Marble stone processing powder residue as chemical adjuvant for the biologic treatment of acid mine drainage

Marble stone powder (calcite tailing) is a residual material resulting from the cutting and polishing of marble stone. Its use as adjuvant for the biologic treatment of acid mine drainage is studied. The performance of a combined chemical/biologic packed bed reactor containing a sulphate-reducing bacteria inoculum and calcite tailing as neutralising agent was compared with the individual neutralisation and biologic steps. Unlike the individual steps, the combined column is efficient in terms of metals and sulphate removal and acidity neutralisation, producing treated water suitable for irrigation. This work emphasizes on the key role played by waste calcite tailing in the inlet of the reactor. By thus adjusting pH to values adequate for sulphate-reducing bacteria water suitable for irrigation can be obtained in a single equipment with low energy demand. This goal would be impossible without the neutralisation action.     

Metal removal by sulphate-reducing bacteria from natural and constructed wetlands

The use of wetlands is a promising technology to treat acid mine drainage, yet there is little understanding of the fundamental biological processes involved. They are considered to centre on the complex anaerobic ecology within sediments and involve the removal of metals by sulphate-reducing bacteria (SRB). These bacteria generate hydrogen sulphide and cause precipitation of metals from solution as the insoluble metal sulphide. Sulphate-reducing bacteria have been isolated from natural and constructed wetlands receiving acid mine drainage. Sulphide production by isolates and removal of the metals iron, manganese and zinc were measured, as well as utilization of a range of carbon sources. Marked ecological differences between the wetlands were reflected in population composition of SRB enrichments, and these consortia displayed significant differences in sulphide generation and rates of metal removal from solution. Rates of metal removal did not correlate with sulphide generation in all cultures, suggesting the involvement of other biological mechanisms of metal removal. Differences in substrate utilization have highlighted the need for further investigation of carbon flow and potential carbon sources within constructed wetlands.     

Immunofluorescence of sulphate-reducing bacteria

An indirect fluorescent antibody technique was used as a method of rapidly assessing and identifying sulphate-reducing bacteria. Five specific antisera and one polyvalent serum were raised and tested against 44 strains of the genera Desulfovibrio and Desulfotomaculum along with 4 control organisms. Immunofluorescence was found to be mainly strain specific with the sulphate-reducing bacteria although weak fluorescence was seen both within and between recognised groups. A polyvalent antiserum was successfully used to detect sulphate-reducing bacteria. No interference from 4 control organisms was found.     

Biological sulphate reduction using food industry wastes as carbon sources

Biological treatment with dissimilatory sulphate-reducing bacteria has been considered the most promising alternative for decontamination of sulphate rich effluents. These wastewaters are usually deficient in electron donors and require their external addition to achieve complete sulphate reduction. The aim of the present study was to investigate the possibility of using food industry wastes (a waste from the wine industry and cheese whey) as carbon sources for dissimilatory sulphate-reducing bacteria. The results show that these wastes can be efficiently used by these bacteria provided that calcite tailing is present as a neutralizing and buffer material. A 95 and 50 % sulphate reduction was achieved within 20 days of experiment by a consortium of dissimilatory sulphate-reducing bacteria grown on media containing waste from the wine industry or cheese whey respectively. Identification of the dissimilatory sulphate-reducing bacteria community using the dsr gene revealed the presence of the species Desulfovibrio fructosovorans, Desulfovibrio aminophilus and Desulfovibrio desulfuricans. The findings of the present study emphasise the potential of using wastes from the wine industry as carbon source for dissimilatory sulphate-reducing bacteria, combined with calcite tailing, in the development of cost effective and environmentally friendly bioremediation processes.     

