Effects of Spilled Oil on Bacterial Communities of Mediterranean Coastal Anoxic Sediments Chronically Subjected to Oil Hydrocarbon Contamination

The effects of spilled oil on sedimentary bacterial communities were examined in situ at 20 m water depth in a Mediterranean coastal area. Sediment collected at an experimental site chronically subjected to hydrocarbon inputs was reworked into PVC cores with or without a massive addition of crude Arabian light oil (∼20 g kg⁻¹ dry weight). Cores were reinserted into the sediment and incubated in situ at the sampling site (20 m water depth) for 135 and 503 days. The massive oil contamination induced significant shifts in the structure of the indigenous bacterial communities as shown by ribosomal intergenic spacer analysis (RISA). The vertical heterogeneity of the bacterial communities within the sediment was more pronounced in the oiled sediments particularly after 503 days of incubation. Response to oil of the deeper depth communities (8–10 cm) was slower than that of superficial depth communities (0–1 and 2–4 cm). Analysis of the oil composition by gas chromatography revealed a typical microbial alteration of n-alkanes during the experiment. Predominant RISA bands in oiled sediments were affiliated to hydrocarbonoclastic bacteria sequences. In particular, a 395-bp RISA band, which was the dominant band in all the oiled sediments for both incubation times, was closely related to hydrocarbonoclastic sulfate-reducing bacteria (SRB). These bacteria may have contributed to the main fingerprint changes and to the observed biodegradation of n-alkanes. This study provides useful information on bacterial dynamics in anoxic contaminated infralittoral sediments and highlights the need to assess more precisely the contribution of SRB to bioremediation in oil anoxic contaminated areas.     

Phylogeography of sulfate-reducing bacteria among disturbed sediments, disclosed by analysis of the dissimilatory sulfite reductase genes (dsrAB)

Sediment samples were collected worldwide from 16 locations on four continents (in New York, California, New Jersey, Virginia, Puerto Rico, Venezuela, Italy, Latvia, and South Korea) to assess the extent of the diversity and the distribution patterns of sulfate-reducing bacteria (SRB) in contaminated sediments. The SRB communities were examined by terminal restriction fragment (TRF) length polymorphism (TRFLP) analysis of the dissimilatory sulfite reductase genes (dsrAB) with NdeII digests. The fingerprints of dsrAB genes contained a total of 369 fluorescent TRFs, of which <20% were present in the GenBank database. The global sulfidogenic communities appeared to be significantly different among the anthropogenically impacted (petroleum-contaminated) sites, but nearly all were less diverse than pristine habitats, such as mangroves. A global SRB indicator species of petroleum pollution was not identified. However, several dsrAB gene sequences corresponding to hydrocarbon-degrading isolates or consortium members were detected in geographically widely separated polluted sites. Finally, a cluster analysis of the TRFLP fingerprints indicated that many SRB microbial communities were most similar on the basis of close geographic proximity (tens of kilometers). Yet, on larger scales (hundreds to thousands of kilometers) SRB communities could cluster with geographically widely separated sites and not necessarily with the site with the closest proximity. These data demonstrate that SRB populations do not adhere to a biogeographic distribution pattern similar to that of larger eukaryotic organisms, with the greatest species diversity radiating from the Indo-Pacific region. Rather, a patchy SRB distribution is encountered, implying an initially uniform SRB community that has differentiated over time.     

Culture-Dependent and -Independent Methods Capture Different Microbial Community Fractions in Hydrocarbon-Contaminated Soils

Bioremediation is a cost-effective and sustainable approach for treating polluted soils, but our ability to improve on current bioremediation strategies depends on our ability to isolate microorganisms from these soils. Although culturing is widely used in bioremediation research and applications, it is unknown whether the composition of cultured isolates closely mirrors the indigenous microbial community from contaminated soils. To assess this, we paired culture-independent (454-pyrosequencing of total soil DNA) with culture-dependent (isolation using seven different growth media) techniques to analyse the bacterial and fungal communities from hydrocarbon-contaminated soils. Although bacterial and fungal rarefaction curves were saturated for both methods, only 2.4% and 8.2% of the bacterial and fungal OTUs, respectively, were shared between datasets. Isolated taxa increased the total recovered species richness by only 2% for bacteria and 5% for fungi. Interestingly, none of the bacteria that we isolated were representative of the major bacterial OTUs recovered by 454-pyrosequencing. Isolation of fungi was moderately more effective at capturing the dominant OTUs observed by culture-independent analysis, as 3 of 31 cultured fungal strains ranked among the 20 most abundant fungal OTUs in the 454-pyrosequencing dataset. This study is one of the most comprehensive comparisons of microbial communities from hydrocarbon-contaminated soils using both isolation and high-throughput sequencing methods.     

