Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments

Differences in methylmercury (CH₃Hg) production normalized to the sulfate reduction rate (SRR) in various species of sulfate-reducing bacteria (SRB) were quantified in pure cultures and in marine sediment slurries in order to determine if SRB strains which differ phylogenetically methylate mercury (Hg) at similar rates. Cultures representing five genera of the SRB (Desulfovibrio desulfuricans, Desulfobulbus propionicus, Desulfococcus multivorans, Desulfobacter sp. strain BG-8, and Desulfobacterium sp. strain BG-33) were grown in a strictly anoxic, minimal medium that received a dose of inorganic Hg 120 h after inoculation. The mercury methylation rates (MMR) normalized per cell were up to 3 orders of magnitude higher in pure cultures of members of SRB groups capable of acetate utilization (e.g., the family Desulfobacteriaceae) than in pure cultures of members of groups that are not able to use acetate (e.g., the family Desulfovibrionaceae). Little or no Hg methylation was observed in cultures of Desulfobacterium or Desulfovibrio strains in the absence of sulfate, indicating that Hg methylation was coupled to respiration in these strains. Mercury methylation, sulfate reduction, and the identities of sulfate-reducing bacteria in marine sediment slurries were also studied. Sulfate-reducing consortia were identified by using group-specific oligonucleotide probes that targeted the 16S rRNA molecule. Acetate-amended slurries, which were dominated by members of the Desulfobacterium and Desulfobacter groups, exhibited a pronounced ability to methylate Hg when the MMR were normalized to the SRR, while lactate-amended and control slurries had normalized MMR that were not statistically different. Collectively, the results of pure-culture and amended-sediment experiments suggest that members of the family Desulfobacteriaceae have a greater potential to methylate Hg than members of the family Desulfovibrionaceae have when the MMR are normalized to the SRR. Hg methylation potential may be related to genetic composition and/or carbon metabolism in the SRB. Furthermore, we found that in marine sediments that are rich in organic matter and dissolved sulfide rapid CH₃Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH₃Hg formation and for developing remediation strategies for Hg-contaminated sediments.     

Sulfate-reducing bacteria in tubes constructed by the marine infaunal polychaete Diopatra cuprea

Marine infaunal burrows and tubes greatly enhance solute transport between sediments and the overlying water column and are sites of elevated microbial activity. Biotic and abiotic controls of the compositions and activities of burrow and tube microbial communities are poorly understood. The microbial communities in tubes of the marine infaunal polychaete Diopatria cuprea collected from two different sediment habitats were examined. The bacterial communities in the tubes from a sandy sediment differed from those in the tubes from a muddy sediment. The difference in community structure also extended to the sulfate-reducing bacterial (SRB) assemblage, although it was not as pronounced for this functional group of species. PCR-amplified 16S rRNA gene sequences recovered from Diopatra tube SRB by clonal library construction and screening were all related to the family Desulfobacteriaceae. This finding was supported by phospholipid fatty acid analysis and by hybridization of 16S rRNA probes specific for members of the genera Desulfosarcina, Desulfobacter, Desulfobacterium, Desulfobotulus, Desulfococcus, and Desulfovibrio and some members of the genera Desulfomonas, Desulfuromonas, and Desulfomicrobium with 16S rRNA gene sequences resolved by denaturing gradient gel electrophoresis. Two of six SRB clones from the clone library were not detected in tubes from the sandy sediment. The habitat in which the D. cuprea tubes were constructed had a strong influence on the tube bacterial community as a whole, as well as on the SRB assemblage.     

Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B(12) in some and perhaps many incomplete-oxidizing SRB strains.     