Molecular analysis of a sulphate-reducing consortium used to treat metal-containing effluents

A sulphate-reducing consortium used in a bioprocess to remove toxic metals from solution as insoluble sulphides, was characterised using molecular (PCR-based) and traditional culturing techniques. After prolonged cultivation under anoxic biofilm-forming conditions, the mixed culture contained a low diversity of sulphate-reducing bacteria, dominated by one strain closely related to Desulfomicrobium norvegicum, identified by three independent PCR-based analyses. The genetic targets used were the 16S rRNA gene, the 16S-23S rRNA gene intergenic spacer region and the disulfite reductase (dsr) gene, which is conserved amongst all known sulphate-reducing bacteria. This organism was also isolated by conventional anaerobic techniques, confirming its presence in the mixed culture. A surprising diversity of other non-sulphate-reducing facultative and obligate anaerobes were detected, supporting a model of the symbiotic/commensal nature of carbon and energy fluxes in such a mixed culture while suggesting the physiological capacity for a wide range of biotransformations by this stable microbial consortium.     

Effect of molybdate ions on methanation of simulated and natural waste-waters

Summary Sulphate in concentrations of 500 and 1000 mg SO₄-S/l did not inhibit methanation of synthetic waste-water (acetate + methanol + glucose) by sludge from a digester treating neutral spent sulphite process effluents. The role of sulphate reducers in the conversion of those substrates was minor although sulphate-reducing bacteria were present with a viable count similar to that of methane-producing bacteria in the sludge. Neutral spent sulphite liquor was partially converted to methane (40% of chemical oxygen demand) under these conditions.Molybdate (20 mM) inhibited methanation of both synthetic waste-water and neutral spent sulphite liquor. Acetate accumulated in glucose plus molybdate media. Molybdate had a direct inhibitory effect on enriched acetoclastic methane-producing bacteria. Molybdate was bacteriocidic to sulphate-reducing bacteria and bacteriostatic to methane-producing bacteria.     

Utilization of cathodic hydrogen by hydrogen-oxidizing bacteria

Summary Hydrogen is consumed by methanogenic, sulphate-reducing, and homoacetogenic bacteria and members of these bacterial groups are able to grow chemolithotrophically with hydrogen as sole energy source. Cathodic hydrogen consumption by sulphate-reducing bacteria has been proposed as one of the factors in the anaerobic corrosion of metals. Desulfovibrio spp. were able to utilize cathodic hydrogen from mild steel as the only source of energy for growth with sulphate or nitrate as terminal electron acceptor. Other hydrogen-oxidizing bacteria such as Methanospirillum hungatei, Acetobacterium woodii and Wolinella succinogenes were also able to utilize cathodic hydrogen from mild steel for energy generation and growth. Weight loss studies of mild steel coupons under different growth conditions of Desulfovibrio spp. indicated that hydrogen removal alone is not the cause of corrosion and the depolarization phenomenon probably plays a role only in the initiation of the anaerobic microbial corrosion process.     

Aluminum and sulphate removal by a highly Al-resistant dissimilatory sulphate-reducing bacteria community

A highly Al-resistant dissimilatory sulphate-reducing bacteria community was isolated from sludge of the wetland of Urgeiri?a mine (community W). This community showed excellent sulphate removal at the presence of Al3?. After 27 days of incubation, 73, 86 and 81% of sulphate was removed in the presence of 0.48, 0.90 and 1.30 mM of Al3?, respectively. Moreover, Al3? was simultaneously removed: 55, 85 and 78% of metal was removed in the presence of 0.48, 0.90 and 1.30 mM of Al3?, respectively. The dissociation of aluminium-lactate soluble complexes due to lactate consumption by dissimilatory sulphate-reducing bacteria can be responsible for aluminum removal, which probably precipitates as insoluble aluminium hydroxide. Phylogenetic analysis of 16S rRNA gene showed that this community was mainly composed by bacteria closely related to Desulfovibrio desulfuricans. However, bacteria affiliated to Proteus and Ralstonia were also present in the community.     

