A novel n-alkane-degrading bacterium as a minor member of p-xylene-degrading sulfate-reducing consortium

A p-xylene-degrading, sulfate-reducing enrichment culture was characterized by analyzing the response of its members to changes in the available substrate. The culture was inoculated into media containing other substrates, resulting in the establishment of benzoate-, acetate-, and lactate-utilizing enrichment cultures. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the enriched cultures targeting 16S rRNA genes showed quite simple band patterns. The predominant band from the benzoate-utilizing enrichment culture was identical to that from the original enrichment culture utilizing p-xylene. A single, dominant DGGE band was observed in common from the acetate- and lactate-utilizing enrichment cultures. A novel sulfate-reducing bacterium, strain PL12, was isolated from the lactate-utilizing enrichment culture. The 16S rRNA gene sequence of strain PL12 was identical to that of the dominant DGGE band in the acetate- and lactate-utilizing enrichment cultures and distinct from the dominant sequences in the original p-xylene-degrading and benzoate-utilizing enrichment cultures. Phylogenetic analysis of the 16S rRNA gene sequences showed that the isolate belonged to the family Desulfobacteraceae in the class Deltaproteobacteria. The isolated strain PL12 could utilize n-hexane and n-decane as substrates, but could not utilize benzoate, p-xylene and other aromatic hydrocarbons. These results suggest that the p-xylene degradation observed in the original enrichment culture was performed by the dominant bacterium corresponding to DGGE band pXy-K-13 (Nakagawa et al. 2008). The novel strain PL12 might have been utilizing metabolites of p-xylene.     

Biostimulation and bioaugmentation for on-site treatment of weathered diesel fuel in Arctic soil

Bioremediation of weathered diesel fuel in Arctic soil at low temperature was studied both on-site in small-scale biopiles and in laboratory microcosms. The field study site was on Ellesmere Island (82°30'N, 62°20'W). Biostimulation was by fertilization with phosphorous and nitrogen. Bioaugmentation was with an enrichment culture originating from the field site. In biopiles, total petroleum hydrocarbons (TPH) were reduced from 2.9 to 0.5 mg/g of dry soil over a period of 65 days. In microcosms at 7 °C, TPH were reduced from 2.4 to 0.5 mg/g of dry soil over a period of 90 days. Inoculation had no effect on hydrocarbon removal in biopiles or in microcosms. Maximum TPH removal rates in the biopiles were approximately 90 μg of TPH g^–1 of soil day^–1, occurring during the first 14 days when ambient temperature ranged from 0 to 10 °C. The fate of three phylotypes present in the inoculum was monitored using most-probable-number PCR, targeting 16S rRNA genes. Populations of all three phylotypes increased more than 100-fold during incubation of both uninoculated and inoculated biopiles. The inoculum increased the initial populations of the phylotypes but did not significantly affect their final populations. Thus, biostimulation on site enriched populations that were also selected in laboratory enrichment cultures. Electronic Publication     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

Molinate biodegradation in soils: natural attenuation versus bioaugmentation

The aims of the present study were to assess the potential of natural attenuation or bioaugmentation to reduce soil molinate contamination in paddy field soils and the impact of these bioremediation strategies on the composition of soil indigenous microbiota. A molinate mineralizing culture (mixed culture DC) was used as inoculum in the bioaugmentation assays. Significantly higher removal of molinate was observed in bioaugmentation than in natural attenuation microcosms (63 and 39 %, respectively) after 42 days of incubation at 22 °C. In the bioaugmentation assays, the impact of Gulosibacter molinativorax ON4^T on molinate depletion was observed since the gene encoding the enzyme responsible for the initial molinate breakdown (harboured by that actinobacterium) was only detected in inoculated microcosms. Nevertheless, the exogenous mixed culture DC did not overgrow as the heterotrophic counts of the bioaugmentation microcosms were not significantly different from those of natural attenuation and controls. Moreover, the actinobacterial clone libraries generated from the bioaugmentation microcosms did not include any 16S rRNA gene sequences with significant similarity to that of G. molinativorax ON4^T. The multivariate analysis of the 16S rRNA DGGE patterns of the soil microcosm suggested that the activity of mixed culture DC did not affect the soil bacterial community structure since the DGGE patterns of the bioaugmentation microcosms clustered with those of natural attenuation and controls. Although both bioremediation approaches removed molinate without indigenous microbiota perturbation, the results suggested that bioaugmentation with mixed culture DC was more effective to treat soils contaminated with molinate.     