High overall diversity and dominance of microdiverse relationships in salt marsh sulphate-reducing bacteria

The biogeochemistry of North Atlantic salt marshes is characterized by the interplay between the marsh grass Spartina and sulphate-reducing bacteria (SRB), which mineralize the diverse carbon substrates provided by the plants. It was hypothesized that SRB populations display high diversity within the sediment as a result of the rich spatial and chemical structuring provided by Spartina roots. A 2000-member 16S rRNA gene library, prepared with delta-proteobacterial SRB-selective primers, was analysed for diversity patterns and phylogenetic relationships. Sequence clustering detected 348 16S rRNA sequence types (ribotypes) related to delta-proteobacterial SRB, and it was estimated that a total of 623 ribotypes were present in the library. Similarity clustering showed that approximately 46% of these sequences fell into groups with < 1% divergence; thus, microheterogeneity accounts for a large portion of the observable genetic diversity. Phylogenetic comparison revealed that sequences most frequently recovered were associated with the Desulfobacteriaceae and Desulfobulbaceae families. Sequences from the Desulfovibrionaceae family were also observed, but were infrequent. Over 80% of the delta-proteobacterial ribotypes clustered with cultured representatives of Desulfosarcina, Desulfococcus and Desulfobacterium genera, suggesting that complete oxidizers with high substrate versatility dominate. The large-scale approach demonstrates the co-existence of numerous SRB-like sequences and reveals an unexpected amount of microdiversity.     

Unexpected population distribution in a microbial mat community: sulfate-reducing bacteria localized to the highly oxic chemocline in contrast to a eukaryotic preference for anoxia.

The distribution and abundance of sulfate-reducing bacteria (SRB) and eukaryotes within the upper 4 mm of a hypersaline cyanobacterial mat community were characterized at high resolution with group-specific hybridization probes to quantify 16S rRNA extracted from 100-microm depth intervals. This revealed a preferential localization of SRB within the region defined by the oxygen chemocline. Among the different groups of SRB quantified, including members of the provisional families "Desulfovibrionaceae" and "Desulfobacteriaceae," Desulfonema-like populations dominated and accounted for up to 30% of total rRNA extracted from certain depth intervals of the chemocline. These data suggest that recognized genera of SRB are not necessarily restricted by high levels of oxygen in this mat community and the possibility of significant sulfur cycling within the chemocline. In marked contrast, eukaryotic populations in this community demonstrated a preference for regions of anoxia.     

Evidence for syntrophic butyrate metabolism under sulfate-reducing conditions in a hydrocarbon-contaminated aquifer

The importance of syntrophy in the degradation of butyrate in an aquifer where sulfate reduction was shown to be an important terminal electron-accepting process was assessed. Hydrocarbon-contaminated aquifer sediments coupled butyrate degradation to sulfate reduction and methane production. Butyrate degradation in methanogenic microcosms was inhibited by the addition of 2-bromoethanesulfonic acid, and was restored by the addition of 10 mM sulfate and a hydrogen- and formate-using sulfate reducer, but not by the addition of 10 mM sulfate alone. Molybdate addition inhibited butyrate degradation in sulfate-reducing microcosms. The addition of CO, which inhibits hydrogenases, to sulfate-reducing microcosms inhibited butyrate metabolism and caused the hydrogen partial pressure to increase to levels that would make syntrophic butyrate degradation via sulfate reduction energetically unfavorable (-5 to +3 kJ mol(-1) ). DNA extracted from the most probable number cultures and contaminated sediments contained sequences related to members of the families Syntrophomonadaceae and Syntrophaceae, whose members are known to syntrophically degrade fatty acids, as well as sequences related to uncultured Firmicutes, Desulfobulbaceae, Desulfobacteriaceae, and Desulfovibrionaceae. These data show that contaminated sediments degraded butyrate syntrophically coupled to methane production and sulfate reduction.     