Methanogenesis and sulfate reduction in timber and drainage water from a gold mine

Biogenesis of methane in the heartwood of diseased trees has been shown, but never in timber in service. Studies were undertaken to establish whether methan‐ogens and sulfate‐reducers were present in wooden pit props and drainage water from underground sites in a gold mine. The predominant methanogen in the mine ecosystem was tentatively identified as Methanobacterium bryantii. The sulfate‐reducers comprised Desulfovibrio desulfuricans and Desulfotomaculum antarcticum. Most probable numbers (MPN) of bacteria indicated that 3.5 × 10⁵ methanogenic and 7.9 × 10³ sulfate‐reducing bacteria were present per milliliter of stagnant drainage water. MPN values per gram of timber were lower for methanogens but comparable for sulfate‐reducers. Laboratory model systems predicted a maximum rate of methan‐ogenesis of 2.3 mL methane/g wood per day; however, rates would never attain this value because of nutrient limitations and environmental restrictions. Analysis of gas samples extracted from sealed areas of the gold mine verified the presence of methane.     

Related assemblages of sulphate-reducing bacteria associated with ultradeep gold mines of South Africa and deep basalt aquifers of Washington State

We characterized the diversity of sulphate-reducing bacteria (SRB) associated with South African gold mine boreholes and deep aquifer systems in Washington State, USA. Sterile cartridges filled with crushed country rock were installed on two hydrologically isolated and chemically distinct sites at depths of 3.2 and 2.7 km below the land surface (kmbls) to allow development of biofilms. Enrichments of sulphate-reducing chemolithotrophic (H2) and organotrophic (lactate) bacteria were established from each site under both meso- and thermophilic conditions. Dissimilatory sulphite reductase (Dsr) and 16S ribosomal RNA (rRNA) genes amplified from DNA extracted from the cartridges were most closely related to the Gram-positive species Desulfotomaculum thermosapovorans and Desulfotomaculum geothermicum, or affiliated with a novel deeply branching clade. The dsr sequences recovered from the Washington State deep aquifer systems affiliated closely with the South African sequences, suggesting that Gram-positive sulphate-reducing bacteria are widely distributed in the deep subsurface.     

Methanogenic and Sulphate-Reducing Microbial Communities in Deep Groundwater of Crystalline Rock Fractures in Olkiluoto,Finland

The long-term safety of final disposal of spent nuclear fuel in the deep geosphere is dependent on stability of biogeochemical conditions at the disposal site. Microbial processes, such as sulphate reduction and methanogenesis, may have profound effects on site biogeochemistry. In this study, sulphate-reducing bacteria and methane-producing archaea were investigated at depths ranging from 68 to 545 m in crystalline rock fractures at an intended spent nuclear fuel disposal site in Olkiluoto, Finland. Denaturing gradient gel electrophoresis detected diverse sulphate-reducing bacterial communities in all samples. Although the number of dsrB gene copies was below 10³ copies ml^?1 in all analyzed samples according to real-time quantitative PCR, their abundance was highest in samples that had the highest sulphate concentrations. Several distinct mcrA gene fragments were also recovered from most of the analyzed samples by cloning, although the number of methanogens was lower than that of sulphate-reducing bacteria when measured by mcrA-targeted quantitative PCR. The detected gene fragments were most closely related to sequences obtained from aquatic and deep subsurface environments. Results imply that sulphate reduction, methanogenesis, and anaerobic methane oxidation may all take place in the Olkiluoto deep geobiosphere.     

