Abstract Characterization of functional and phylogenetic genes was carried out on a bacterial consortium, enriched from a water treatment system of an oilfield, that could use phenanthrene as the sole carbon source. The mixed culture degraded 130 mg phenanthrene l−1 in 16 days, which is significantly faster than previously reported pure cultures. The existence of catabolic genes (nahAc, C23O) in the mixed culture was quantitated by most probable number PCR. The plasmid encoding phenanthrene catabolic genes increased relative to the chromosome genes. Heterogeneous bacteria were present according to both PCR denaturing gradient gel electrophoresis and cloning methods, suggesting the possible existence of cooperation between different biochemical PAH-transforming pathways.

Abstract A yeast strain AEH was isolated from oil contaminated soil and identified by analysis of 18S and 26S ribosomal DNA sequences asPichia anomala. Strain AEH was capable of degrading naphthalene, phenanthrene and chrysene, singly, and benzo(a)pyrene in combination. The yeast degraded 5.36 mg naphthalene l−1 within 2 days, and 5.04 mg phenanthrene l−1 and 1.54 mg chrysene 1−1 within 10 days. When a mixture of the four polycyclic aromatic hydrocarbons (PAHs) was treated at a concentration between 2.98 mg l−1 and 6.89 mg l−1, degradation rates were delayed for naphthalene and phenanthrene (3.79 mg l−1 and, 4.20 mg l−1 within 10 days, respectively), but enhanced for chrysene and benzo(a)pyrene (3.37 mg l−1 and, 1.91 mg l−1 within 10 days, respectively). In a binary system, all of the other 3 PAHs could be utilized as the carbon source for the cometabolic degradation of benzo(a)pyrene with naphthale ne as the best one.

Abstract Slurry-phase reactors have been used to investigate the biodegradation feasibility of polycyclic aromatic hydrocarbons (PAHs) in weathered crude oil, by mixed culture containing five PAHs-degrading yeast strains. Yeasts were isolated from the oily soil by enrichment culture, using phenanthrene as a sole carbon source, and identified based on the 26S ribosomal DNA (rDNA) sequence. Yeast strains belonged to the genera Candida, Pichia, Rhodotorula and Sporidiobolus. The experiment was carried out for a period of 6 weeks at room temperature with a solid : liquid ratio of 50% w/w. The results showed that high removal efficiency was obtained for all PAHs, including low molecular weight (LMW) and high molecular weight (HMW) compounds (89.3–98.6% and 66.3–89.4% within 6 weeks, respectively). The higher removal efficiency for HMW-PAHs obtained in this work suggested that yeast strains mixture could play an important role to reclaim oil-contaminated sites. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S rRNA genes was used to follow the changes of yeast populations during the slurry reactor process. The results of DGGE indicated that Candida maltosa-like and Pichia guilliermondii were the dominant species but Rhodotorula dairenensis appeared as a weak band and Sporidiobolus salmonicolor and Pichia anomala disappeared during the study. Moreover, the results showed that all of the five strains, including the two belonging to the same genus, could be differentiated from each other in the DGGE profile. Sequences of yeast isolates reported in this study have been deposited in the GenBank database under Accession Nos DQ302753, DQ303392, DQ303393, DQ350841 and DQ432635.

Abstract This study focused on the changes of soil microbial diversity and potential inhibitory effects of heavy metals on soil enzymatic activities at different application rates of Cd and/or Pb. The soil used for experiments was collected from Beijing and classified as endoaquepts. Pots containing 500 g of the soil with different Cd and/or Pb application rates were incubated for a period of 0, 2, 9, 12 weeks in a glasshouse and the soil samples were analyzed for individual enzymes, including catalase, alkaline phosphatase and dehydrogenase, and the changes of microbial community structure. Results showed that heavy metals slightly inhibited the enzymatic activities in all the samples spiked with heavy metals. The extent of inhibition increased significantly with increasing level of heavy metals, and varied with the incubation periods. The soil bacterial community structure, as determined by polymerase chain reaction-denaturing gradient gel electrophoresis techniques, was different in the contaminated samples as compared to the control. The highest community change was observed in the samples amended with high level of Cd. Positive correlations were observed among the three enzymatic activities, but negative correlations were found between the amounts of the heavy metals and the enzymatic activities.

