Co-existence of physiologically similar sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single organic electron donor.
ABSTRACT: A combination of culture-dependent and independent methods was used to study the co-existence of different sulfate-reducing bacteria (SRB) in an upflow anaerobic sludge bed reactor treating sulfate-rich wastewater. The wastewater was fed with ethanol as an external electron donor. Twenty six strains of SRB were randomly picked and isolated from the highest serial dilution that showed growth (i.e. 10(8)). Repetitive enterobacterial palindromic polymerase chain reaction and whole cell protein profiling revealed a low genetic diversity, with only two genotypes among the 26 strains obtained in the pure culture. The low genetic diversity suggests the absence of micro-niches within the reactor, which might be due to a low spatial and temporal micro-heterogeneity. The total 16S rDNA sequencing of two representative strains L3 and L7 indicated a close relatedness to the genus Desulfovibrio. The two strains differed in as many as five physiological traits, which might allow them to occupy distinct niches and thus co-exist within the same habitat. Whole cell hybridisation with fluorescently labeled oligonucleotide probes was performed to characterise the SRB community in the reactor. The isolated strains Desulfovibrio L3 and Desulfovibrio L7 were the most dominant SRB, representing 30-35% and 25-35%, respectively, of the total SRB community. Desulfobulbus-like bacteria contributed for 20-25%, and the Desulfobacca acetoxidans-specific probe targeted approximately 15-20% of the total SRB. The whole cell hybridisation results thus revealed a consortium of four different species of SRB that can be enriched and maintained on a single energy source in a full-scale sulfidogenic reactor.
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.
Project description:In this study, the sulfate-reducing bacteria, (SRB) were identified and reported for the first time through analysis of functional gene dsrAB, from the DNA of sediment samples collected from 10 sites of the Chilika lake. The finding illustrates Forty six Operational Taxonomic Units (OTUs), identified from the DGGE which were obtained from the 10 sediment samples. Of these, 34 OTUs exhibited around 78-96% sequence similarity and 12 OTUs showed 97 to 100% sequence similarity to the dsrAB gene of reported type strains of SRB. The sequence information obtained revealed the presence and distribution of diverse types of SRB which include phylotypes related to Desulfovibrio, Desulfonatronovibrio, Desulfomicrobium, Desulfobotulous and Desulfobacca. Upon comparison of dsrAB gene sequences of SRB obtained through this study with those collected from the GenBank, and through the dendrogram constructed, it was observed that except 13 OTUs that clustered closely with the reported type strains, all other 36 OTUs clustered distantly and had no representative member of SRB. This indicated the presence of phylogenetically diverse groups of SRB inhabiting the lake Chilika.
Project description:Recently, sulfate-reducing granular sludge has been developed for application in sulfate-laden water and wastewater treatment. However, little is known about biomass stratification and its effects on the bioprocesses inside the granular bioreactor. A comprehensive investigation followed by a verification trial was therefore conducted in the present work. The investigation focused on the performance of each sludge layer, the internal hydrodynamics and microbial community structures along the height of the reactor. The reactor substratum (the section below baffle 1) was identified as the main acidification zone based on microbial analysis and reactor performance. Two baffle installations increased mixing intensity but at the same time introduced dead zones. Computational fluid dynamics simulation was employed to visualize the internal hydrodynamics. The 16S rRNA gene of the organisms further revealed that more diverse communities of sulfate-reducing bacteria (SRB) and acidogens were detected in the reactor substratum than in the superstratum (the section above baffle 1). The findings of this study shed light on biomass stratification in an SRB granular bioreactor to aid in the design and optimization of such reactors.
Project description:Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.
