Characterization of a Marine Microbial Community Used for Enhanced Sulfate Reduction and Copper Precipitation in a Two-Step Process.

Marine microorganisms that are obtained from hydrothermal vent sediments present a great metabolic potential for applications in environmental biotechnology. However, the work done regarding their applications in engineered systems is still scarce. Hence, in this work, the sulfate reduction process carried out by a marine microbial community in an upflow anaerobic sludge blanket (UASB) reactor was investigated for 190 days under sequential batch mode. The effects of 1000 to 5500 mg L-1 of SO4-2 and the chemical oxygen demand (COD)/SO4-2 ratio were studied along with a kinetic characterization with lactate as the electron donor. Also, the feasibility of using the sulfide produced in the UASB for copper precipitation in a second column was studied under continuous mode. The system presented here is an alternative to sulfidogenesis, particularly when it is necessary to avoid toxicity to sulfide and competition with methanogens. The bioreactor performed better with relatively low concentrations of sulfate (up to 1100 mg L-1) and COD/SO4-2 ratios between 1.4 and 3.6. Under the continuous regime, the biogenic sulfide was sufficient to precipitate copper at a removal rate of 234 mg L-1 day-1. Finally, the identification of the microorganisms in the sludge was carried out; some genera of microorganisms identified were Desulfitobacterium and Clostridium.

Concentration effects on biotic and abiotic processes in the removal of 1,1,2-trichloroethane and vinyl chloride using carbon-amended ZVI.

A permeable reactive barrier, consisting of both zero valent iron (ZVI) and a biodegradable organic carbon, was evaluated for the remediation of 1,1,2-trichloroethane (1,1,2-TCA) contaminated groundwater. During an 888 day laboratory column study, degradation rates initially stabilized with a degradation half-life of 4.4±0.4 days. Based on the accumulation of vinyl chloride (VC) and limited production of 1,1-dichloroethene (1,1-DCE) and 1,2-dichloroethane (1,2-DCA), the dominant degradation pathway was likely abiotic dichloroelimination to form VC. Degradation of VC was not observed based on the accumulation of VC and limited ethene production. After a step reduction in the influent concentration of 1,1,2-TCA from 170±20 mg L(-1) to 39±11 mg L(-1), the degradation half-life decreased 5-fold to 0.83±0.17 days. The isotopic enrichment factor of 1,1,2-TCA also changed after the step reduction from -14.6±0.7‰ to -0.72±0.12‰, suggesting a possible change in the degradation mechanism from abiotic reductive degradation to biodegradation. Microbiological data suggested a co-culture of Desulfitobacterium and Dehalococcoides was responsible for the biodegradation of 1,1,2-TCA to ethene.

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.

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.

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase.

COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptors such as nitrate or fumarate, producing dimethylglycine and CO2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. In addition, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. They are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.

A Desulfitobacterium sp. strain PR reductively dechlorinates both 1,1,1-trichloroethane and chloroform.

1,1,1-Trichloroethane (TCA) and chloroform are two notorious groundwater pollutants. Here we report the isolation and characterization of Desulfitobacterium sp. strain PR that rapidly dechlorinates both compounds. In pyruvate-amended medium, strain PR reductively dechlorinates ∼ 1.0 mM TCA completely to monochloroethane within 15 days. Under the same conditions, strain PR dechlorinates ∼ 1.2 mM chloroform to predominantly dichloromethane (∼ 1.14 mM) and trace amount of monochloromethane (∼ 0.06 mM) within 10 days. Strain PR shares 96.7% 16S rRNA gene sequence similarity with its closest relative - Desulfitobacterium metallireducens strain 853-15; however, it distinguishes itself from known Desulfitobacterium strains by its inability of utilizing several of their commonly shared substrates such as lactate, thiosulfate and sulfite. A reductive dehalogenase gene (ctrA) in strain PR was identified to be responsible for dechlorination of both TCA and chloroform, showing a maximum expression level of 5.95 ∼ 6.25 copies of transcripts cell(-1) . CtrA shares 94% amino acid sequence identity with CfrA in Dehalobacter sp. strain CF50 and DcrA in Dehalobacter sp. strain DCA. Interestingly, strain PR could tolerate high aqueous concentrations (up to 0.45 mM) of trichloroethene, another groundwater pollutant that often coexists with TCA/chloroform. As the first chloroform-respiring and the second TCA-respiring isolate that has been identified, Desulfitobacterium sp. strain PR may prove useful in remediation of halogenated alkanes with trihalomethyl (-CX3) groups.

Global transcriptome analysis of the tetrachloroethene-dechlorinating bacterium Desulfitobacterium hafniense Y51 in the presence of various electron donors and terminal electron acceptors.