Enrichment of dissimilatory Fe(III)-reducing bacteria from groundwater of the Siklós BTEX-contaminated site (Hungary)

Dissimilatory iron-reducing bacteria are commonly found in microbial communities of aromatic hydrocarbon-contaminated subsurface environments where they often play key role in the degradation of the contaminants. The Siklós benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated area is one of the best characterized petroleum hydrocarbon-contaminated sites of Hungary. Continuous monitoring of the microbial community in the center of the contaminant plume indicated the presence of an emerging Geobacter population and a Rhodoferax phylotype highly associated with aromatic hydrocarbon-contaminated subsurface environments. The aim of the present study was to make an initial effort to enrich Rhodoferax-related and other dissimilatory iron-reducing bacteria from this environment. Accordingly, four slightly different freshwater media were used to enrich Fe(III) reducers, differing only in the form of nitrogen source (organic, inorganic nitrogen or gaseous headspace nitrogen). Although enrichment of the desired Rhodoferax phylotype was not succeeded, Geobacter-related bacteria were readily enriched. Moreover, the different nitrogen sources caused the enrichment of different Geobacter species. Investigation of the diversity of benzylsuccinate synthase gene both in the enrichments and in the initial groundwater sample indicated that the Geobacter population in the center of the contaminant plume may not play a significant role in the anaerobic degradation of toluene.     

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.     

Effect of nitrate injection on the bacterial community in a water-oil tank system analyzed by PCR-DGGE

Sulfide production by sulfate-reducing bacteria (SRB) is a major concern for the petroleum industry since it is toxic and corrosive, and causes plugging due to the formation of insoluble iron sulfides (reservoir souring). In this study, PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) using two sets of primers based on the 16S rRNA gene and on the aps gene (adenosine-5-phosphosulfate reductase) was used to track changes in the total bacterial and SRB communities, respectively, present in the water-oil tank system on an offshore platform in Brazil in which nitrate treatment was applied for 2 months (15 nitrate injections). PCR-DGGE analysis of the total bacterial community showed the existence of a dominant population in the water-oil tank, and that the appearance and/or the increase of intensity of some bands in the gels were not permanently affected by the introduction of nitrate. On the other hand, the SRB community was stimulated following nitrate treatment. Moreover, sulfide production did not exceed the permissible exposure limit in the water-oil separation tank studied treated with nitrate. Therefore, controlling sulfide production by treating the produced water tank with nitrate could reduce the quantity of chemical biocides required to control microbial activities.     

Activity and diversity of sulfate-reducing bacteria in a petroleum hydrocarbon-contaminated aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO(4)(2-) reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day(-1) for sulfate reduction and from 0.13 to 0.60 day(-1) for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

Phylogeography of Sulfate-Reducing Bacteria among Disturbed Sediments, Disclosed by Analysis of the Dissimilatory Sulfite Reductase Genes (dsrAB)

Sediment samples were collected worldwide from 16 locations on four continents (in New York, California, New Jersey, Virginia, Puerto Rico, Venezuela, Italy, Latvia, and South Korea) to assess the extent of the diversity and the distribution patterns of sulfate-reducing bacteria (SRB) in contaminated sediments. The SRB communities were examined by terminal restriction fragment (TRF) length polymorphism (TRFLP) analysis of the dissimilatory sulfite reductase genes (dsrAB) with NdeII digests. The fingerprints of dsrAB genes contained a total of 369 fluorescent TRFs, of which <20% were present in the GenBank database. The global sulfidogenic communities appeared to be significantly different among the anthropogenically impacted (petroleum-contaminated) sites, but nearly all were less diverse than pristine habitats, such as mangroves. A global SRB indicator species of petroleum pollution was not identified. However, several dsrAB gene sequences corresponding to hydrocarbon-degrading isolates or consortium members were detected in geographically widely separated polluted sites. Finally, a cluster analysis of the TRFLP fingerprints indicated that many SRB microbial communities were most similar on the basis of close geographic proximity (tens of kilometers). Yet, on larger scales (hundreds to thousands of kilometers) SRB communities could cluster with geographically widely separated sites and not necessarily with the site with the closest proximity. These data demonstrate that SRB populations do not adhere to a biogeographic distribution pattern similar to that of larger eukaryotic organisms, with the greatest species diversity radiating from the Indo-Pacific region. Rather, a patchy SRB distribution is encountered, implying an initially uniform SRB community that has differentiated over time.     