Survival and growth of Salmonella Enteritidis PT 30 in almond orchard soils

Aims:  To evaluate factors potentially contributing to the long-term persistence of Salmonella enterica serovar Enteritidis phage type (PT) 30 in an almond orchard. Methods and Results:  Surface and subsurface soil temperatures, and air temperatures in a radiation shelter, were recorded during a 12-month period, and were used to identify relevant storage temperatures (20 or 35°C) for microcosms of two different soil types (clay and sandy loams) with moisture levels near saturation or near field capacity. Salmonella Enteritidis PT 30 was inoculated into the microcosms at 6 log CFU g⁻¹ dry weight. Between 14 and 180 days of incubation, counts of S. Enteritidis PT 30 decreased rapidly at 35°C and were significantly different (P < 0·05) from counts at 20°C, regardless of the soil type or moisture level. Salmonella was detected by enrichment of 10-g samples from all microcosms after 180 days of incubation at 20°C, but from none of the microcosms held at 35°C. To measure the potential for the growth of S. Enteritidis PT 30 in clay loam soil, an aqueous extract of almond hulls (containing 1·6% mono and disaccharides) or equivalent volume of water was added 7 days after inoculation. Significant (P < 0·05) growth of S. Enteritidis PT 30 was observed within 8 or 24 h of adding hull extract, but not water, to soil. Conclusions:  Opportunities may exist for S. Enteritidis PT 30 to survive for an extended time in almond orchard soils and to grow in these soils where hull nutrients are released. Significance and Impact of the Study:  Temperature has a significant impact on the long-term survival of S. Enteritidis PT 30 in soil, and nutrients leached from almond hulls may result in Salmonella growth. These factors should be considered in the design of Good Agricultural Practices for almonds.     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Molecular analysis of methanogenic archaeal communities in managed and natural upland pasture soils

Grassland management influences soil archaeal communities, which appear to be dominated by nonthermophilic crenarchaeotes. To determine whether methanogenic Archaea associated with the Euryarchaeota lineage are also present in grassland soils, anaerobic microcosms containing both managed (improved) and natural (unimproved) grassland rhizosphere soils were incubated for 28 days to encourage the growth of anaerobic Archaea. The contribution of potential methanogenic organisms to the archaeal community was assessed by the molecular analysis of RNA extracted from soil, using primers targeting all Archaea and Euryarchaeota. Archaeal RT‐PCR products were obtained from all anaerobic microcosms. However, euryarchaeal RT‐PCR products (of putative methanogen origin) were obtained only from anaerobic microcosms of improved soil, their presence coinciding with detectable methane production. Sequence analysis of excised denaturing gradient gel electrophoresis (DGGE) bands revealed the presence of euryarchaeal organisms that could not be detected before anaerobic enrichment. These data indicate that nonmethanogenic Crenarchaeota dominate archaeal communities in grassland soil and suggest that management practices encourage euryarchaeal methanogenic activity.     

