Successional development of sulfate-reducing bacterial populations and their activities in a wastewater biofilm growing under microaerophilic conditions.
ABSTRACT: A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 microM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 microm from the surface during all stages of biofilm development. The first sulfide production of 0.32 micromol of H(2)S m(-2) s(-1) was detected below ca. 500 microm in the 3rd week and then gradually increased to 0.70 micromol H(2)S m(-2) s(-1) in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 x 10(9) cells cm(-3) and 3.6 x 10(9) cells cm(-3), respectively, in the surface 400 microm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.
Project description:We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [(14)C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H(2)-utilizing and (14)CO(2)-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H(2)-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H(2) utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.
Project description:Simultaneous production of sulfide and methane by anaerobic sewer biofilms has recently been observed, suggesting that sulfate-reducing bacteria (SRB) and methanogenic archaea (MA), microorganisms known to compete for the same substrates, can coexist in this environment. This study investigated the community structures and activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 ?m) using a combination of microelectrode measurements, molecular techniques, and mathematical modeling. It was seen that sulfide was mainly produced in the outer layer of the biofilm, between the depths of 0 and 300 ?m, which is in good agreement with the distribution of SRB population as revealed by cryosection-fluorescence in situ hybridization (FISH). SRB had a higher relative abundance of 20% on the surface layer, which decreased gradually to below 3% at a depth of 400 ?m. In contrast, MA mainly inhabited the inner layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of 200 ?m and 700 ?m, respectively, from the biofilm surface layer. High-throughput pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly affiliated with five genera, Desulfobulbus, Desulfomicrobium, Desulfovibrio, Desulfatiferula, and Desulforegula, while about 90% of the MA population belonged to the genus Methanosaeta. The spatial organizations of SRB and MA revealed by pyrosequencing were consistent with the FISH results. A biofilm model was constructed to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit between model predictions and the experimental data indicate that the coexistence and spatial structure of SRB and MA in the biofilm resulted from the microbial types and their metabolic transformations and interactions with substrates.
Project description:Sulfate-reducing bacteria (SRB) have been studied extensively in the petroleum industry due to their role in corrosion, but very little is known about sulfur-oxidizing bacteria (SOB), which drive the oxidization of sulfur-compounds produced by the activity of SRB in petroleum reservoirs. Here, we surveyed the community structure, diversity and abundance of SRB and SOB simultaneously based on 16S rRNA, dsrB and soxB gene sequencing, and quantitative PCR analyses, respectively in petroleum reservoirs with different physicochemical properties. Similar to SRB, SOB were found widely inhabiting the analyzed reservoirs with high diversity and different structures. The dominant SRB belonged to the classes Deltaproteobacteria and Clostridia, and included the Desulfotignum, Desulfotomaculum, Desulfovibrio, Desulfobulbus, and Desulfomicrobium genera. The most frequently detected potential SOB were Sulfurimonas, Thiobacillus, Thioclava, Thiohalomonas and Dechloromonas, and belonged to Betaproteobacteria, Alphaproteobacteria, and Epsilonproteobacteria. Among them, Desulfovibrio, Desulfomicrobium, Thioclava, and Sulfurimonas were highly abundant in the low-temperature reservoirs, while Desulfotomaculum, Desulfotignum, Thiobacillus, and Dechloromonas were more often present in high-temperature reservoirs. The relative abundances of SRB and SOB varied and were present at higher proportions in the relatively high-temperature reservoirs. Canonical correspondence analysis also revealed that the SRB and SOB communities in reservoirs displayed high niche specificity and were closely related to reservoir temperature, pH of the formation brine, and sulfate concentration. In conclusion, this study extends our knowledge about the distribution of SRB and SOB communities in petroleum reservoirs.
Project description: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.