Abstract This paper reports on the investigation of concentration levels of PAHs, community structure, as well as the abundance of PAH-related catabolic genes including upper-pathway dioxygenase genes (nahAc and phnAc) and down-pathway catechol dioxygenase genes (C12O and C23O) in a successive anoxic and aerobic treatment of produced water from the Jidong Oilfield, China. 93% of total PAHs were removed, almost equally contributed by the anoxic and aerobic units. However, PAHs of more than 3 benzene rings remained almost unchanged. The signals for phnAc and C12O were undetectable in this biological system, whereas the existence of nahAc and C23O was confirmed in the system and the copies of the two genes in the aerobic tank were 2 or 3 orders higher than those in the influent water sample. The different behavior of C23O demonstrated that mineralization of PAHs might mainly occur in the aerobic unit. The existence of nahAc and C23O genes in the influent and the high similarity of genotype between the influent and the two sludge samples suggested that bacteria existing in the influent contributed to PAH removal and bacteria harboring PAH catabolic genes were enriched in the sludge.

Abstract Polycyclic aromatic hydrocarbons (PAHs) belong to a class of toxic environmental pollutants and PAH exposure causes public health risks and raises environmental concerns. Identification of the key microorganisms that play a role in pollutant degradation processes is relevant to the development of optimal in situ bioremediation strategies. In the current study, three yeast strains were isolated from oilcontaminated soil by enrichment technique in mineral basal salts (MBS) medium supplemented with phenanthrene as a sole carbon source. Out of these, strain AH70 was selected for PAHs degradation, because of its fast growth on agar plate coated by PAHs as sole source of carbon and energy. The yeast was identified by molecular genetics technique based on sequence analysis of the variable D1/D2 domain of the large subunit (26S) ribosomal DNA. Subsequent 26S rRNA gene sequencing showed 100% base sequence homology and it was identified as Candida viswanathii. The degradation of PAHs by this yeast was confirmed by GC-MS analyses. The yeast was capable of degrading a mixture of low and high molecular weight PAHs and degradation efficiency was found as 89.76% for naphthalene, 77.21% for phenanthrene, 60.77% for pyrene and 55.53% for benzo(a)pyrene at the end of 10 days.