Project description:Bacteria of the genus Desulfovibrio belong to the group of Sulphate Reducing Bacteria (SRB). SRB generate significant liabilities in the petroleum industry, mainly due to their ability to microbiologically induce corrosion, biofilm formation and H<sub>2</sub>S production. Bacteriophages are an alternative control method for SRB, whose information for this group of bacteria however, is scarce. The present study developed a workflow for the identification of complete prophages in Desulfovibrio. Poly-lysogenesis was shown to be common in Desulfovibrio. In the 47 genomes analyzed 53 complete prophages were identified. These were classified within the order Caudovirales, with 69.82% belonging to the Myoviridade family. More than half the prophages identified have genes coding for lysozyme or holin. Four of the analyzed bacterial genomes present prophages with identity above 50% in the same strain, whose comparative analysis demonstrated the existence of colinearity between the sequences. Of the 17 closed bacterial genomes analyzed, 6 have the CRISPR-Cas system classified as inactive. The identification of bacterial poly-lysogeny, the proximity between the complete prophages and the possible inactivity of the CRISPR-Cas in closed bacterial genomes analyzed allowed the choice of poly-lysogenic strains with prophages belonging to the Myoviridae family for the isolation of prophages and testing of related strains for subsequent studies.
Project description:Biological sulfate-reducing bacteria (SRB) may be effective in removing toxic lead and mercury ions (Pb(II) and Hg(II)) from wet flue gas desulfurization (FGD) wastewater through anaerobic sulfite reduction. To confirm this hypothesis, a sulfite-reducing up-flow anaerobic sludge blanket reactor was set up to treat FGD wastewater at metal loading rates of 9.2?g/m(3)-d Pb(II) and 2.6?g/m(3)-d Hg(II) for 50 days. The reactor removed 72.5?±?7% of sulfite and greater than 99.5% of both Hg(II) and Pb(II). Most of the removed lead and mercury were deposited in the sludge as HgS and PbS. The contribution of cell adsorption and organic binding to Pb(II) and Hg(II) removal was 20.0?±?0.1% and 1.8?±?1.0%, respectively. The different bioavailable concentration levels of lead and mercury resulted in different levels of lethal toxicity. Cell viability analysis revealed that Hg(II) was less toxic than Pb(II) to the sludge microorganisms. In the batch tests, increasing the Hg(II) feeding concentration increased sulfite reduction rates. In conclusion, a sulfite-reducing reactor can efficiently remove sulfite, Pb(II) and Hg(II) from FGD wastewater.
Project description:A comparative study of the kinetic characteristics (specific activity, initial and maximum rate, and affinity for substrates) of key enzymes of assimilatory sulfate reduction (APS reductase and dissimilatory sulfite reductase) in cell-free extracts of sulphate-reducing bacteria (SRB) from various biotopes was performed. The material for the study represented different strains of SRB from various ecotopes. Microbiological (isolation and cultivation), biochemical (free cell extract preparation) and chemical (enzyme activity determination) methods served in defining kinetic characteristics of SRB enzymes. The determined affinity data for substrates (i.e., sulfite) were 10 times higher for SRB strains isolated from environmental (soil) ecotopes than for strains from the human intestine. The maximum rate of APS reductase reached 0.282-0.862 µmol/min×mg-1 of protein that is only 10 to 28% higher than similar initial values. The maximum rate of sulfite reductase for corrosive relevant collection strains and SRB strains isolated from heating systems were increased by 3 to 10 times. A completely different picture was found for the intestinal SRB Vmax in the strains Desulfovibrio piger Vib-7 (0.67 µmol/min × mg-1 protein) and Desulfomicrobium orale Rod-9 (0.45 µmol/min × mg-1 protein). The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction-sulfite reductase, but not APS reductase. The data obtained from the activity of sulfate reduction enzymes indicated the adaptive plasticity of SRB strains that is manifested in the change in enzymatic activity.
Project description:We studied the peptide-degrading anaerobic communities of methanogenic reactors from two mesophilic full-scale modified upflow anaerobic sludge blanket (UASB) reactors treating brewery wastewater in Colombia. Most probable number (MPN) counts varied between 7.1 x 10(8) and 6.6 × 10(9) bacteria/g volatile suspended solids VSS (Methanogenic Reactor 1) and 7.2 × 10(6) and 6.4 × 10(7) bacteria/g (VSS) (Methanogenic Reactor 2). Metabolites detected in the highest positive MPN dilutions in both reactors were mostly acetate, propionate, isovalerate and, in some cases, negligible concentrations of butyrate. Using the highest positive dilutions of MPN counts, 50 dominant strains were isolated from both reactors, and 12 strains were selected for sequencing their 16S rRNA gene based on their phenotypic characteristics. The small-subunit rRNA gene sequences indicated that these strains were affiliated to the families Propionibacteriaceae, Clostridiaceae and Syntrophomonadaceae in the low G + C gram-positive group and Desulfovibrio spp. in the class ?-Proteobacteria. The main metabolites detected in the highest positive dilutions of MPN and the presence of Syntrophomonadaceae indicate the effect of the syntrophic associations on the bioconversion of these substrates in methanogenic reactors. Additionally, the potential utilization of external electron acceptors for the complete degradation of amino acids by Clostridium strains confirms the relevance of these acceptors in the transformation of peptides and amino acids in these systems.