Identity and abundance of active sulfate-reducing bacteria in deep tidal flat sediments determined by directed cultivation and CARD-FISH analysis

The identity and abundance of potentially active sulfate-reducing bacteria (SRB) in several metre deep sediments of a tidal sand flat in the German Wadden Sea were assessed by directed cultivation and cultivation-independent CARD-FISH analysis (catalysed reporter deposition fluorescence in situ hybridization). Presumably abundant SRB from different sediment layers between 0.5 and 4 m depth were selectively enriched in up to million-fold diluted cultures supplemented with lactate, acetate or hydrogen. Partial 16S rRNA gene sequences obtained from highest dilution steps showing sulfide formation indicated growth of deltaproteobacterial SRB belonging to the Desulfobulbaceae and the Desulfobacteraceae as well as of members of the Firmicutes. Subsequent isolation resulted in 10 novel phylotypes of both litho- and organotrophic sulfate-reducing Deltaproteobacteria. Molecular pre-screening identified six isolates as members of the Desulfobulbaceae, sharing highest identities with either candidatus 'Desulfobacterium corrodens' (95-97%) or Desulfobacterium catecholicum (98%), and four isolates as members of Desulfobacteraceae, being related to either Desulfobacter psychrotolerans (98%) or Desulfobacula phenolica (95-97%). Relatives of D. phenolica were exlusively isolated from 50 and 100 cm deep sediments with 10 and 2 mM of pore water sulfate respectively. In contrast, relatives of D. corrodens, D. psychrotolerans and D. catecholicum were also obtained from layers deeper than 100 cm and with less than 2 mM sulfate. The high in situ abundance of members of both families in sediment layers beneath 50 cm could be confirmed via CARD-FISH analysis performed with a set of six SRB-specific oligonucleotide probes. Moreover, SRB represented a numerically significant fraction of the microbial community throughout the sediment (up to 7%) and reached even higher cell numbers in deep, sulfate-poor layers than in the sulfate-rich surface sediment. This relatively large community size of potentially active SRB in deep sandy sediments might on the one hand be a result of their syntrophic association with other anaerobes. Our results furthermore support the hypothesis that enhanced advective pore water transport might supply nutrients to microbial communities in deep sandy sediments and point to their so far unrecognized contribution to biogeochemical processes in Wadden Sea sediments.     

Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities

Here, we describe a three-step nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to detect sulfate-reducing bacteria (SRB) in complex microbial communities from industrial bioreactors. In the first step, the nearly complete 16S rRNA gene was amplified using bacterial primers. Subsequently, this product was used as a template in a second PCR with group-specific SRB primers. A third round of amplification was conducted to obtain fragments suitable for DGGE. The largest number of bands was observed in DGGE patterns of products obtained with primers specific for the Desulfovibrio-Desulfomicrobium group, indicating a large diversity of these SRBs. In addition, members of other phylogenetic SRB groups, i.e., Desulfotomaculum, Desulfobulbus, and Desulfococcus-Desulfonema-Desulfosarcina, were detected. Bands corresponding to Desulfobacterium and Desulfobacter were not detected in the bioreactor samples. Comparative sequence analysis of excised DGGE bands revealed the identity of the community members. The developed three-step PCR-DGGE strategy is a welcome tool for studying the diversity of sulfate-reducing bacteria.     

Diversity of bacteria and Archaea in sulphate-reducing enrichment cultures inoculated from serial dilution of Zostera noltii rhizosphere samples

We have analysed the diversity of culturable sulphate-reducing bacteria (SRB) in Zostera noltii colonized sediments from Bassin d'Arcachon (France). Four organic substrates have been tested as well as the combination of H2 and CO2 to select for lithotrophic SRB. All energy sources were supplied in parallel cultures that were amended with yeast extract plus NH4+ and prepared without a source of combined nitrogen, the latter to select for diazotrophic SRB. The 10 different enrichment media were inoculated from serial dilution of rhizosphere samples. The highest dilution cultures yielding positive growth (i.e. 10-7) were studied by molecular techniques (16S rDNA clone libraries, RISA and ARDRA). Lactate as a single organic substrate in combination with a source of combined nitrogen resulted in selection of members of the Desulfovibrionaceae. Surprisingly, when lactate was added without a source of combined nitrogen, Desulfobacteriaceae were selected. A strong influence of the presence or absence of combined nitrogen was also observed for the substrates sucrose and fructose. Whereas the liquid culture growing on sucrose and NH4+ systematically yielded 16S rDNA clones related to an environmental unidentified green sulphur bacterium (OPS185), on plates we were able to isolate a SRB related to Desulfovibrio dechloracetivorans, which likely represents a non-described species. Under diazotrophic conditions, sucrose selected for SRB clones related to the cluster formed by Desulfovibrio zosterae, Desulfovibrio salexigens and Desulfovibrio bastinii. The corresponding isolate obtained on plates showed only low sequence similarity with this closest neighbour (93.8%), and we suggest that it also represents a non-described species. Surprisingly, a 16S rDNA sequence corresponding to an archaeon, i.e. a non-extremophile Crenoarchaeota, was retrieved from several of the SRB enrichment cultures even after subsequent transfers.     