Abstract Background, aim, and scope In the soil environment, polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) are of great environmental and human health concerns due to their widespread occurrence, persistence, and carcinogenic properties. Bioremediation of contaminated soil is a cost-effective, environmentally friendly, and publicly acceptable approach to address the removal of environmental contaminants. However, bioremediation of contaminants depends on plant–microbe interactions in the rhizosphere. The microorganisms that can mineralize various PAHs have PAH dioxygenase genes like nahAc, phnAc, and pdo1. To understand the fate of pyrene in rhizospheric and non-rhizospheric soils in the presence or absence of Pb, pyrene biodegradation, bacterial community structure, and dioxygenase genes were investigated in a pot experiment. Methods Soil was amended with Pb (a representative heavy metal), pyrene, and a Pb/pyrene mixture. After 8 weeks of aging, one set of pot microcosms was cultivated with rye grass (Lolium multiflorum L) seedlings, while another set was not cultivated for the purpose of comparing rhizosphere and non-rhizosphere pyrene degradation. Pyrene was extracted from freeze-dried soil and plant samples using a Soxhlet extraction method and the extracts were dried to 1 mL under gentle nitrogen flow and analyzed using gas chromatograph mass spectrometry (Agilent 6890, USA). Soil DNA was extracted from triplicate samples and the DGGE was performed using a Bio-Rad Dcode™ Universal Mutation Detection System (Bio-Rad, USA). PAH dioxygenase genes, including nahAc, phnAc, and pdo1, were detected using PCR amplification. Similarly, pyrene degraders were also investigated using plate counting technique. Results Biodegradation rates recorded over an 18-week period showed that rye grass promoted significant (P  0.05) pyrene degradation. Pyrene removal efficiency from rhizospheric soils was 59.1 ± 2.1% and 68.7 ± 2.3% in pyrene- and Pb/pyrene-amended soils, respectively. The results indicate that pyrene dissipation was significantly (P  0.05) higher in Pb/pyrene-amended soils than only-pyrene-amended soils. The plant growth promoted the degradation of pyrene and accounted for 12.1% to 17.0% of dissipation enhancement in the rhizospheric soils. In this study, the DGGE profiles revealed a shift in soil bacterial community structure in all amended soils, with a higher number and greater complexity of banding patterns in Pb/pyrene-amended samples than in either Pb- or pyrene-amended samples. In the control and Pb-amended soil, pdo1 and nahAc genes were not detected throughout the incubation period but were detected in the pyrene- and Pb/pyrene-amended soils. However, phnAc genes were not detected in either amended or non-amended soils throughout the incubation period. The addition of pyrene had a dramatic effect on the number of pyrene degraders. Discussion Plants contribute to the degradation of PAHs by increasing the size of microbial population, promoting microbial activity, and modifying microbial community diversity in the rhizosphere. In this study, the presence of plants significantly promoted the degradation of pyrene in the soil due to enhanced bacterial community size and increased the number of pyrene degraders. Similarly, the results of this study have also clearly shown that pyrene remaining in soils only accounted for about 1/3 of the total pyrene addition, suggesting that most of the pyrene added could be removed by plant and/or microbial degradation. HMs and PAHs interaction towards degradation of PAHs can be both negative and positive depending on type and concentration of both HMs and PAHs. In both rhizospheric and non-rhizospheric soils, the pyrene degradation was in line with the changes of bacterial structure, increasing number of pyrene degraders, and the prevalence of dioxygenase genes (nahAc and pdo1). This work represents the first report that Pb can affect the dissipation of pyrene and functional genes were detected in both rhizospheric and non-rhizospheric soils amended with pyrene and Pb/pyrene. Conclusions Pyrene removal efficiency for rhizospheric soils was higher than for non-rhizospheric soils and pyrene dissipation was accelerated in the presence of Pb in both rhizospheric and non-rhizospheric soils. The bacterial community structure was changed and the addition of pyrene indicated dramatic effects on the number of pyrene degraders. The catabolic genes, including nahAc and pdo1, which are responsible for HMW-PAH degradation, were confirmed in both rhizosphere and non-rhizosphere soils amended with pyrene or Pb/pyrene. Recommendations and perspectives Our findings suggest that the interaction between bacterial community and plant roots could influence the PAH degradation both in the presence and absence of HMs in the contaminated soils.