Project description:The concentration of sulphate present in wastewater can vary from 10 to 500 mg SO<sub>4</sub><sup>2-</sup>/L. During anaerobic conditions, sulphate is reduced to sulphide by sulphate-reducing bacteria (SRB). Sulphide generation is undesired in wastewater treatment plants (WWTPs). Previous research indicated that SRB are inhibited by the presence of electron acceptors (such as O<sub>2</sub>, NO<sub>3</sub> and NO<sub>2</sub>). However, the contact times and concentrations used in those studies are by far higher than occur in WWTPs. Since sulphide can influence the biological nitrogen and phosphorus removal processes, this research aimed to understand how the different electron acceptors commonly present in biological nutrient removal (BNR) systems can affect the proliferation of SRB. For this purpose, a culture of SRB was enriched in a sequencing batch reactor (approx. 88% of the total bacteria population). Once enriched, the SRB were exposed for 2 h to typical concentrations of electron acceptors like those observed in BNR systems. Their activity was assessed using three different types of electron donors (acetate, propionate and lactate). Oxygen was the most inhibiting electron acceptor regardless the carbon source used. After exposure to oxygen and when feeding acetate, an inactivation time in the sulphate reduction activity was observed for 1.75 h. Once the sulphate reduction activity resumed, only 60% of the original activity was recovered. It is suggested that the proliferation of SRB is most likely to occur in BNR plants with an anaerobic fraction higher than 15% and operating at sludge retention times higher than 20 days (at a temperature of 20 °C). These results can be used to implement strategies to control the growth of sulphate reducers that might compete for organic carbon with phosphate-accumulating organisms.
Project description:Nitrate is an inhibitor of sulfate-reducing bacteria (SRB). In petroleum production sites, amendments of nitrate and nitrite are used to prevent SRB production of sulfide that causes souring of oil wells. A better understanding of nitrate stress responses in the model SRB, Desulfovibrio vulgaris Hildenborough and Desulfovibrio alaskensis G20, will strengthen predictions of environmental outcomes of nitrate application. Nitrate inhibition of SRB has historically been considered to result from the generation of small amounts of nitrite, to which SRB are quite sensitive. Here we explored the possibility that nitrate might inhibit SRB by a mechanism other than through nitrite inhibition. We found that nitrate-stressed D. vulgaris cultures grown in lactate-sulfate conditions eventually grew in the presence of high concentrations of nitrate, and their resistance continued through several subcultures. Nitrate consumption was not detected over the course of the experiment, suggesting adaptation to nitrate. With high-throughput genetic approaches employing TnLE-seq for D. vulgaris and a pooled mutant library of D. alaskensis, we determined the fitness of many transposon mutants of both organisms in nitrate stress conditions. We found that several mutants, including homologs present in both strains, had a greatly increased ability to grow in the presence of nitrate but not nitrite. The mutated genes conferring nitrate resistance included the gene encoding the putative Rex transcriptional regulator (DVU0916/Dde_2702), as well as a cluster of genes (DVU0251-DVU0245/Dde_0597-Dde_0605) that is poorly annotated. Follow-up studies with individual D. vulgaris transposon and deletion mutants confirmed high-throughput results. We conclude that, in D. vulgaris and D. alaskensis, nitrate resistance in wild-type cultures is likely conferred by spontaneous mutations. Furthermore, the mechanisms that confer nitrate resistance may be different from those that confer nitrite resistance.