Mercury Methylation by the Methanogen Methanospirillum hungatei

Methylmercury (MeHg), a neurotoxic substance that accumulates in aquatic food chains and poses a risk to human health, is synthesized by anaerobic microorganisms in the environment. To date, mercury (Hg) methylation has been attributed to sulfate- and iron-reducing bacteria (SRB and IRB, respectively). Here we report that a methanogen, Methanospirillum hungatei JF-1, methylated Hg in a sulfide-free medium at comparable rates, but with higher yields, than those observed for some SRB and IRB. Phylogenetic analyses showed that the concatenated orthologs of the Hg methylation proteins HgcA and HgcB from M. hungatei are closely related to those from known SRB and IRB methylators and that they cluster together with proteins from eight other methanogens, suggesting that these methanogens may also methylate Hg. Because all nine methanogens with HgcA and HgcB orthologs belong to the class Methanomicrobia, constituting the late-evolving methanogenic lineage, methanogenic Hg methylation could not be considered an ancient metabolic trait. Our results identify methanogens as a new guild of Hg-methylating microbes with a potentially important role in mineral-poor (sulfate- and iron-limited) anoxic freshwater environments.     

Mercury Methylation Independent of the Acetyl-Coenzyme A Pathway in Sulfate-Reducing Bacteria

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B₁₂ in some and perhaps many incomplete-oxidizing SRB strains.     

Mercury methylation in Sphagnum moss mats and its association with sulfate-reducing bacteria in an acidic Adirondack forest lake wetland

Processes leading to the bioaccumulation of methylmercury (MeHg) in northern wetlands are largely unknown. We have studied various ecological niches within a remote, acidic forested lake ecosystem in the southwestern Adirondacks, NY, to discover that mats comprised of Sphagnum moss were a hot spot for mercury (Hg) and MeHg accumulation (190.5 and 18.6 ng g?1 dw, respectively). Furthermore, significantly higher potential methylation rates were measured in Sphagnum mats as compared with other sites within Sunday Lake's ecosystem. Although MPN estimates showed a low biomass of sulfate-reducing bacteria (SRB), 2.8 × 10? cells mL?1 in mat samples, evidence consisting of (1) a twofold stimulation of potential methylation by the addition of sulfate, (2) a significant decrease in Hg methylation in the presence of the sulfate reduction inhibitor molybdate, and (3) presence of dsrAB-like genes in mat DNA extracts, suggested that SRB were involved in Hg methylation. Sequencing of dsrB genes indicated that novel SRB, incomplete oxidizers including Desulfobulbus spp. and Desulfovibrio spp., and syntrophs dominated the sulfate-reducing guild in the Sphagnum moss mat. Sphagnum, a bryophyte dominating boreal peatlands, and its associated microbial communities appear to play an important role in the production and accumulation of MeHg in high-latitude ecosystems.     