Combined Approach for Characterization of Uncultivated Magnetotactic Bacteria from Various Aquatic Environments

Both magnetic collection and “race track” purification techniques were highly effective for selective enrichment of magnetotactic bacteria (MTB) from complex communities, as suggested by amplified ribosomal DNA restriction analysis and denaturing gradient gel electrophoresis combined with sequence analysis of 16S rRNA genes. Using these purification methods, the occurrence and diversity of MTB in microcosms from various marine and freshwater environments were assayed by using a combined microscopic, molecular, and cultivation approach. Most microcosms were dominated by magnetotactic cocci. Consistently, the majority of retrieved 16S RNA sequences were affiliated with a distinct cluster in the Alphaproteobacteria. Within this lineage the levels of sequence divergence were <1 to 11%, indicating genus-level diversity between magnetotactic cocci from various microcosms, as well as between MTB from different stages of succession of the same microcosms. The community composition in microscosms underwent drastic succession during incubation, and significant heterogeneities were observed between microcosms from the same environmental sources. A novel magnetotactic rod (MHB-1) was detected in a sediment sample from a lake in northern Germany by fluorescence in situ hybridization. MHB-1 falls into the Nitrospira phylum, displaying 91% 16S rRNA sequence similarity to “Magnetobacterium bavaricum.” In extensive cultivation attempts, we failed to isolate MHB-1, as well as most other MTB present in our samples. However, although magnetotactic spirilla were not frequently observed in the enrichments, 10 novel isolates of the genus Magnetospirillum which had not routinely been isolated in pure culture before were obtained.     

Verification of Degradation of <Emphasis Type="Italic">n</Emphasis>-Alkanes in Diesel Oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strain WatG in Soil Microcosms

Degradation of n-alkanes in diesel oil by Pseudomonas aeruginosa strain WatG (WatG) was verified in soil microcosms. The total petroleum hydrocarbon (TPH) degradation level in two bioaugmentation samples was 51% and 46% for 1 week in unsterilized and sterilized soil microcosms, respectively. The TPH degradation in the biostimulation was of control level (15%). The TPH degradation in aeration-limited samples was clearly reduced when compared with that in aeration-unlimited ones under both sterilized and unsterilized conditions. Addition of WatG into soil microcosms was accompanied by dirhamnolipid production only in the presence of diesel oil. These findings suggest that degradation of n-alkanes in diesel oil in soil microcosms would be facilitated by bioaugmentation of WatG, with production of dirhamnolipid, and also by participation of biostimulated indigenous soil bacteria.     

Continuous Enrichment Culture and Molecular Monitoring to Investigate the Microbial Diversity of Thermophiles Inhabiting Deep-Sea Hydrothermal Ecosystems

The microflora developing during a continuous enrichment culture from a hydrothermal chimney sample was investigated by molecular methods. The culture was performed in a gas-lift bioreactor under anaerobic conditions, at 90°C and pH 6.5, on a complex medium containing sulfur as the terminal electron acceptor. Archaeal and bacterial diversity was studied. Microorganisms affiliated with the genera Pyrococcus, Marinitoga, and Bacillus were detected through DGGE analysis of 16S rDNA. Additional sequences phylogenetically related to Thermococcus and -Proteobacteria were detected by cloning and sequencing of 16S rDNA from two samples of the enrichment culture. In comparison, the sequences retrieved from cloning analysis from an enrichment culture performed in a flask (batch condition) using the same culture medium showed that only members of the genus Thermococcus were cultivated. Therefore, continuous enrichment culture using the gas-lift bioreactor can be considered as an efficient and improved method for investigating microbial communities originating from deep-sea hydrothermal vents.     