Desulfitobacterium hafniense Y51 is a dechlorinating bacterium that encodes an unusually large set of O-demethylase paralogs and specialized respiratory systems including specialized electron donors and acceptors. To use this organism in bioremediation of tetrachloroethene (PCE) or trichloroethene (TCE) pollution, expression patterns of its 5,060 genes were determined under different conditions using 60-mer probes in DNA microarrays. PCE, TCE, fumarate, nitrate, and dimethyl sulfoxide (DMSO) respiration all sustain the growth of strain Y51. Global transcriptome analyses were thus performed using various electron donor and acceptor couples (respectively, pyruvate and either fumarate, TCE, nitrate, or DMSO, and vanillate/fumarate). When TCE is used as terminal electron acceptor, resulting in its detoxification, a series of electron carriers comprising a cytochrome bd-type quinol oxidase (DSY4055-4056), a ferredoxin (DSY1451), and four Fe-S proteins (DSY1626, DSY1629, DSY0733, DSY3309) are upregulated, suggesting that the products of these genes are involved in PCE oxidoreduction. Interestingly, the PCE dehalogenase cluster (pceABCT) is constitutively expressed in the media tested, with pceT being upregulated and pceC downregulated in pyruvate/TCE-containing medium. In addition, another dehalogenation enzyme (DSY1155 coding for a putative chlorophenol reductive dehalogenase), is induced 225-fold in that medium, despite not being involved in PCE respiration. Remarkably since the reducing equivalents formed during pyruvate conversion to acetyl-CoA are channeled to electron acceptors including halogenated compounds, pyruvate induces expression of a pyruvate:ferredoxin oxidoreductase. This study paves the way to understanding the physiology of D. hafniense, optimizing this microbe as a bioremediation agent, and designing bioarray sensors to monitor the presence of dechlorinating organisms in the environment.

Reductive dehalogenation of brominated ethenes by Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S.

Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S are anaerobes that can utilize tetrachloroethene (PCE) as an electron acceptor in their energy metabolism. The end-product of PCE reduction for both organisms is cis-1,2-dichloroethene, which is formed via trichloroethene as the intermediate. The bacteria were able to dehalogenate cis- and trans-1,2-dibromoethene (cDBE and tDBE) in growing cultures and cell extracts. Dibromoethene supported growth of both organisms. The organisms debrominated cDBE and tDBE to vinyl bromide (VB); D. hafniense PCE-S also produced ethene in addition to VB. The PCE reductive dehalogenases (PCE dehalogenases) of S. multivorans and D. hafniense PCE-S mediated the debromination of tribromoethene (TBE) and both isomers of 1,2-DBE, indicating that this enzyme was responsible for the reductive dehalogenation of brominated ethenes. cDBE, tDBE, 1,1-DBE and VB were formed upon TBE debromination; VB was the major end-product. The PCE dehalogenase of D. hafniense PCE-S also formed ethene. With the purified enzymes from both organisms the kinetic properties of dehalogenation of brominated alkenes were studied and compared with those of their chlorinated analogues.

Effects of chloromethanes on growth of and deletion of the pce gene cluster in dehalorespiring Desulfitobacterium hafniense strain Y51.

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 microM, but not carbon dichloride even at 1 mM.

Exposure to sulfide causes populations shifts in sulfate-reducing consortia.

The shift in the community structure of a mixed culture of sulfate-reducing bacteria (SRB) at 0.5, 0.75, 1, and 1.5 kg m(-3) sulfide loadings was investigated in an anaerobic continuous bioreactor used for treatment of sulfate-containing wastewater by fluorescence in situ hybridization (FISH), using SRB species-specific and group-specific 16S rRNA-targeting probes. Hybridization analysis using these 16S rRNA-targeted oligonucleotide probes revealed that sulfide was toxic for Desulfonema, Desulfobulbus spp. and the Desulfobacteriaceae group, although it was not toxic for Desulfobacter, Desulfotomaculum, Desulfobacterium spp. or the Desulfovibrionaceae group. On the other hand, only a high concentration of sulfide of 1.5 kg m(-3) was found to be toxic for the Desulfococcus group in the bioreactor. When the sulfide in the feed was 1.00 kg m(-3) the sulfate-reducing capacity of the system decreased, and this decrease was more pronounced when the inlet sulfide was further increased to 1.5 kg m(-3).

[Detection of SRPs in injection water of Shenli Oil Field by FISH].

Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes was applied for analyzing the structure of sulfate reducing prokaryotes (SRPs) community in injection water of Shengli Oil Field. Eight probes and their various combinations were used to detect SRPs in the water. Results showed SRPs detected in the water were diverse, which followed in 4 bacterial phyla and 1 archaeal phylum. Total amount of SRPs was 2.86 x 10(4) cells/mL, accounting for 20% of total cells of microorganisms in the water of the Oil Field. Desulfovibrio and Desulfotomaculum cells were about 8.71% (+/- 4.45%) and 12.15% (+/- 3.90%) of the total microbial cells respectively, being dominant in the water. The relative amounts of SRPs belonging to Desulfobacterales and Syntrophobacterales, Thermodesulfobacteriales, and Thermodesulfovibro to total microbial cells in the water were 7.59% (+/- 2.92%), 3.57% (+/- 1.39%) and 2.32% (+/- 0.80%) respectively. In addition, SRPs belonging to Archaeoglobus were also detected with the amount of 4.29% (+/- 1.75%) of total microbial cells, which tells that archeal SRPs are also very important sulfate reducing microorganisms in the injection water of oil field.