长庆油田延9低渗透油藏微生物群落研究

目的:研究长庆油田延9低渗透油藏微生物群落,为实施微生物提高原油采收率提供指导和依据。方法:长庆油田延9油藏三口不同油井(柳28-46、柳28-47和柳27-45)的油水样品建立16S rDNA克隆文库进行研究。结果:构建了柳28-46、柳28-47和柳27-45油井样品的微生物基因克隆文库,其分类操作单元(OUT)数分别为21、20和20个;序列分析比对表明,3口井的共同的优势微生物菌群为铜绿假单胞菌(Pseudomonas aeruginos),分别占各文库的32.8%、32%和42.9%,它是最常见最主要的采油功能菌之一。此外硫酸盐还原菌(SRB)和铁细菌也处于优势地位,它们是原油开采中的有害菌。结论:延9低渗透油藏微生物群落和其潜在功能的分析为开展微生物提高石油采收率应用提供了良好的基础资料。     

Diversity and characterization of sulfate-reducing bacteria in groundwater at a uranium mill tailings site.

Microbially mediated reduction and immobilization of U(VI) to U(IV) plays a role in both natural attenuation and accelerated bioremediation of uranium-contaminated sites. To realize bioremediation potential and accurately predict natural attenuation, it is important to first understand the microbial diversity of such sites. In this paper, the distribution of sulfate-reducing bacteria (SRB) in contaminated groundwater associated with a uranium mill tailings disposal site at Shiprock, N.Mex., was investigated. Two culture-independent analyses were employed: sequencing of clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) gene fragments and phospholipid fatty acid (PLFA) biomarker analysis. A remarkable diversity among the DSR sequences was revealed, including sequences from delta-Proteobacteria, gram-positive organisms, and the Nitrospira division. PLFA analysis detected at least 52 different mid-chain-branched saturate PLFA and included a high proportion of 10me16:0. Desulfotomaculum and Desulfotomaculum-like sequences were the most dominant DSR genes detected. Those belonging to SRB within delta-Proteobacteria were mainly recovered from low-uranium (< or =302 ppb) samples. One Desulfotomaculum-like sequence cluster overwhelmingly dominated high-U (>1,500 ppb) sites. Logistic regression showed a significant influence of uranium concentration over the dominance of this cluster of sequences (P = 0.0001). This strong association indicates that Desulfotomaculum has remarkable tolerance and adaptation to high levels of uranium and suggests the organism's possible involvement in natural attenuation of uranium. The in situ activity level of Desulfotomaculum in uranium-contaminated environments and its comparison to the activities of other SRB and other functional groups should be an important area for future research.     

Changes in sulfate-reducing bacterial populations during the onset of black band disease

Factors that facilitate the onset of black band disease (BBD) of corals remain elusive, though anoxic conditions under the complex microbial mat and production of sulfide are implicated in necrosis of underlying coral tissues. This study investigated the diversity and quantitative shifts of sulfate-reducing bacterial (SRB) populations during the onset of BBD using real-time PCR (RT-PCR) and cloning approaches targeting the dissimilatory (bi)sulfite reductase (dsrA) gene. A quantitativePCR (qPCR) assay targeting the 16S rRNA gene also provided an estimate of total bacteria, and allowed the relative percentage of SRB within the lesions to be determined. Three Montipora sp. coral colonies identified with lesions previously termed cyanobacterial patches (CPs) (comprising microbial communities unlike those of BBD lesions), were tagged and followed through time as CP developed into BBD. The dsrA-targeted qPCR detected few copies of the gene in the CP samples (<65 per ng DNA), though copy numbers increased in BBD lesions (>2500 per ng DNA). SRB in CP samples were less than 1% of the bacterial population, though represented up to 7.5% of the BBD population. Clone libraries also demonstrated a shift in the dominant dsrA sequences as lesions shifted from CP into BBD. Results from this study confirm that SRB increase during the onset of BBD, likely increasing sulfide concentrations at the base of the microbial mat and facilitating the pathogenesis of BBD.     

