Microbial Community Structure and Functions in Ethanol-Fed Sulfate Removal Bioreactors for Treatment of Mine Water.
ABSTRACT: Sulfate-rich mine water must be treated before it is released into natural water bodies. We tested ethanol as substrate in bioreactors designed for biological sulfate removal from mine water containing up to 9 g L-1 sulfate, using granular sludge from an industrial waste water treatment plant as inoculum. The pH, redox potential, and sulfate and sulfide concentrations were measured twice a week over a maximum of 171 days. The microbial communities in the bioreactors were characterized by qPCR and high throughput amplicon sequencing. The pH in the bioreactors fluctuated between 5.0 and 7.7 with the highest amount of up to 50% sulfate removed measured around pH 6. Dissimilatory sulfate reducing bacteria (SRB) constituted only between 1% and 15% of the bacterial communities. Predicted bacterial metagenomes indicated a high prevalence of assimilatory sulfate reduction proceeding to formation of l-cystein and acetate, assimilatory and dissimilatory nitrate reduction, denitrification, and oxidation of ethanol to acetaldehyde with further conversion to ethanolamine, but not to acetate. Despite efforts to maintain optimal conditions for biological sulfate reduction in the bioreactors, only a small part of the microorganisms were SRB. The microbial communities were highly diverse, containing bacteria, archaea, and fungi, all of which affected the overall microbial processes in the bioreactors. While it is important to monitor specific physicochemical parameters in bioreactors, molecular assessment of the microbial communities may serve as a tool to identify biological factors affecting bioreactor functions and to optimize physicochemical attributes for ideal bioreactor performance.
Project description:Two continuous-flow bench-scale bioreactor systems populated by mixed communities of acidophilic sulfate-reducing bacteria were constructed and tested for their abilities to promote the selective precipitation of transition metals (as sulfides) present in synthetic mine waters, using glycerol as electron donor. The objective with the first system (selective precipitation of copper from acidic mine water containing a variety of soluble metals) was achieved by maintaining a bioreactor pH of ? 2.2-2.5. The second system was fed with acidic (pH 2.5) synthetic mine water containing 3 mM of both zinc and ferrous iron, and varying concentrations (0.5-30 mM) of aluminium. Selective precipitation of zinc sulfide was possible by operating the bioreactor at pH 4.0 and supplementing the synthetic mine water with 4 mM glycerol. Analysis of the microbial populations in the bioreactors showed that they changed with varying operational parameters, and novel acidophilic bacteria (including one sulfidogen) were isolated from the bioreactors. The acidophilic sulfidogenic bioreactors provided 'proof of principle' that segregation of metals present in mine waters is possible using simple online systems within which controlled pH conditions are maintained. The modular units are versatile and robust, and involve minimum engineering complexity.
Project description:Sulfate is present in foods, beverages, and drinking water. Its reduction and concentration in the gut depend on the intestinal microbiome activity, especially sulfate-reducing bacteria (SRB), which can be involved in inflammatory bowel disease (IBD). Assimilatory sulfate reduction (ASR) is present in all living organisms. In this process, sulfate is reduced to hydrogen sulfide and then included in cysteine and methionine biosynthesis. In contrast to assimilatory sulfate reduction, the dissimilatory process is typical for SRB. A terminal product of this metabolism pathway is hydrogen sulfide, which can be involved in gut inflammation and also causes problems in industries (due to corrosion effects). The aim of the review was to compare assimilatory and dissimilatory sulfate reduction (DSR). These processes occur in some species of intestinal bacteria (e.g., Escherichia and Desulfovibrio genera). The main attention was focused on the description of genes and their location in selected strains. Their coding expression of the enzymes is associated with anabolic processes in various intestinal bacteria. These analyzed recent advances can be important factors for proposing possibilities of metabolic pathway extension from hydrogen sulfide to cysteine in intestinal SRB. The switch from the DSR metabolic pathway to the ASR metabolic pathway is important since toxic sulfide is not produced as a final product.
Project description:The effect of sulfate addition on the stability of, and microbial community behavior in, low-temperature anaerobic expanded granular sludge bed-based bioreactors was investigated at 15°C. Efficient bioreactor performance was observed, with chemical oxygen demand (COD) removal efficiencies of >90%, and a mean SO(2-) 4 removal rate of 98.3%. In situ methanogensis appeared unaffected at a COD: SO(2-) 4 influent ratio of 8:1, and subsequently of 3:1, and was impacted marginally only when the COD: SO(2-) 4 ratio was 1:2. Specific methanogenic activity assays indicated a complex set of interactions between sulfate-reducing bacteria (SRB), methanogens and homoacetogenic bacteria. SO(2-) 4 addition resulted in predominantly acetoclastic, rather than hydrogenotrophic, methanogenesis until >600 days of SO(2-) 4-influenced bioreactor operation. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes. Fluorescence in situ hybridizations (FISH), qPCR and microsensor analysis were combined to investigate the distribution of microbial groups, and particularly SRB and methanogens, along the structure of granular biofilms. qPCR data indicated that sulfidogenic genes were present in methanogenic and sulfidogenic biofilms, indicating the potential for sulfate reduction even in bioreactors not exposed to SO(2-) 4. Although the architecture of methanogenic and sulfidogenic granules was similar, indicating the presence of SRB even in methanogenic systems, FISH with rRNA targets found that the SRB were more abundant in the sulfidogenic biofilms. Methanosaeta species were the predominant, keystone members of the archaeal community, with the complete absence of the Methanosarcina species in the experimental bioreactor by trial conclusion. Microsensor data suggested the ordered distribution of sulfate reduction and sulfide accumulation, even in methanogenic granules.
