Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments.

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Identification of Distinct Communities of Sulfate-Reducing Bacteria in Oil Fields by Reverse Sample Genome Probing

Thirty-five different standards of sulfate-reducing bacteria, identified by reverse sample genome probing and defined as bacteria with genomes showing little or no cross-hybridization, were in part characterized by Southern blotting, using 16S rRNA and hydrogenase gene probes. Samples from 56 sites in seven different western Canadian oil field locations were collected and enriched for sulfate-reducing bacteria by using different liquid media containing one of the following carbon sources: lactate, ethanol, benzoate, decanoate, propionate, or acetate. DNA was isolated from the enrichments and probed by reverse sample genome probing using master filters containing denatured chromosomal DNAs from the 35 sulfate-reducing bacterial standards. Statistical analysis of the microbial compositions at 44 of the 56 sites indicated the presence of two distinct communities of sulfate-reducing bacteria. The discriminating factor between the two communities was the salt concentration of the production waters, which were either fresh water or saline. Of 34 standards detected, 10 were unique to the fresh water and 18 were unique to the saline oil field environment, while only 6 organisms were cultured from both communities.     

Effect of sulfate on methanogenic communities that degrade unsaturated and saturated long-chain fatty acids (LCFA)

Anaerobic bacteria involved in the degradation of long-chain fatty acids (LCFA), in the presence of sulfate as electron acceptor, were studied by combined cultivation-dependent and molecular techniques. The bacterial diversity in four mesophilic sulfate-reducing enrichment cultures, growing on oleate (C_18:1, unsaturated LCFA) or palmitate (C_16:0, saturated LCFA), was studied by denaturing gradient gel electrophoresis (DGGE) profiling of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments. These enrichment cultures were started using methanogenic inocula in order to assess the competition between methanogenic communities and sulfate-reducing bacteria. Phylogenetic affiliation of rRNA gene sequences corresponding to predominant DGGE bands demonstrated that members of the Syntrophomonadaceae , together with sulfate reducers mainly belonging to the Desulfovibrionales and Syntrophobacteraceae groups, were present in the sulfate-reducing enrichment cultures. Subculturing of LCFA-degrading methanogenic cultures in the presence of sulfate resulted in the inhibition of methanogenesis and, after several transfers, archaea could no longer be detected by real-time PCR. Competition for hydrogen and acetate was therefore won by sulfate reducers, but acetogenic syntrophic bacteria were the only known LCFA-degrading organisms present after subculturing with sulfate. Principal component analysis of the DGGE profiles from methanogenic and sulfate-reducing oleate- and palmitate-enrichment cultures showed a greater influence of the substrate than the presence or absence of sulfate, indicating that the bacterial communities degrading LCFA in the absence/presence of sulfate are rather stable.     

Alkaliphilic anaerobic community at pH 10

Relict or ancient microbial communities in extreme environment might be analogous to the centers of origin of bacterial diversity. A bacterial community of an alkaline lake was investigated, and the diversity of bacteria found there indicates that both conditions of autonomy and phylogenetic variety are fulfilled for anaerobic bacteria developing at pH 10±0.2. Major functional groups in the trophic network were present. Representatives of proteolytic, bacteriolytic, cellulolytic, saccharolytic, dissipotrophic, acetogenic, sulfate-reducing, methanogenic bacteria were isolated.     

Enumeration of selected anaerobic bacterial groups in cecal and colonic contents of growing-finishing pigs

Selected anaerobic bacterial groups in cecal and colonic contents of clinically healthy pigs fed a corn-soybean meal production diet were determined at sacrifice after 4, 8, and 11 weeks on feed, corresponding to intervals within the growing-finishing growth period. By using ruminal fluid-based media, the densities of the culturable anaerobic population; the cellulolytic, pectin-fermenting, pectin-hydrolyzing, xylan-fermenting; and the xylan-hydrolyzing, sulfate-reducing, and methanogenic bacterial populations were estimated. An analysis of variance was performed on these bacterial group variables to examine the effects of phase (weeks on feed), site (cecum or colon), or the interaction of phase with site. The population of total anaerobic bacteria was twice as dense in the colon as it was in the cecum (2 x 10(10) versus 1 x 10(10)/g [wet weight]; P = 0.001). The proportion of cellulolytic bacteria was lower at 4 weeks on feed than at 8 or 11 weeks (23 versus 32%; P = 0.026), while the proportion of pectin-fermenting bacteria depended on the interaction of phase with site (P = 0.021). The numbers of sulfate-reducing bacteria were significantly higher in the colon than in the cecum (6 x 10(7) versus 3 x 10(7); P = 0.014), as were methanogenic bacteria (19 x 10(7) versus 0.6 x 10(7); P = 0.0002). The remaining bacterial groups were stable with respect to phase and site. The results suggest that except for density differences, the microbial communities of the pig cecum and colon are similar in composition throughout the growing-finishing phase.     