Bacterial activities in the sediment of Lake Velencei,Hungary

Lake Velencei (Hungary) is one of the westernmost shallow soda lakes, extending from Eastern Europe to the Carpatian basin. The spatial and temporal distribution of the sediment microbiota, the metabolic potential of bacterial communities and the species composition of the genera Bacillus and Clostridium, as well as sulphate-reducing bacteria (SRB) were investigated regarding the close interactions between the lake sediment and the overlaying water column, the special water chemical parameters, and the extensive reed coverage of the lake. Aerobic microbial activities were tested with community-level physiological profiling (CLPP) using BIOLOG microplates. The quantification of the anaerobic fermentative and sulphate-reducing bacteria was done by the MPN (Most Probable Number) method. The cultivation of bacteria adapted to the special physico-chemical characteristics of the lake was carried out employing selective media. Multivariate analysis of CLPP data indicated that the microbial communities of the sediment separated from that of the water and showed seasonal variations of the utilised carbon sources. The results of the MPN demonstrated that the counts of the fermentative and sulphate-reducing bacteria in the reed rhizosphere were about one order higher than in the sediment. Among the isolated bacterial strains, a large number were characterised as facultative or obligate alkaliphilic and also moderately halophilic. The partial sequencing of 16S rDNA of the selected representatives resulted in species of aerobic bacteria, such as Bacillus pseudofirmus, B. halmapalus, B. cohnii, B. (Marinibacillus) marinus, and anaerobes, such as Clostridium putrificum – sporogenes, C. scatologenes, C. bifermentans, Desulfotomaculum guttoideum, Desulfovibrio alcoholivorans, and Desulfovibrio burkinensis.

     

Characterization of the core microbiota of the drainage and surrounding soil of a Brazilian copper mine

The core microbiota of a neutral mine drainage and the surrounding high heavy metal content soil at a Brazilian copper mine were characterized by 16S rDNA pyrosequencing. The core microbiota of the drainage was dominated by the generalist genus Meiothermus. The soil samples contained a more heterogeneous bacterial community, with the presence of both generalist and specialist bacteria. Both environments supported mainly heterotrophic bacteria, including organisms resistant to heavy metals, although many of the bacterial groups identified remain poorly characterized. The results contribute to the understanding of bacterial communities in soils impacted by neutral mine drainage, for which information is scarce, and demonstrate that heavy metals can play an important role in shaping the microbial communities in mine environments.     

Iron-coproporphyrin III is a natural cofactor in bacterioferritin from the anaerobic bacterium Desulfovibrio desulfuricans

A bacterioferritin was recently isolated from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 [Romão et al. (2000) Biochemistry 39, 6841–6849]. Although its properties are in general similar to those of the other bacterioferritins, it contains a haem quite distinct from the haem B, found in bacterioferritins from aerobic organisms. Using visible and NMR spectroscopies, as well as mass spectrometry analysis, the haem is now unambiguously identified as iron-coproporphyrin III, the first example of such a prosthetic group in a biological system. This unexpected finding is discussed in the framework of haem biosynthetic pathways in anaerobes and particularly in sulphate-reducing bacteria.     

Isolation of new types of sulphate-reducing bacteria from estuarine and marine sediments using chemostat enrichments

Anaerobic chemostat enrichments have been successfully employed to isolate acetate-utilizing, sulphate-reducing bacteria from sediments in the Tay estuary. Several different morphologically and nutritionally versatile types of sulphate-reducing bacteria have been isolated by this means which has proved especially useful when they are present in low numbers. The principal advantage of this method is that strict anaerobes such as sulphate-reducing bacteria can be isolated in a controlled and reproducible enrichment system at low growth rates.     

Genetic Diversity among Thermophilic Bacteria Isolated from Geothermal Sites by Using Two PCR Typing Methods

Microbial communities thriving at two hot springs, Hammam Pharaon (Pharaoh's Bath) and Oyoun Mossa (Moses springs), in Egypt was studied by cultural and molecular methods. Thirteen morphologically distinct strains of facultative anaerobic thermophilic bacterial isolates have been characterized and identified using phenotypic and genotypic characters including RAPD-PCR, ERIC-PCR typing, plasmid analysis and 16S rRNA sequencing. All isolates produced plasmid DNA with various sizes ranging from 0.7 kb to a larger plasmid 7.2 kb. The bacterial strains could tolerate a temperature range between 45 to 85°C and a pH between 4–11. Also, sulphate-reducing bacteria (SRB) in the thermal springs were investigated with combined biochemical and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore-forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 30 to 50°C and could use pyruvate, lactate and ethanol as electron donors. The dissimilatory sulphite reductase (DSR) gene sequences of eleven representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris. 16S rRNA gene partial sequence results indicated the presence of novel or existing species of Bacillus (one species), Anoxybacillus (four species) and Geobacillus (eight species). In this study phenotypic and genotypic diversity were applied for the first time to differentiate thermophilic bacteria of such geothermal sites in Sinai, Egypt.     