Project description:Sodium bisulfite (SBS) is used as an oxygen scavenger to decrease corrosion in pipelines transporting brackish subsurface water used in the production of bitumen by steam-assisted gravity drainage. Sequencing 16S rRNA gene amplicons has indicated that SBS addition increased the fraction of the sulfate-reducing bacteria (SRB) Desulfomicrobium, as well as of Desulfocapsa, which can also grow by disproportionating sulfite into sulfide, sulfur, and sulfate. SRB use cathodic H2, formed by reduction of aqueous protons at the iron surface, or use low potential electrons from iron and aqueous protons directly for sulfate reduction. In order to reveal the effects of SBS treatment in more detail, metagenomic analysis was performed with pipe-associated solids (PAS) scraped from a pipe section upstream (PAS-616P) and downstream (PAS-821TP) of the SBS injection point. A major SBS-induced change in microbial community composition and in affiliated hynL genes for the large subunit of [NiFe] hydrogenase was the appearance of sulfur-metabolizing Epsilonproteobacteria of the genera Sulfuricurvum and Sulfurovum. These are chemolithotrophs, which oxidize sulfide or sulfur with O2 or reduce sulfur with H2. Because O2 was absent, this class likely catalyzed reduction of sulfur (S(0)) originating from the metabolism of bisulfite with cathodic H2 (or low potential electrons and aqueous protons) originating from the corrosion of steel (Fe(0)). Overall this accelerates reaction of of S(0) and Fe(0) to form FeS, making this class a potentially powerful contributor to microbial corrosion. The PAS-821TP metagenome also had increased fractions of Deltaproteobacteria including the SRB Desulfomicrobium and Desulfocapsa. Altogether, SBS increased the fraction of hydrogen-utilizing Delta- and Epsilonproteobacteria in brackish-water-transporting pipelines, potentially stimulating anaerobic pipeline corrosion if dosed in excess of the intended oxygen scavenger function.
Project description:Pitting corrosion due to microbial activity is the most severe type of corrosion that occurs in ship hull. Since biogenic sulfide produced by sulfate-reducing bacteria (SRB) is involved in the acceleration of pitting corrosion of marine vessels, so it is important to collect information about SRB community involved in maritime vessel failure. We investigated the SRB community on corroded hull portion of the ship. With the use of common cultural method and 16S rDNA sequencing, ten bacteria with sulfate reduction ability were isolated and identified. They belonged to both traditional (Desulfovibrio, Desulfotomaculum) and non-traditional (Citrobacter) sulfate-reducing bacteria. All the isolates were able to produce a high amount of sulfide. However, only traditional isolates were showing the amplification for the SRB-specific gene, dsrAB. Further studies on corrosion potential of these two groups of bacteria showed that in spite of high sulfide and biofilm production by non-traditional SRB, they are less aggressive towards the mild steel compare to the traditional group.
Project description:Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low delta(34)S values relative to delta(34)S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of approximately 2), although possibly within less acidic microenvironments. Interestingly, delta(34)S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.
Project description:<h4>Introduction</h4>Increased numbers of sulfate-reducing bacteria (SRB) are often found in the feces of people and animals with inflammatory bowel disease. The final products of their metabolism are hydrogen sulfide and acetate, which are produced during dissimilatory sulfate reduction process.<h4>Objectives</h4>The aim of the study was to monitor processes concerning sulfate reduction microbial metabolisms, including: the main microbial genera monitoring and their hydrogen sulfide production in the intestines of healthy and not healthy individuals, phylogenetic analysis of SRB isolates, cluster analysis of SRB physiological and biochemical parameters, SRB growth kinetic parameters calculation, same as the application of the two-factor dispersion analysis for finding relationship between SRB biomass accumulation, temperature and pH. Feces samples from healthy people and patients with colitis were used for isolation of sulfate-reducing microbial communities.<h4>Methods</h4>Microbiological, biochemical, biophysical, molecular biology methods, and statistical processing of the results have been used for making an evaluation of gained results.<h4>Results</h4>Two dominant SRB morphotypes differed in colony size and quantitative ratio in the feces of healthy and colitis patients were observed and identified. In the feces of healthy people, 93% of SRB of morphotype I prevailed <i>(Desulfovibrio)</i> while morphotype II made only 7% <i>(Desulfomicrobium)</i>; in the feces of patients with colitis, the ratio of these morphotypes was 99:1, respectively. Hydrogen sulfide concentrations are also higher in the feces of people with colitis and certain synergy effects exist among acetate produced by SRB.<h4>Conclusions</h4>The study results brought important findings concerning colony environments with developed colitis and these findings can lead to the development of possible risk indicators of ulcerative colitis prevalence.