Enumeration of selected anaerobic bacterial groups in cecal and colonic contents of growing-finishing pigs.

Selected anaerobic bacterial groups in cecal and colonic contents of clinically healthy pigs fed a corn-soybean meal production diet were determined at sacrifice after 4, 8, and 11 weeks on feed, corresponding to intervals within the growing-finishing growth period. By using ruminal fluid-based media, the densities of the culturable anaerobic population; the cellulolytic, pectin-fermenting, pectin-hydrolyzing, xylan-fermenting; and the xylan-hydrolyzing, sulfate-reducing, and methanogenic bacterial populations were estimated. An analysis of variance was performed on these bacterial group variables to examine the effects of phase (weeks on feed), site (cecum or colon), or the interaction of phase with site. The population of total anaerobic bacteria was twice as dense in the colon as it was in the cecum (2 x 10(10) versus 1 x 10(10)/g [wet weight]; P = 0.001). The proportion of cellulolytic bacteria was lower at 4 weeks on feed than at 8 or 11 weeks (23 versus 32%; P = 0.026), while the proportion of pectin-fermenting bacteria depended on the interaction of phase with site (P = 0.021). The numbers of sulfate-reducing bacteria were significantly higher in the colon than in the cecum (6 x 10(7) versus 3 x 10(7); P = 0.014), as were methanogenic bacteria (19 x 10(7) versus 0.6 x 10(7); P = 0.0002). The remaining bacterial groups were stable with respect to phase and site. The results suggest that except for density differences, the microbial communities of the pig cecum and colon are similar in composition throughout the growing-finishing phase.     

Response Characteristics between Sulfate-Reducing Bacteria and Environmental Factors in Petroleum Hydrocarbon-Contaminated Field of Northeast China

Lithology samples were collected from six sites of a petroleum hydrocarbon-contaminated site in northeast China along a contamination plume. The sulfate-reducing bacteria (SRB) diversity of all samples was analyzed by PCR-DGGE technology. The Shannon-Wiener indexes (1.004–3.665), Simpson indexes (0.516–0.907) and Pielou indexes (0.996–1.004) of all samples were used to characterize the abundances, advantages and evenness of the microbial communities. Additionally, Canoco for Windows 4.5 was utilized to analyze the correlation between dominant SRB and environmental factors. The results showed that the abundance, advantages and evenness of the sulfate-reducing bacterial community changed regularly along the contamination plume direction. The microbial homology of the samples was not high (0.25–0.80), and the dominant bacteria exhibited heteroplasmy. Additionally, the dominant bacteria were identified as uncultured bacteria. Canonical correspondence analysis (CCA) analysis showed that the distribution and structure of SRB communities were not obviously correlated with the concentration of total petroleum hydrocarbons (TPH), dissolved oxygen (DO) and other environmental factors. The results presented herein provide evidence of natural bioremediation in petroleum hydrocarbon-contaminated fields and information that will be useful to bioremediation of other contaminated fields.     

Competition and coexistence of sulfate-reducing bacteria,acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol⁻¹, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol⁻¹, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.     

Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture

The aromatic hydrocarbon biphenyl is a widely distributed environmental pollutant. Whereas the aerobic degradation of biphenyl has been extensively studied, knowledge of the anaerobic biphenyl-oxidizing bacteria and their biochemical degradation pathway is scarce. Here, we report on an enrichment culture that oxidized biphenyl completely to carbon dioxide under sulfate-reducing conditions. The biphenyl-degrading culture was dominated by two distinct bacterial species distantly affiliated with the Gram-positive genus Desulfotomaculum . Moreover, the enrichment culture has the ability to grow with benzene and a mixture of anthracene and phenanthrene as the sole source of carbon, but here the microbial community composition differed substantially from the biphenyl-grown culture. Biphenyl-4-carboxylic acid was identified as an intermediate in the biphenyl-degrading culture. Moreover, 4-fluorobiphenyl was converted cometabolically with biphenyl because in addition to the biphenyl-4-carboxylic acid, a compound identified as its fluorinated analog was observed. These findings are consistent with the general pattern in the anaerobic catabolism of many aromatic hydrocarbons where carboxylic acids are found to be central metabolites.     