Activity and Diversity of Sulfate-Reducing Bacteria in a Petroleum Hydrocarbon-Contaminated Aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO₄²⁻ reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day⁻¹ for sulfate reduction and from 0.13 to 0.60 day⁻¹ for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

Microbial characterization of a JP-4 fuel-contaminated site using a combined lipid biomarker/polymerase chain reaction--denaturing gradient gel electrophoresis (PCR-DGGE)-based approach

The impact of pollution on soil microbial communities and subsequent bioremediation can be measured quantitatively in situ using direct, non-culture- dependent techniques. Such techniques have advantages over culture-based methods, which often account for less than 1% of the extant microbial community. In 1988, a JP-4 fuel spill contaminated the glacio-fluvial aquifer at Wurtsmith Air Force Base, Michigan, USA. In this study, lipid biomarker characterization of the bacterial and eukaryotic communities was combined with polymerase chain reaction– denaturing gradient gel electrophoresis (PCR–DGGE) analysis of the eubacterial community to evaluate correlation between contaminant (JP-4 fuel) concentration and community structure shifts. Vadose, capillary fringe and saturated zone samples were taken from cores within and up- and down-gradient from the contaminant plume. Lipid biomarker analysis indicated that samples from within the plume contained increased biomass, with large proportions of typically Gram-negative bacteria. Outside the plume, lipid profiles indicated low-biomass microbial communities compared with those within the initial spill site. 16S rDNA sequences derived from DGGE profiles from within the initial spill site suggested dominance of the eubacterial community by a limited number of phylogenetically diverse organisms. Used in tandem with pollutant quantification, these molecular techniques should facilitate significant improvements over current assessment procedures for the determination of remediation end-points.     

Measurement of Oxygen Demand of Petroleum Hydrocarbon-Contaminated Groundwater

The standard biological oxygen demand (BOD) test was modified for application to petroleum hydrocarbon-contaminated groundwater. The goal was to assess the potential oxygen demand of plume constituents as part of a field trial investigating oxygen-enhanced in situ bioremediation. Modifications to standard BOD protocol included the use of an adapted microbial population developed from site groundwater and methods to minimize both the loss of volatile compounds and the exposure of samples to air. Results from this study indicated that the measured oxygen demand was significantly greater than the oxygen demand estimated solely by stoichiometric calculations from the concentrations of the analytes of typical regulatory concern, that is, benzene, toluene, ethylbenzene, and xylenes (BTEX). This is not surprising, because the petroleum hydrocarbon sources typically contain many organic contaminants other than BTEX, as well as potentially oxidizable natural dissolved organic matter and inorganic species typically present in hydrocarbon plumes. However, in practice, estimation of the total oxygen demand of a contaminated groundwater by exhaustive analyses of all oxidizable or aerobically degradable species typically will be infeasible. The modified BOD test may be a simple, low-cost, useful tool when assessing the potential for natural attenuation by aerobic biodegradation or designing methods to supply oxygen for enhanced aerobic bioremediation.     

Phospholipid analysis as a tool to study complex microbial communities in marine sediments.

To complement information on microbial communities in marine sediments that can be obtained using microbiological methods, we developed an analytical procedure to trace microbial lipids in environmental samples. We focused on analyzing intact phospholipids as these membrane constituents are known to be biomarkers for viable cells. Analysis of intact phospholipids from a fractionated and preconcentrated sediment extract was achieved using liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). The combined analysis of phospholipid types and their fatty acid substituents allowed a differentiation between various groups of microorganisms living in the sediment. For comparison three strains of marine sulfate-reducing bacteria (SRB) were analysed for their lipid content.     

Microbial community analysis of two field-scale sulfate-reducing bioreactors treating mine drainage

The microbial communities of two field-scale pilot sulfate-reducing bioreactors treating acid mine drainage (AMD), Luttrell and Peerless Jenny King (PJK), were compared using biomolecular tools and multivariate statistical analyses. The two bioreactors were well suited for this study because their geographic locations and substrate compositions were similar while the characteristics of influent AMD, configuration and degree of exposure to oxygen were distinct. The two bioreactor communities were found to be functionally similar, including cellulose degraders, fermenters and sulfate-reducing bacteria (SRB). Significant differences were found between the two bioreactors in phylogenetic comparisons of cloned 16S rRNA genes and adenosine 5'-phosphosulfate reductase ( apsA ) genes. The apsA gene clones from the Luttrell bioreactor were dominated by uncultured SRB most closely related to Desulfovibrio spp., while those of the PJK bioreactor were dominated by Thiobacillus spp. The fraction of the SRB genus Desulfovibrio was also higher at Luttrell than at PJK as determined by quantitative real-time polymerase chain reaction analysis. Oxygen exposure at PJK is hypothesized to be the primary cause of these differences. This study is the first rigorous phylogenetic investigation of field-scale bioreactors treating AMD and the first reported application of multivariate statistical analysis of remediation system microbial communities applying UniFrac software.     