Project description:Sulfate-reducing bacteria (SRB) participate in microbially induced corrosion (MIC) of equipment and H2S-driven reservoir souring in oil field sites. Successful management of industrial processes requires methods that allow robust monitoring of microbial communities. This study investigated the applicability of denaturing high-performance liquid chromatography (DHPLC) targeting the dissimilatory sulfite reductase ß-subunit (dsrB) gene for monitoring SRB communities in oil field samples from the North Sea, the United States, and Brazil. Fifteen of the 28 screened samples gave a positive result in real-time PCR assays, containing 9 × 10(1) to 6 × 10(5) dsrB gene copies ml(-1). DHPLC and denaturing gradient gel electrophoresis (DGGE) community profiles of the PCR-positive samples shared an overall similarity; both methods revealed the same samples to have the lowest and highest diversity. The SRB communities were diverse, and different dsrB compositions were detected at different geographical locations. The identified dsrB gene sequences belonged to several phylogenetic groups, such as Desulfovibrio, Desulfococcus, Desulfomicrobium, Desulfobulbus, Desulfotignum, Desulfonatronovibrio, and Desulfonauticus. DHPLC showed an advantage over DGGE in that the community profiles were very reproducible from run to run, and the resolved gene fragments could be collected using an automated fraction collector and sequenced without a further purification step. DGGE, on the other hand, included casting of gradient gels, and several rounds of rerunning, excising, and reamplification of bands were needed for successful sequencing. In summary, DHPLC proved to be a suitable tool for routine monitoring of the diversity of SRB communities in oil field samples.
Project description:Here we describe the diversity and activity of sulfate-reducing bacteria (SRB) in sulfidogenic bioreactors by using the simultaneous analysis of PCR products obtained from DNA and RNA of the 16S rRNA and dissimilatory sulfite reductase (dsrAB) genes. We subsequently analyzed the amplified gene fragments by using denaturing gradient gel electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE profiles than in the DNA-based profiles, indicating marked differences in the populations present and in those that were metabolically active at the time of sampling. Comparative sequence analyses of the bands obtained from rRNA and dsrB DGGE profiles were congruent, revealing the same SRB populations. Bioreactors that received either ethanol or isopropanol as an energy source showed the presence of SRB affiliated with Desulfobulbus rhabdoformis and/or Desulfovibrio sulfodismutans, as well as SRB related to the acetate-oxidizing Desulfobacca acetoxidans. The reactor that received wastewater containing a diverse mixture of organic compounds showed the presence of nutritionally versatile SRB affiliated with Desulfosarcina variabilis and another acetate-oxidizing SRB, affiliated with Desulfoarculus baarsii. In addition to DGGE analysis, we performed whole-cell hybridization with fluorescently labeled oligonucleotide probes to estimate the relative abundances of the dominant sulfate-reducing bacterial populations. Desulfobacca acetoxidans-like populations were most dominant (50 to 60%) relative to the total SRB communities, followed by Desulfovibrio-like populations (30 to 40%), and Desulfobulbus-like populations (15 to 20%). This study is the first to identify metabolically active SRB in sulfidogenic bioreactors by using the functional gene dsrAB as a molecular marker. The same approach can also be used to infer the ecological role of coexisting SRB in other habitats.