Anaerobic biodegradation of pentachlorophenol in a contaminated soil inoculated with a methanogenic consortium or with Desulfitobacterium frappieri strain PCP-1

Anaerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil from a wood-treating industrial site was studied in soil slurry microcosms inoculated with a PCP-degrading methanogenic consortium. When the microcosms containing 10%–40% (w/v) soil were inoculated with the consortium, more than 90% of the PCP was removed in less than 30 days at 29 °C. Less-chlorinated phenols, mainly 3-chlorophenol were slowly degraded and accumulated in the cultures. Addition of glucose and sodium formate to the microcosms was not necessary, suggesting that the organic compounds in the soil can sustain the dechlorinating activity. Inoculation of Desulfitobacterium frappieri strain PCP-1 along with a 3-chlorophenol-degrading consortium in the microcosms also resulted in the rapid dechlorination of PCP and the slow degradation of 3-chlorophenol. Competitive polymerase chain reaction experiments showed that PCP-1 was present at the same level throughout the 21-day biotreatment. D. frappieri, strain PCP-1, inoculated into the soil microcosms, was able to remove PCP from soil containing up to 200 mg PCP/kg soil. However, reinoculation of the strain was necessary to achieve more than 95% PCP removal with a concentration of 300 mg and 500 mg PCP/kg soil. These results demonstrate that D. frappieri strain PCP-1 can be used effectively to dechlorinate PCP to 3-chlorophenol in contaminated soils. Received: 14 November 1997 / Received revision: 29 January 1998 / Accepted: 24 February 1998     

Novel Firmicutes Group Implicated in the Dechlorination of Two Chlorinated Xanthones,Analogues of Natural Organochlorines

Although the abundance and diversity of natural organochlorines are well established, much is still unknown about the degradation of these compounds. Triplicate microcosms were used to determine whether, and which, bacterial communities could dechlorinate two chlorinated xanthones (2,7-dichloroxanthone and 5,7-dichloro-1,3-dihydroxylxanthone), analogues of a diverse class of natural organochlorines. According to quantitative-PCR (qPCR) results, several known dechlorinating genera were either not present or not enriched during dechlorination of the xanthones. Denaturing gradient gel electrophoresis, however, indicated that several Firmicutes were enriched in the dechlorinating cultures compared to triplicate controls amended with nonchlorinated xanthones. One such group, herein referred to as the Gopher group, was further studied with a novel qPCR method that confirmed enrichment of Gopher group 16S rRNA genes in the dechlorinating cultures. The enrichment of the Gopher group was again tested with two new sets of triplicate microcosms. Enrichment was observed during chlorinated xanthone dechlorination in one set of these triplicate microcosms. In the other set, two microcosms showed clear enrichment while a third did not. The Gopher group is a previously unidentified group of Firmicutes, distinct from but related to the Dehalobacter and Desulfitobacterium genera; this group also contains clones from at least four unique cultures capable of dechlorinating anthropogenic organochlorines that have been previously described in the literature. This study suggests that natural chlorinated xanthones may be effective biostimulants to enhance the remediation of pollutants and highlights the idea that novel genera of dechlorinators likely exist and may be active in bioremediation and the natural cycling of chlorine.     

Anaerobic degradation of p-xylene in sediment-free sulfate-reducing enrichment culture

Anaerobic degradation of p-xylene was studied with sulfate-reducing enrichment culture. The enrichment culture was established with sediment-free sulfate-reducing consortium on crude oil. The crude oil-degrading consortium prepared with marine sediment revealed that toluene, and xylenes among the fraction of alkylbenzene in the crude oil were consumed during the incubation. The PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene for the p-xylene degrading sulfate-reducing enrichment culture showed the presence of the single dominant DGGE band pXy-K-13 coupled with p-xylene consumption and sulfide production. Sequence analysis of the DGGE band revealed a close relationship between DGGE band pXy-K-13 and the previously described marine sulfate-reducing strain oXyS1 (similarity value, 99%), which grow anaerobically with o-xylene. These results suggest that microorganism corresponding to pXy-K-13 is an important sulfate-reducing bacterium to degrade p-xylene in the enrichment culture.     