Stable carbon isotope fractionation by sulfate-reducing bacteria

Biogeochemical transformations occurring in the anoxic zones of stratified sedimentary microbial communities can profoundly influence the isotopic and organic signatures preserved in the fossil record. Accordingly, we have determined carbon isotope discrimination that is associated with both heterotrophic and lithotrophic growth of pure cultures of sulfate-reducing bacteria (SRB). For heterotrophic-growth experiments, substrate consumption was monitored to completion. Sealed vessels containing SRB cultures were harvested at different time intervals, and delta(13)C values were determined for gaseous CO(2), organic substrates, and products such as biomass. For three of the four SRB, carbon isotope effects between the substrates, acetate or lactate and CO(2), and the cell biomass were small, ranging from 0 to 2 per thousand. However, for Desulfotomaculum acetoxidans, the carbon incorporated into biomass was isotopically heavier than the available substrates by 8 to 9 per thousand. SRB grown lithoautotrophically consumed less than 3% of the available CO(2) and exhibited substantial discrimination (calculated as isotope fractionation factors [alpha]), as follows: for Desulfobacterium autotrophicum, alpha values ranged from 1.0100 to 1.0123; for Desulfobacter hydrogenophilus, the alpha value was 0.0138, and for Desulfotomaculum acetoxidans, the alpha value was 1.0310. Mixotrophic growth of Desulfovibrio desulfuricans on acetate and CO(2) resulted in biomass with a delta(13)C composition intermediate to that of the substrates. The extent of fractionation depended on which enzymatic pathways were used, the direction in which the pathways operated, and the growth rate, but fractionation was not dependent on the growth phase. To the extent that environmental conditions affect the availability of organic substrates (e.g., acetate) and reducing power (e.g., H(2)), ecological forces can also influence carbon isotope discrimination by SRB.     

Effect of growth temperature on cellular fatty acids in sulphate-reducing bacteria

The effect of growth temperature on the cellular fatty acid composition of sulphate-reducing bacteria (SRB) was studied in 12 species belonging to eight genera including psychrophiles and mesophiles. Most of these species were of marine origin. The investigated SRB with the exception of four Desulfobacter species exhibited only a minor increase in the proportion of cis-unsaturated fatty acids (by < or = 5% per 10 degrees C) when the growth temperature was decreased; psychrophiles maintained their typically high content of cis-unsaturated fatty acids (around 75% of total fatty acids) nearly constant. The four Desulfobacter species, however, increased the proportion of cis-unsaturated among total fatty acids significantly (by > or =14% per 10 degrees C; measured in late growth phase) with decreasing growth temperature. The ratio between unsaturated and saturated fatty acids in Desulfobacter species changed not only with the growth temperature, but also with the growth state in batch cultures at constant temperature. Changes of cellular fatty acids were studied in detail with D. hydrogenophilus, the most psychrotolerant (growth range 0-35 degrees C) among the mesophilic SRB examined. Desulfobacter hydrogenophilus also formed cis-9,10-methylenehexadecanoic acid (a cyclopropane fatty acid) and 10-methylhexadecanoic acid. At low growth temperature (12 degrees C), the relative amount of these fatty acids was at least threefold lower; this questions the usefulness of 10-methylhexadecanoic acid as a reliable biomarker of Desulfobacter in cold sediments.     

Demethylation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by marine sulfate-reducing bacteria.