Removal of selenate from sulfate-containing media by sulfate-reducing bacterial biofilms.

A biofilm-selected strain of a Desulfomicrobium sp. removed selenate from solution to sub-micromolar concentrations during growth on lactate (or hydrogen) and sulfate. Under sulfate-limited growth conditions, selenium was enzymatically reduced to selenide. Under excess sulfate conditions, selenate removal was primarily by enzymatic reduction to elemental selenium. Sequestration by biofilms was greater under the latter condition. Experiments with washed cell suspensions showed that high sulfate concentrations inhibited cell-specific selenate reduction, but when growing cells were exposed to selenate, the biomass increase achieved during incubations with abundant sulfate resulted in more rapid selenate removal. The addition of small amounts of sulfite, or thiosulfate, ameliorated this inhibition. Nitrate also inhibited selenate reduction in washed cell suspensions, apparently due to a general oxidizing effect. These results suggest that where biofilm-based sulfate-reducing bacteria (SRB) bioreactors are considered for the treatment of mixed metalliferous wastes that contain selenium oxyanions, adequate selenate removal should be achievable under a range of environmental conditions. The form and fate of the precipitated product will, however, be influenced by the dominant reduction pathway, which is controlled by environmental variables.

Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin.

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B12) was studied. Fractionation was largest during the reaction with cyanocobalamin with alphaC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (alphaC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (alphaC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, alphaC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.

Dehalogenation of the herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodino-4-hydroxybenzonitrile) by Desulfitobacterium chlororespirans.

Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 microM) and DBHB (250 to 260 microM) were used as electron acceptors for growth. Doubling times for growth (means +/- standard deviations for triplicate cultures) on bromoxynil (18.4 +/- 5.2 h) and DBHB (11.9 +/- 1.4 h), determined by rate of [14C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review.

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 microM and as low as 0.06 microM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min(-1) mg protein(-1). The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1.

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22 degrees C, compared to 30 and 37 degrees C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K(m) for 3,5-dicholorophenol was 49.3 +/- 3.1 microM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.

The transformation of inorganic sulfur compounds and the assimilation of organic and inorganic carbon by the sulfur disproportionating bacterium Desulfocapsa sulfoexigens.

The physiology of the sulfur disproportionator Desulfocapsa sulfoexigens was investigated in batch cultures and in a pH-regulated continuously flushed fermentor system. It was shown that a sulphide scavanger in the form of ferric iron was not obligatory and that the control of pH allowed production of more biomass than was possible in carbonate buffered but unregulated batch cultures. Small amounts of sulphite were produced during disproportionation of elemental sulfur and thiosulphate. In addition, it was shown that in the presence of hydrogen, a respiratory type of process is favored before the disproportionation of sulphite, thiosulphate and elemental sulfur. Sulphate reduction was not observed. D. sulfoexigens assimilated inorganic carbon even in the presence of organic carbon sources. Inorganic carbon assimilation was probably catalyzed by the reverse CO-dehydrogenase pathway, which was supported by the constitutive expression of the gene encoding CO-dehydrogenase in cultures grown in the presence of acetate and by the high carbon fractionation values that are indicative of this pathway.

Phenyl methyl ethers: novel electron donors for respiratory growth of Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S.

Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S grew under anoxic conditions with a variety of phenyl methyl ethers as electron donors in combination with fumarate as electron acceptor. The phenyl methyl ethers were O-demethylated to the corresponding phenol compounds. O-demethylation was strictly dependent on the presence of fumarate; no O-demethylation occurred with CO2 as electron acceptor. One mol phenyl methyl ether R-O-CH3 was O-demethylated to R-OH per 3 mol fumarate reduced to succinate. The growth yields with vanillate or syringate plus fumarate were approximately 15 g cells (dry weight) per mol methyl moiety converted. D. hafniense utilized vanillate or syringate as an electron donor for reductive dehalogenation of 3-Cl-4-hydroxyphenylacetate, whereas strain PCE-S was not able to dechlorinate tetrachloroethene with phenyl methyl ethers. Crude extracts of both organisms showed O-demethylase activity in the O-demethylase assay with vanillate or syringate as substrates when the organism was grown on syringate plus fumarate. Besides the homoacetogenic bacteria, only growing cells of Desulfitobacterium frappieri PCP-1 have thus far been reported to be capable of phenyl methyl ether O-demethylation. This present study is the first report of Desulfitobacteria utilizing phenyl methyl ethers as electron donors for fumarate reduction and for growth.

Microbial anaerobic demethylation and dechlorination of chlorinated hydroquinone metabolites synthesized by basidiomycete fungi.

The synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed that Desulfitobacterium hafniense strains DCB2 and PCP1, Desulfitobacterium chlororespirans strain Co23, and Desulfitobacterium dehalogenans JW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.