Spatial distribution patterns of benthic microbial communities along the Pearl Estuary,China

In the present study, benthic microbial communities along the Pearl Estuary, a typical subtropical estuary in China subjected to extensive anthropogenic disturbance, were investigated using 16S rRNA gene-based pyrosequencing. The results showed that microbial communities in freshwater samples were clearly distinct from those in saltwater samples, since the relative sequence abundances of Deltaproteobacteria, Thermoplasmata and Marine Group I (MG-I) were higher in saltwater sediments, whereas Chloroflexi, Spirochaetes, Betaproteobacteria and methanogens were more prevalent in freshwater sediments. In addition, bacterial communities showed vertical stratifications in saltwater sediments, but remained constant with depth in freshwater sediments. The total organic carbon and carbon/nitrogen ratio in sediments correlated significantly with the overall community variations. The predominance of various microorganisms in specific niches led to efforts to identify their functional couplings by exploring their co-occurrence patterns. Using network analysis, strong positive correlations were observed between sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria, and between SRB and nitrite-oxidizing bacteria, indicating the potential interactions of intra-sulfur cycle processes, as well as sulfur and nitrogen cycles, in coastal sediments. Archaeal clades revealed strong and wide correlations between the Miscellaneous Crenarchaeotal Group (MCG) and other groups, suggesting a central role of MCG in the coastal benthic environment. Inversely, MG-I displayed negative correlations with other clades, which might indicate that the lifestyles of heterotrophic and autotrophic clades were mutually exclusive. This study presented a detailed outline of the biogeographic patterns of benthic microbial communities along the Pearl Estuary and provided new information regarding the potential interactions of various biogeochemical cycles in coastal sediments.     

Aerobic Organic Carbon Mineralization by Sulfate-Reducing Bacteria in the Oxygen-Saturated Photic Zone of a Hypersaline Microbial Mat

The sulfate-reducing bacterium strain SRB D2 isolated from the photic zone of a hypersaline microbial mat, from Lake Chiprana, NE Spain, respired pyruvate, alanine, and α-ketoglutarate but not formate, lactate, malate, succinate, and serine at significant rates under fully oxic conditions. Dehydrogenase enzymes of only the former substrates are likely oxygen-tolerant as all substrates supported anaerobic sulfate reduction. No indications were found, however, that aerobic respiration supported growth. Although strain SRB D2 appeared phylogenetically closely related to the oxygen-tolerant sulfate-reducing bacterium Desulfovibrio oxyclinae, substrate spectra were markedly different. Most-probable-number (MPN) estimates of sulfate-reducing bacteria and aerobic heterotrophic bacteria indicated that the latter were numerically dominant in both the photic and aphotic zones of the mat. Moreover, substrate spectra of representative isolates showed that the aerobic heterotrophic bacteria are metabolically more diverse. These findings indicate that sulfate-reducing bacteria in the fully oxic photic zone of mats have to compete with aerobic heterotrophic bacteria for organic substrates. Porewater analysis revealed that total carbohydrates and low-molecular-weight carbon compounds (LMWC) made up substantial fractions of the total dissolved organic carbon (DOC) pool and that nighttime degradation of the former was concomitant with increased concentration of the latter. Our findings indicate that aerobic respiration by sulfate-reducing bacteria contributes to organic carbon mineralization in the oxic zone of microbial mats as daytime porewater LMWC concentrations are above typical half-saturation constants.     

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.     

Response of sulfate-reducing bacteria to an artificial oil-spill in a coastal marine sediment

In situ mesocosm experiments using a calcareous sand flat from a coastal area of the island of Mallorca in the Mediterranean Sea were performed in order to study the response of sulfate-reducing bacteria (SRB) to controlled crude oil contamination, or heavy contamination with naphthalene. Changes in the microbial community caused by the contamination were monitored by a combination of comparative sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, cultivation approaches and metabolic activity rates. Our results showed that crude oil and naphthalene negatively influenced the total microbial community as the natural increase in cell numbers due to the seasonal dynamics was attenuated. However, both contaminants enhanced the sulfate reduction rates, as well as the culturability of SRB. Our results suggested the presence of autochthonous deltaproteobacterial SRBs that were able to degrade crude oil or polycyclic aromatic hydrocarbons such as naphthalene in anaerobic sediment layers.