Bacterial enzymes for dissimilatory sulfate reduction in a marine microbial mat (Black Sea) mediating anaerobic oxidation of methane

Anaerobic oxidation of methane (AOM) with sulfate is catalysed by microbial consortia of archaea and bacteria affiliating with methanogens and sulfate-reducing Deltaproteobacteria respectively. There is evidence that methane oxidation is catalysed by enzymes related to those in methanogenesis, but the enzymes for sulfate reduction coupled to AOM have not been examined. We collected microbial mats with high AOM activity from a methane seep in the Black Sea. The mats consisted mainly of archaea of the ANME-2 group and bacteria of the Desulfosarcina-Desulfococcus group. Cell-free mat extract contained activities of enzymes involved in sulfate reduction to sulfide: ATP sulfurylase (adenylyl : sulfate transferase; Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). We partially purified the enzymes by anion-exchange chromatography. The amounts obtained indicated that the enzymes are abundant in the mat, with Sat accounting for 2% of the soluble mat protein. N-terminal amino acid sequences of purified proteins suggested similarities to the corresponding enzymes of known species of sulfate-reducing bacteria. The deduced amino acid sequence of PCR-amplified genes of the Apr subunits is similar to that of Apr of the Desulfosarcina/Desulfococcus group. These results indicate that the major enzymes involved in sulfate reduction in the Back Sea microbial mats are of bacterial origin, most likely originating from the bacterial partner in the consortium.     

Competition and Coexistence of Sulfate-Reducing and Methanogenic Populations in Anaerobic Biofilms

The microbial population structure and function of natural anaerobic communities maintained in laboratory fixed-bed biofilm reactors were tracked before and after a major perturbation, which involved the addition of sulfate to the influent of a reactor that had previously been fed only glucose (methanogenic), while sulfate was withheld from a reactor that had been fed both glucose and sulfate (sulfidogenic). The population structure, determined by using phylogenetically based oligonucleotide probes for methanogens and sulfate-reducing bacteria, was linked to the functional performance of the biofilm reactors. Before the perturbation, the methanogenic reactor contained up to 25% methanogens as well as 15% sulfate-reducing bacteria, even though sulfate was not present in the influent of this reactor. Methanobacteriales and Desulfovibrio spp. were the most abundant methanogens and sulfate-reducing bacteria, respectively. The presence of sulfate-reducing bacteria (primarily Desulfovibrio spp. and Desulfobacterium spp.) in the absence of sulfate may be explained by their ability to function as proton-reducing acetogens and/or fermenters. Sulfate reduction began immediately following the addition of sulfate consistent with the presence of significant levels of sulfate-reducing bacteria in the methanogenic reactor, and levels of sulfate-reducing bacteria increased to a new steady-state level of 30 to 40%; coincidentally, effluent acetate concentrations decreased. Notably, some sulfate-reducing bacteria (Desulfococcus/Desulfosarcina/Desulfobotulus group) were more competitive without sulfate. Methane production decreased immediately following the addition of sulfate; this was later followed by a decrease in the relative concentration of methanogens, which reached a new steady-state level of approximately 8%. The changeover to sulfate-free medium in the sulfidogenic reactor did not cause a rapid shift to methanogenesis. Methane production and a substantial increase in the levels of methanogens were observed only after approximately 50 days following the perturbation.     

Comparative evaluation of anaerobic bacterial communities associated with roots of submerged macrophytes growing in marine or brackish water sediments

Sediment microbial communities are important for seagrass growth and carbon cycling, however relatively few studies have addressed the composition of prokaryotic communities in seagrass bed sediments. Selective media were used enumerate culturable anaerobic bacteria associated with the roots of the seagrass, Halodule wrightii, the fresh to brackish water plant, Vallisneria americana, and the respective vegetated and unvegetated sediments. H. wrightii roots and sediments had high numbers of sulfate-reducing bacteria whereas iron-reducing bacteria appeared to have a more significant role in V. americana roots and sediments. Numbers of glucose-utilizing but not acetate-utilizing iron reducers were higher on the roots of both plants relative to the vegetated sediments indicating a difference within the iron reducing bacterial community. H. wrightii roots had lower glucose-utilizing iron reducers, and higher acetogenic bacteria than did V. americana roots suggesting different aquatic plants support different anaerobic microbial communities. Sulfur-disproportionating and sulfide-oxidizing bacteria were also cultured from the roots and sediments. These results provide evidence of the potential importance of sulfur cycle bacteria, in addition to sulfate-reducing bacteria, in seagrass bed sediments.     

