiple reductive dehalogenases.

Potential for microbial oxidation of ferrous iron in basaltic glass.

Basaltic glass (BG) is an amorphous ferrous iron [Fe(II)]-containing material present in basaltic rocks, which are abundant on rocky planets such as Earth and Mars. Previous research has suggested that Fe(II) in BG can serve as an energy source for chemolithotrophic microbial metabolism, which has important ramifications for potential past and present microbial life on Mars. However, to date there has been no direct demonstration of microbially catalyzed oxidation of Fe(II) in BG. In this study, three different culture systems were used to investigate the potential for microbial oxidation of Fe(II) in BG, including (1) the chemolithoautotrophic Fe(II)-oxidizing, nitrate-reducing "Straub culture"; (2) the mixotrophic Fe(II)-oxidizing, nitrate-reducing organism Desulfitobacterium frappieri strain G2; and (3) indigenous microorganisms from a streambed Fe seep in Wisconsin. The BG employed consisted of clay and silt-sized particles of freshly quenched lava from the TEB flow in Kilauea, Hawaii. Soluble Fe(II) or chemically reduced NAu-2 smectite (RS) were employed as positive controls to verify Fe(II) oxidation activity in the culture systems. All three systems demonstrated oxidation of soluble Fe(II) and/or structural Fe(II) in RS, whereas no oxidation of Fe(II) in BG material was observed. The inability of the Straub culture to oxidize Fe(II) in BG was particularly surprising, as this culture can oxidize other insoluble Fe(II)-bearing minerals such as biotite, magnetite, and siderite. Although the reason for the resistance of the BG toward enzymatic oxidation remains unknown, it seems possible that the absence of distinct crystal faces or edge sites in the amorphous glass renders the material resistant to such attack. These findings have implications with regard to the idea that Fe(II)-Si-rich phases in basalt rocks could provide a basis for chemolithotrophic microbial life on Mars, specifically in neutral-pH environments where acid-promoted mineral dissolution and utilization of dissolved Fe(II) as an energy source is not likely to take place.

Bioremediation of 1,2-dichloroethane contaminated groundwater: Microcosm and microbial diversity studies.

In this study, the effectiveness of bioremediating 1,2-dichloroethane (DCA)-contaminated groundwater under different oxidation-reduction processes was evaluated. Microcosms were constructed using indigenous bacteria and activated sludge as the inocula and cane molasses and a slow polycolloid-releasing substrate (SPRS) as the primary substrates. Complete DCA removal was obtained within 30 days under aerobic and reductive dechlorinating conditions. In anaerobic microcosms with sludge and substrate addition, chloroethane, vinyl chloride, and ethene were produced. The microbial communities and DCA-degrading bacteria in microcosms were characterized by 16S rRNA-based denatured-gradient-gel electrophoresis profiling and nucleotide sequence analyses. Real-time polymerase chain reaction was applied to evaluate the variations in Dehalococcoides spp. and Desulfitobacterium spp. Increase in Desulfitobacterium spp. indicates that the growth of Desulfitobacterium might be induced by DCA. Results indicate that DCA could be used as the primary substrate under aerobic conditions. The increased ethene concentrations imply that dihaloelimination was the dominate mechanism for DCA biodegradation.

Detoxification of 1,1,2-trichloroethane to ethene by desulfitobacterium and identification of its functional reductase gene.

1,1,2-trichloroethane (1,1,2-TCA) has become a common groundwater pollutant due to historically extensive utilization, improper disposal, as well as from incomplete dechlorination of 1,1,2,2-tetrachloroethane. Currently, limited information is available on microbial detoxification of 1,1,2-TCA. Desulfitobacterium sp. strain PR, which was isolated from an anaerobic bioreactor maintained to dechlorinate chloroethenes/ethanes, exhibited the capacity to dechlorinate 1,1,1-trichloroethane and chloroform. In this study, the dechlorinating ability of strain PR was further explored. Strain PR showed the capability to dechlorinate 1,1,2-TCA (~1.12 mM) predominantly to 1,2-dichloroethane (1,2-DCA) and chloroethane, and to trace amounts of vinyl chloride and ethene within 20 days. Strain PR coupled growth with dechlorination of 1,1,2-TCA to 1,2-DCA, while no cell growth was observed with dechlorination of 1,2-DCA to chloroethane. Later, through transcriptomic and enzymatic analysis, the reductive dehalogenase CtrA, which was previously reported to be responsible for 1,1,1-trichloroethane and chloroform dechlorination, was identified as the 1,1,2-TCA reductive dehalogenase. Since trichloroethene (TCE) is usually co-contaminated with 1,1,2-TCA, a co-culture containing Dehalococcoides mccartyi strain 11a capable of detoxifying TCE and 1,2-DCA and strain PR was established. Interestingly, this co-culture dechlorinated 1,1,2-TCA and TCE to the non-toxic end-product ethene within 48 days without chloroethane production. This novel pathway avoids production of the carcinogenic intermediate dechlorination product vinyl chloride, providing a more environmentally friendly strategy to treat 1,1,2-TCA.

Substrate hydrophobicity and cell composition influence the extent of rate limitation and masking of isotope fractionation during microbial reductive dehalogenation of chlorinated ethenes.

This study investigated the effect of intracellular microscale mass transfer on microbial carbon isotope fractionation of tetrachloroethene (PCE) and trichloroethene (TCE). Significantly stronger isotope fractionation was observed for crude extracts vs intact cells of Sulfurospirillum multivorans, Geobacter lovleyi, Desulfuromonas michiganensis, Desulfitobacterium hafniense strain PCE-S, and Dehalobacter restrictus. Furthermore, carbon stable isotope fractionation was stronger for microorganisms with a Gram-positive cell envelope compared to those with a Gram-negative cell envelope. Significant differences were observed between model organisms in cellular sorption capacity for PCE (S. multivorans-K(d-PCE) = 0.42-0.51 L g(-1); D. hafniense-K(d-PCE) = 0.13 L g(-1)), as well as in envelope hydrophobicity (S. multivorans 33.0° to 72.2°; D. hafniense 59.1° to 60.8°) when previously cultivated with fumarate or PCE as electron acceptor, but not for TCE. Cell envelope properties and the tetrachloroethene reductive dehalogenase (PceA-RDase) localization did not result in significant effects on observed isotope fractionation of TCE. For PCE, however, systematic masking of isotope effects as a result of microscale mass transfer limitation at microbial membranes was observed, with carbon isotope enrichment factors of -2.2‰, -1.5 to -1.6‰, and -1.0‰ (CI95% < ± 0.2‰) for no membrane, hydrophilic outer membrane, and outer + cytoplasmic membrane, respectively. Conclusively, rate-limiting mass transfer barriers were (a) the outer membrane or cell wall and (b) the cytoplasmic membrane in case of a cytoplasmic location of the RDase enzyme. Overall, our results indicate that masking of isotope fractionation is determined by (1) hydrophobicity of the degraded compound, (2) properties of the cell envelope, and (3) the localization of the reacting enzyme.

Genomic, proteomic, and biochemical analysis of the organohalide respiratory pathway in Desulfitobacterium dehalogenans.

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.