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: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: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:Nitrate injection into oil reservoirs can prevent and remediate souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB). Nitrate stimulates nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) and heterotrophic nitrate-reducing bacteria (hNRB) that compete with SRB for degradable oil organics. Up-flow, packed-bed bioreactors inoculated with water produced from an oil field and injected with lactate, sulfate, and nitrate served as sources for isolating several NRB, including Sulfurospirillum and Thauera spp. The former coupled reduction of nitrate to nitrite and ammonia with oxidation of either lactate (hNRB activity) or sulfide (NR-SOB activity). Souring control in a bioreactor receiving 12.5 mM lactate and 6, 2, 0.75, or 0.013 mM sulfate always required injection of 10 mM nitrate, irrespective of the sulfate concentration. Community analysis revealed that at all but the lowest sulfate concentration (0.013 mM), significant SRB were present. At 0.013 mM sulfate, direct hNRB-mediated oxidation of lactate by nitrate appeared to be the dominant mechanism. The absence of significant SRB indicated that sulfur cycling does not occur at such low sulfate concentrations. The metabolically versatile Sulfurospirillum spp. were dominant when nitrate was present in the bioreactor. Analysis of cocultures of Desulfovibrio sp. strain Lac3, Lac6, or Lac15 and Sulfurospirillum sp. strain KW indicated its hNRB activity and ability to produce inhibitory concentrations of nitrite to be key factors for it to successfully outcompete oil field SRB.
Project description:Woodchip bioreactors are increasingly used to remove nitrate (NO<sub>3</sub> <sup>-</sup>) from agricultural drainage water in order to protect aquatic ecosystems from excess nitrogen. Nitrate removal in woodchip bioreactors is based on microbial processes, but the microbiomes and their role in bioreactor efficiency are generally poorly characterized. Using metagenomic analyses, we characterized the microbiomes from 3 full-scale bioreactors in Denmark, which had been operating for 4-7 years. The microbiomes were dominated by <i>Proteobacteria</i> and especially the genus <i>Pseudomonas</i>, which is consistent with heterotrophic denitrification as the main pathway of NO<sub>3</sub> <sup>-</sup> reduction. This was supported by functional gene analyses, showing the presence of the full suite of denitrification genes from NO<sub>3</sub> <sup>-</sup> reductases to nitrous oxide reductases. Genes encoding for dissimilatory NO<sub>3</sub> <sup>-</sup> reduction to ammonium were found only in minor proportions. In addition to NO<sub>3</sub> <sup>-</sup> reducers, the bioreactors harbored distinct functional groups, such as lignocellulose degrading fungi and bacteria, dissimilatory sulfate reducers and methanogens. Further, all bioreactors harbored genera of heterotrophic iron reducers and anaerobic iron oxidizers (<i>Acidovorax</i>) indicating a potential for iron-mediated denitrification. Ecological indices of species diversity showed high similarity between the bioreactors and between the different positions along the flow path, indicating that the woodchip resource niche was important in shaping the microbiome. This trait may be favorable for the development of common microbiological strategies to increase the NO<sub>3</sub> <sup>-</sup> removal from agricultural drainage water.
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:In river-lake systems, sediment and water column are two distinct habitats harboring different bacterial communities which play a crucial role in biogeochemical processes. In this study, we employed Phylogenetic Investigation of Communities by Reconstruction of Unobserved States to assess the potential functions and functional redundancy of the bacterial communities in sediment and water in a eutrophic river-lake ecosystem, Poyang Lake in China. Bacterial communities in sediment and water had distinct potential functions of carbon, nitrogen, and sulfur metabolisms as well as phosphorus cycle, while the differences between rivers and the lake were inconspicuous. Bacterial communities in sediment had a higher relative abundance of genes associated with carbohydrate metabolism, carbon fixation pathways in prokaryotes, methane metabolism, anammox, nitrogen fixation, and dissimilatory sulfate reduction than that of water column. Bacterial communities in water column were higher in lipid metabolism, assimilatory nitrate reduction, dissimilatory nitrate reduction, phosphonate degradation, and assimilatory sulfate reduction than that of sediment bacterial communities. Furthermore, the variations in functional composition were closely associated to the variations in taxonomic composition in both habitats. In general, the bacterial communities in water column had a lower functional redundancy than in sediment. Moreover, comparing to the overall functions, bacterial communities had a lower functional redundancy of nitrogen metabolism and phosphorus cycle in water column and lower functional redundancy of nitrogen metabolism in sediment. Distance-based redundancy analysis and mantel test revealed close correlations between nutrient factors and functional compositions. The results suggested that bacterial communities in this eutrophic river-lake system of Poyang Lake were vulnerable to nutrient perturbations, especially the bacterial communities in water column. The results enriched our understanding of the bacterial communities and major biogeochemical processes in the eutrophic river-lake ecosystems.