The initial step in the anaerobic degradation of the algal osmolyte dimethylsulfoniopropionate (DMSP) in anoxic marine sediments involves either a cleavage to dimethylsulfide and acrylate or a demethylation to 3-S-methylmercaptopropionate. Thus far, only one anaerobic bacterial strain has been shown to carry out the demethylation, namely, Desulfobacterium sp. strain PM4. The aims of the present work were to study how common this property is among certain groups of anaerobic bacteria and to obtain information on the affinities for DMSP of DMSP-demethylating strains. Screening of several pure cultures of sulfate-reducing and acetogenic bacteria showed that Desulfobacterium vacuolatum DSM 3385 and Desulfobacterium niacini DSM 2059 are also able to demethylate DMSP; a very slow demethylation of DMSP was observed with a salt-tolerant strain of Eubacterium limosum. From a 10(5) dilution of intertidal sediment a new marine DMSP-demethylating sulfate-reducing bacterium (strain WN) was isolated. Strain WN was a short, gram-negative, nonmotile rod that grew on betaine, sarcosine, palmitate, H2 plus CO2, and several alcohols, organic acids, and amino acids. Extracts of betaine-grown cells had hydrogenase, formate dehydrogenase, and CO dehydrogenase activities but no alpha-ketoglutarate oxidoreductase activity, indicating the presence of the acetyl coenzyme A-CO dehydrogenase pathway. Analysis of the 16S rRNA gene sequence of strain WN revealed a close relationship with Desulfobacter hydrogenophilus, Desulfobacter latus, and Desulfobacula toluolica. Strain PM4 was shown to group with Desulfobacterium niacini. The K(m) of strain WN for DMSP, as derived from substrate progress curves in cell suspensions, was approximately 10 microM. A similar value was found for D. niacini PM4.     

Methylmercury and methane production potentials in North Carolina Piedmont stream sediments

Methylated mercury (MeHg) can be produced by all microbes possessing the genes hgcA and hgcB, which can include sulfate-reducing bacteria (SRB), iron-reducing bacteria (FeRB), methane-producing archaea (MPA), and other anaerobic microbes. These microbial groups compete for substrates, including hydrogen and acetate. When sulfate is in excess, SRB can outcompete other anaerobic microbes. However, low concentrations of sulfate, which often occur in stream sediments, are thought to reduce the relative importance of SRB. Although SRB are regarded as the primary contributors of MeHg in many aquatic environments, their significance may not be universal, and stream sediments are poorly studied with respect to microbial Hg methylation. We evaluated suppression of methanogenesis by SRB and the potential contributions from SRB, MPA and other MeHg producing microbes (including FeRB) to the production of MeHg in stream sediments from the North Carolina Piedmont region. Lower methanogenesis rates were observed when SRB were not inhibited, however, application of a sulfate-reduction inhibitor stimulated methanogenesis. Greater MeHg production occurred when SRB were active. Other MeHg producing microbes (i.e., FeRB) contributed significantly less MeHg production than SRB. MPA produced MeHg in negligible amounts. Our results suggest that SRB are responsible for the majority of MeHg production and suppress methanogenesis in mid-order stream sediments, similar to other freshwater sediments. Further investigation is needed to evaluate the generality of these findings to streams in other regions, and to determine the mechanisms regulating sulfate and electron acceptor availability and other potential factors governing Hg methylation and methane production in stream sediments.     

Diversity of Sulfate-Reducing Bacteria Inhabiting the Rhizosphere of Phragmites australis in Lake Velencei (Hungary) Revealed by a Combined Cultivation-based and Molecular approach

The community structure of sulfate-reducing bacteria (SRB) associated with reed (Phragmites australis) rhizosphere in Lake Velencei (Hungary) was investigated by using cultivation-based and molecular methods. The cultivation methods were restricted to recover lactate-utilizing species with the exclusion of Desulfobacter and some Desulfobacterium species presumably not being dominant members of the examined community. The most-probable-number (MPN) estimations of lactate-utilizing SRB showed that the cell counts in reed rhizosphere were at least one order of magnitude higher than that in the bulk sediment. The number of endospores was low compared to the total SRB counts. From the highest positive dilution of MPN series, 47 strains were isolated and grouped by restriction fragment length polymorphism (RFLP) analysis of the amplified 16S ribosomal RNA (rRNA) and dsrAB (dissimilatory sulfite reductase) genes. Contrary to the physiological diversity of the isolates, the combined results of RFLP analysis revealed higher diversity at species as well as at subspecies level. Based on the partial 16S rRNA sequences, the representative strains were closely affiliated with the genera Desulfovibrio and Desulfotomaculum. The partial dsrAB sequences of the clones, recovered after isolation and PCR amplification of the community DNA, were related to hitherto uncultured species of the genera Desulfovibrio and Desulfobulbus. Nevertheless, the representative of the second largest clone group was shown to be closely affiliated with the sequenced dsrAB gene of a strain isolated from the same environment and identified as Desulfovibrio alcoholivorans. Another clone sequence was closely related to a possible novel species also isolated within the scope of this work.     