Analysis of the microbial community in moderately acidic drainage from the Yanahara pyrite mine in Japan

Acid rock drainage (ARD) originating from the Yasumi-ishi tunnel near the main tunnel of the Yanahara mine in Japan was characterized to be moderately acidic (pH 4.1) and contained iron at a low concentration (51?mg/L). The composition of the microbial community was determined by sequence analysis of 16S rRNA genes using PCR and denaturing gradient gel electrophoresis. The analysis of the obtained sequences showed their similarity to clones recently detected in other moderately acidic mine drainages. Uncultured bacteria related to Ferrovum- and Gallionella-like clones were dominant in the microbial community. Analyses using specific primers for acidophilic iron- or sulfur-oxidizing bacteria, Acidithiobacillus ferrooxidans, Leptospirillum spp., Acidithiobacillus caldus, Acidithiobacillus thiooxidans, and Sulfobacillus spp. revealed the absence of these bacteria in the microbial community in ARD from the Yasumi-ishi tunnel. Clones affiliated with a member of the order Thermoplasmatales were detected as the dominant archaea in the ARD microbial population.     

Sulphate-reducing bacteria in paper machine waters and in suction roll perforations

Summary To define some aspects of the biological corrosion sulphate-reducing bacteria were studied in paper machine waters and in plugged perforations of a suction roll. The desulphuricants were most active on passive fiber recipients. Most bacteria found in fiber plugs taken from the perforations of suction rolls belonged to the genus Desulfovibrio. Desulphuricants were found mainly at the outer ends of plugged perforations, where corrosion of the roll metal is most evident.     

Changes in the Bacterial Community of Soil from a Neutral Mine Drainage Channel

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments.     

Distribution of sulphur-cycle bacteria in the Ems-Dollard estuary

Summary A quantitative study of the sulphur cycle in the tidal flat-sediments of the Eems-Dollard estuary was started by determining the distribution of two physiologically different groups of bacteria: sulphate-reducing and sulphide-oxidizing bacteria. Viable counts of these bacterial groups were determined by most probable number techniques.The highest numbers of aerobic sulphide-oxidizing bacteria were found in the upper 2 cm of the sediment. A rapid decrease was observed with increasing depth.The anaerobic sulphate-reducing bacteria showed different distribution-patterns with depth. Frequently high numbers of these bacteria were found above the redox-discontinuity-layer. This may be attributed to the presence of anaerobic micro-pockets in this largely aerobic top-layer of the sediment.The horizontal distribution of the sulphide-oxidizing bacteria appeared to be highly correlated with sediment parameters such as organic carbon and clay content of the sediment. The sulphate-reducing bacteria showed only a small linear correlation with these parameters.By means of polyfactor-analysis mathematical models were made with bacterial numbers as the dependent variables and with some environmental parameters as independent variables. The parameters used in this models could explain the variance of the viable counts for approximately 70%. The clay content of the sediment and the number of sulphate reducing bacteria appeared to determine to a large extent the variance in numbers of sulphide-oxidizing bacteria. There are indications that a great deal of the sulphide-oxidizing bacteria might be mixotrophic.For the explanation of the variance in numbers of sulphate-reducing bacteria the most important parameters were the clay content of the sediment, the number of aerobic heterotrophic bacteria and temperature (or season). Therefore the numbers of these organisms were varying throughout the year. It is assumed that the heterotrophic bacteria supply the sulphate-reducing bacteria with organic substrates.