Phylogenetic diversity of microorganisms associated with the deep-water sponge Baikalospongia intermedia

The diversity of bacteria associated with deep-water sponge Baikalospongia intermedia was evaluated by sequence analysis of 16S rRNA genes from two sponge samples collected in Lake Baikal from depths of 550 and 1204 m. A total of 64 operational taxonomic units, belonging to nine bacterial phyla, Proteobacteria (classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria), Actinobacteria, Planctomycetes, Cloroflexi, Verrucomicrobia, Acidobacteria, Chlorobi, and Nitrospirae, including candidate phylum WS5, were identified. Phylogenetic analysis showed that the examined communities contained phylotypes exhibiting homology to uncultured bacteria from different lake ecosystems, freshwater sediments, soil and geological formations. Moreover, a number of phylotypes were relative to psychrophilic, methane-oxidizing, sulfate-reducing bacteria, and to microorganisms resistant to the influence of heavy metals. It is noted that the unusual habitation conditions of deep-water sponges contribute to the taxonomic diversity of associated bacteria and have an influence on the presence of functionally important microorganisms in bacterial communities.     

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.     

不同pH值条件下硫酸盐还原菌组成及硫酸盐还原机制分析

【目的】从海洋沉积物中富集获得硫酸盐还原菌群,改变pH值进行培养,分析pH值对硫酸盐还原性质的影响,明确菌群组成和进行硫酸盐还原功能基因预测,探究硫酸盐还原机制。【方法】分析硫酸盐还原菌群在不同pH值条件下的硫酸盐还原率,在此基础上,利用高通量测序技术和PICRUSt软件分析硫酸盐还原菌群优势菌组成及硫酸盐还原相关基因相对丰度。【结果】硫酸盐还原菌群在不同pH值培养条件下的生长和硫酸盐还原率出现显著变化(P<0.01),在pH 5.0时达到峰值,分别为0.34±0.01和96.52%±0.44%。高通量测序数据显示,pH 5.0时菌群丰富度和多样性最高,优势菌属为假单胞菌(Pseudomonas)和芽孢杆菌(Bacillus),相对丰度较高的基因为同化性硫酸盐还原相关基因。【结论】硫酸盐还原菌富集生长的最适pH 5.0,在此条件下的高硫酸盐还原率由同化性硫酸盐还原途径主导,为揭示硫酸盐还原机制提供了实验支持,并拓宽了硫酸盐还原菌实践应用方面的种质资源。     

Investigation of the sulfate-reducing bacterial community in the aerobic water and chemocline zone of the Black Sea by the fish technique

Fluorescent in situ hybridization (FISH) was used to analyze the abundance and phylogenetic composition of sulfate-reducing bacteria in the aerobic waters and in the oxic/anoxic transitional zone (chemocline) of the Black Sea, where biogenic formation of reduced sulfur compounds was detected by radioisotope techniques. Numerous sulfate-reducing bacteria of the genera Desulfotomaculum (30.5% of detected bacterial cells), Desulfovibrio (29.6%), and Desulfobacter (6.7%) were revealed in the aerobic zone at a depth of 30 m, while Desulfomicrobium-related bacteria (33.5%) were prevalent in the upper chemocline waters at 150-m depth. Active cells of sulfate-reducing bacteria were much more abundant in the samples collected in summer than in the winter samples from the deep-sea zone. The presence of physiologically active sulfate reducers in oxic and chemocline waters of the Black Sea correlates with the hydrochemical data on the presence of reduced sulfur compounds in the aerobic water column.     

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.     

Extracellular metabolites of hydrocarbon-oxidizing bacteria as a substrate for sulfate reduction

The relationship between bacterial oxidation of hydrocarbons and sulfate reduction was studied in the experimental system with liquid paraffin was used as a source of organic compounds inoculated with silt taken from a reservoir. Pseudomonads dominated in the hydrocarbon-oxidizing silt bacteriocenosis. However, Rhodococcus and Arthrobacteria amounted to not more than 3%. Arthrobacteria dominated the microbial association formed in the paraffin film of the model system. Sulfate-reducing bacteria were represented by genera Desulfomonas, Desulfotomaculum, and Desulfovibrio. The growth of sulfate-reducing bacteria in media containing with paraffin, successive products of its oxidation (cetyl alcohol, stearate, and acetate), and extracellular metabolites of hydrocarbon-reducing bacteria was studied. The data showed that sulfate-reducing bacteria did not use paraffin or cetyl alcohol as growth substrates. However, active growth of sulfate-reducing bacteria was observed in the presence of stearate and extracellular water-soluble or lipid metabolites of Arthrobacteria.     

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.