Anaerobic microbial methylation of inorganic tin in estuarine sediment slurries

Estuarine sediment slurries and microorganisms were examined for the ability to methylate inorganic tin. Under controlled redox conditions, tin was methylated only in oxygen-free sediment slurries. Monomethyltin usually comprised greater than 90% of the alkyltin products formed, although dimethyltin was also produced. Autoclaved anoxic sediments did not produce organotins. Several bacterial cultures, most notably sulfate-reducing bacteria isolated from anoxic estuarine sediments, formed monoand dimethyltin from inorganic tin in the absence of sediment. The results suggest that inorganic tin methylation in estuarine environments is an anaerobic process catalyzed primarily by sulfate-reducing microorganisms.     

Use of a three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria

A mixed culture of human fecal bacteria was grown for 120 days in a three-stage continuous culture system. To reproduce some of the nutritional and pH characteristics of the large gut, each vessel had a different operating volume (0.3, 0.5, and 0.8 liter) and pH (6.0, 6.5, and 7.0). A mixture of polysaccharides and proteins was used as carbon and nitrogen sources. Measurements of H2, CH4, S2-, sulfate reduction rates, sulfate-reducing bacteria (SRB), and volatile fatty acids were made throughout the experiment. After 48 days of running, porcine gastric mucin (5.8 g/day) was independently fed to vessel 1 of the multichamber system. The mucin was extensively degraded as evidenced by the stimulation of volatile fatty acid production. In the absence of mucin, sulfate-reducing activity was comparatively insignificant and methanogenesis was the major route for the disposal of electrons. The reverse occurred upon the addition of mucin; sulfate reduction predominated and methanogenesis was completely inhibited. This was attributed to release of sulfate from the mucin which enabled SRB to outcompete methanogenic bacteria for H2. SRB stimulated by mucin were acetate-utilizing Desulfobacter spp., lactate- and H2-utilizing Desulfovibrio spp., and propionate-utilizing Desulfobulbus spp. When the mucin pump was switched off, the multichamber system reverted to a state close to its original equilibrium. These data provide further evidence that sulfated polysaccharides such as mucin may be a source of sulfate for SRB in the human large gut.     

Use of a three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria.

A mixed culture of human fecal bacteria was grown for 120 days in a three-stage continuous culture system. To reproduce some of the nutritional and pH characteristics of the large gut, each vessel had a different operating volume (0.3, 0.5, and 0.8 liter) and pH (6.0, 6.5, and 7.0). A mixture of polysaccharides and proteins was used as carbon and nitrogen sources. Measurements of H2, CH4, S2-, sulfate reduction rates, sulfate-reducing bacteria (SRB), and volatile fatty acids were made throughout the experiment. After 48 days of running, porcine gastric mucin (5.8 g/day) was independently fed to vessel 1 of the multichamber system. The mucin was extensively degraded as evidenced by the stimulation of volatile fatty acid production. In the absence of mucin, sulfate-reducing activity was comparatively insignificant and methanogenesis was the major route for the disposal of electrons. The reverse occurred upon the addition of mucin; sulfate reduction predominated and methanogenesis was completely inhibited. This was attributed to release of sulfate from the mucin which enabled SRB to outcompete methanogenic bacteria for H2. SRB stimulated by mucin were acetate-utilizing Desulfobacter spp., lactate- and H2-utilizing Desulfovibrio spp., and propionate-utilizing Desulfobulbus spp. When the mucin pump was switched off, the multichamber system reverted to a state close to its original equilibrium. These data provide further evidence that sulfated polysaccharides such as mucin may be a source of sulfate for SRB in the human large gut.