Characterization of an alkane-degrading methanogenic enrichment culture from production water of an oil reservoir after 274 days of incubation

Oil reservoirs represent special habitats for the activity of anaerobic microbial communities in the transformation of organic compounds. To understand the function of microbial communities in oil reservoirs under anaerobic conditions, an alkane-degrading methanogenic enrichment culture was established and analyzed. Results showed that a net 538 ??mol of methane higher than the controls were produced over 274 days of incubation in microcosms amended with alkanes and a decrease in the alkanes profile was also observed. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that the archaeal phylotypes were mostly related to members of the orders Methanobacteriales and Methanosarcinales. The bacterial clone library was composed of sequences affiliated with the Firmicutes, Proteobacteria, Deferribacteres, and Bacteroidetes. However, most of the bacterial clones retrieved from the enrichment cultures showed low similarity to 16S rRNA gene sequences of the cultured members, indicating that the enrichment cultures contained novel bacterial species. Though alkane-degrading methanogenic enrichment consortium has rarely been reported from petroleum reservoirs, our results indicated that oilfield production water harbors a microbial community capable of syntrophic conversion of n-alkanes to methane, which sheds light on the bio-utilization of marginal oil reservoirs for enhanced energy recovery.     

Bioremediation of Crude Oil–Contaminated Soil By Immobilized Bacteria on an Agroindustrial Waste—Sunflower Seed Husks

This study reports the immobilization and performance of a hydrocarbon-degrading Rhodococcus sp. strain (designated as QBTo) on sunflower seed husks (SH) for the bioremediation of soils polluted with crude oil. The SH performance as inoculants carrier was compared with peat, which is a vegetal material traditionally used in carrier-based inoculants production. The stability of the immobilized culture under storage conditions was assessed by viability at different times when stored at 25°C and 10°C. The catabolic activity of immobilized and free QTBo cells introduced into sandy loam soil, freshly contaminated with crude oil, was studied in microcosms. A higher number of viable QTBo cells were recovered from the inoculants formulated with SH (QTBo-SH) after prolonged storage at 10°C and 25°C. The microcosms amended with QTBo-SH inoculants showed a removal of about 66% of total petroleum hydrocarbons (TPH), whereas in those inoculated with QTBo-peat inoculants, the decrease was of about 47%. In the control microcosms (noninoculated) and liquid culture–amended soils, the TPH removal was about 28%. SH is a waste of edible oil industry, nontoxic, and biodegradable and has demonstrated to confer to the immobilized cultures greater potential to survive not only during storage but also in the soil environment, improving bioremediation process.     

Microcosm Enrichment of Biphenyl-Degrading Microbial Communities from Soils and Sediments

A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 × 10⁹ and 2 × 10¹¹ CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxybiphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences. The R. opacus isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used. In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to Alcaligenes sp., Terrabacter sp., and Bacillus thuringiensis on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives. Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species R. opacus, independent of the pollution history of the original sample. R. opacus, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.     

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.     

In vitro study of Prebiotic Properties of Levan-type Exopolysaccharides from Lactobacilli and Non-digestible Carbohydrates Using Denaturing Gradient Gel Electrophoresis

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.     

Anthracene biodegradation under nitrate-reducing condition and associated microbial community changes

Anaerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) and degraders in the subsurface environment have aroused increasing attention. Molecular techniques are especially useful when isolates are hard to obtain. Nitrate-reducing microcosms inoculated with aquifer sediment were constructed to investigate anthracene biodegradation. The associated microbial community changes were characterized using terminal restriction fragment length polymorphism analysis (TRFLP) in combination with 16S rRNA gene clone library analysis. A nearly complete removal of anthracene was achieved after an eighty day incubation under the nitrate-reducing condition. The two molecular techniques revealed a significant shift of microbial community structure, coupled with anthracene biodegradation. Species of genera Paracoccus, Herbaspirillum, Azotobacter, and Rhodococcus were grouped into four major operational taxonomic units (OTUs) in the library that was constructed with the microcosm sample on day 80. The enrichment of these genera might have links to anthracene biodegradation under the nitrate-reducing condition. Microbial consortia likely played a part in anthracene degradation.