Abstract Background, aim, and scope Soil contamination with heavy metals occurs as a result of both anthropogenic and natural activities. Heavy metals could have long-term hazardous impacts on the health of soil ecosystems and adverse influences on soil biological processes. Soil enzymatic activities are recognized as sensors towards any natural and anthropogenic disturbance occurring in the soil ecosystem. Similarly, microbial biomass carbon (MBC) is also considered as one of the important soil biological activities frequently influenced by heavy metal contamination. The polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) has recently been used to investigate changes in soil microbial community composition in response to environmental stresses. Soil microbial community structure and activities are difficult to elucidate using single monitoring approach; therefore, for a better insight and complete depiction of the soil microbial situation, different approaches need to be used. This study was conducted in a greenhouse for a period of 12 weeks to evaluate the changes in indigenous microbial community structure and activities in the soil amended with different application rates of Cd, Pb, and Cd/Pb mix. In a field environment, soil is contaminated with single or mixed heavy metals; so that, in this research, we used the selected metals in both single and mixed forms at different application rates and investigated their toxic effects on microbial community structure and activities, using soil enzyme assays, plate counting, and advanced molecular DGGE technique. Soil microbial activities, including acid phosphatase (ACP), urease (URE), and MBC, and microbial community structure were studied. Materials and methods A soil sample (0–20 cm) with an unknown history of heavy metal contamination was collected and amended with Cd, Pb, and Cd/Pb mix using the CdSO4 and Pb(NO3)2 solutions at different application rates. The amended soils were incubated in the greenhouse at 25 ± 4°C and 60% water-holding capacity for 12 weeks. During the incubation period, samples were collected from each pot at 0, 2, 9, and 12 weeks for enzyme assays, MBC, numeration of microbes, and DNA extraction. Fumigation–extraction method was used to measure the MBC, while plate counting techniques were used to numerate viable heterotrophic bacteria, fungi, and actinomycetes. Soil DNAs were extracted from the samples and used for DGGE analysis. Results ACP, URE, and MBC activities of microbial community were significantly lower (p  0.05) in the metal-amended samples than those in the control. The enzyme inhibition extent was obvious between different incubation periods and varied as the incubation proceeded, and the highest rate was detected in the samples after 2 weeks. However, the lowest values of ACP and URE activities (35.6% and 36.6% of the control, respectively) were found in the Cd3/Pb3-treated sample after 2 weeks. Similarly, MBC was strongly decreased in both Cd/Pb-amended samples and highest reduction (52.4%) was detected for Cd3/Pb3 treatment. The number of bacteria and actinomycetes were significantly decreased in the heavy metal-amended samples compared to the control, while fungal cells were not significantly different (from 2.3% to 23.87%). In this study, the DGGE profile indicated that the high dose of metal amendment caused a greater change in the number of bands. DGGE banding patterns confirmed that the addition of metals had a significant impact on microbial community structure. Discussion In soil ecosystem, heavy metals exhibit toxicological effects on soil microbes which may lead to the decrease of their numbers and activities. This study demonstrated that toxicological effects of heavy metals on soil microbial community structure and activities depend largely on the type and concentration of metal and incubation time. The inhibition extent varied widely among different incubation periods for these enzymes. Furthermore, the rapid inhibition in microbial activities such as ACP, URE, and MBC were observed in the 2 weeks, which should be related to the fact that the microbes were suddenly exposed to heavy metals. The increased inhibition of soil microbial activities is likely to be related to tolerance and adaptation of the microbial community, concentration of pollutants, and mechanisms of heavy metals. The DGGE profile has shown that the structure of the bacterial community changed in amended heavy metal samples. In this research, the microbial community structure was highly affected, consistent with the lower microbial activities in different levels of heavy metals. Furthermore, a great community change in this study, particularly at a high level of contamination, was probably a result of metal toxicity and also unavailability of nutrients because no nutrients were supplied during the whole incubation period. Conclusions The added concentrations of heavy metals have changed the soil microbial community structure and activities. The highest inhibitory effects on soil microbial activities were observed at 2 weeks of incubation. The bacteria were more sensitive than actinomycetes and fungi. The DGGE profile indicated that bacterial community structure was changed in the Cd/Pb-amended samples, particularly at high concentrations. Recommendations and perspectives The investigation of soil microbial community structure and activities together could give more reliable and accurate information about the toxic effects of heavy metals on soil health.

Abstract To investigate the effects of physiological properties on polycyclic aromatic compound (PAH) degradation, the surface tension and emulsification activities, and cell surface hydrophobicity of five PAH-degrading yeast isolates were compared to Saccharomyces cerevisiae from cultures grown with glucose, hexadecane, or naphthalene as carbon sources. The cell surface hydrophobicity values for the five yeast strains were significantly higher than for S. cerevisiae for all culture conditions, although these were highest with hexadecane and naphthalene. Strains with higher hydrophobicity showed higher rates of naphthalene and phenanthrene degradation, indicating that increased cell hydrophobicity might be an important strategy in PAH degradation for the five strains. Emulsification activities increased for all five yeast strains with naphthalene culturing, although no relationship existed between emulsification activity and PAH degradation rate. Surface tensions were not markedly reduced with naphthalene culturing.

Abstract Microbial characteristics in the anaerobic tank of a full-scale produced water treatment plant capable of anaerobic hydrocarbon removal were analyzed and compared to those in the influent produced water using cultivation-independent molecular methods. Clones related to methanogens including the methylotrophic Methanomethylovorans thermophila and hydrogen- and the formate-utilizing Methanolinea tarda were in abundance in both samples, but greater numbers of M. tarda-like clones were detected in the biofilm library. Both DGGE and cloning analysis results indicated that the archaea in the biofilm were derived from the influent produced water. Bacterial communities in the influent and biofilm samples were significantly different. Epsilonproteobacteria was the dominant bacterial group in the influent while Nitrospira and Deltaproteobacteria were the predominant groups in the biofilm. Many clones related to syntrophic bacteria were found among the Deltaproteobacteria. One Deltaproteobacteria clone was related to Syntrophus, which is commonly found in methanogenic hydrocarbon-degrading consortia. A number of Deltaproteobacteria clones were assigned to the clone cluster group TA, members of which predominate in various methanogenic consortia that degrade aromatic compounds. These results suggest that a microbial community associated with methanogenic hydrocarbon degradation may have been established in the biofilm.