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.

Abstract The effects of seasonal change in temperature on the chemical compositions of water and bacterial diversity in copper mining wastewater reservoir (CMWR) located in Jiangxi province, China, was investigated. Wastewater samples were collected collected in December 2008 and May 2009 from different points of CMWR and analyzed for anions, heavy metals, and microbial community structure using standard procedures. Most of the parameters exceeded the limits set by the Chinese government. However, the concentrations of some selected parameters such as pH, BOD, DO, and DOC and heavy metals were significantly (P=0.05) varied and exhibited a reduction from the inlet to the outlet of CMWR. Bacterial diversity was studied by the combined polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and cloning approach. The PCR-DGGE profiles showed the presence of 10 common bands in the seasonal samples of the reservoir indicating the high similarities among the bacterial populations existing during the two seasons. Furthermore, the DGGE profiles also evidenced the existence of some unique bands suggesting that the differences in bacterial diversity may be caused by the different environmental conditions. 22 major bands from the DGGE profiles were further reamplified, cloned, and sequenced. The results of sequencing analysis indicated the presence of Rheinheimera sp., Acidithiobacillus ferrooxidans, Afipia sp. and Burkholderia sp. as the dominant bacterial species in the CMWR samples. The Afipia sp. and Burkholderia sp. were found in summer samples only while most of the other species were common in both the seasons. Finally, the Copper mine wastewater was deficient in nutrients but enriched with the bacterial diversity of the extremophiles.

Abstract Forty yeast strains isolated from soils taken from different locations in Egypt were tested for their P-solubilizing activities on the basis of analyzing the clear zone around colonies growing on a tricalcium phosphate medium after incubation for 5 days at 25degreesC, denoted as the solubilization index (SI). Nine isolates that exhibited P-solubilization potential with an SI ranging from 1.19 to 2.76 were genetically characterized as five yeasts belonging to the genus Saccharomyces cerevisiae and four non-Saccharomyces, based on a PCR analysis of the ITS1-26S region amplied by SC1/SC2 species-specific primers. The highest Psolubilization efficiency was demonstrated by isolate PSY- 4, which was identified as Saccharomyces cerevisiae by a sequence analysis of the variable D1/D2 domain of the 26S rDNA. The effects of single and mixed inoculations with yeast PSY-4 and Bacillus polymyxa on the P-uptake and growth of corn were tested in a greenhouse experiment using different levels of a phosphorus chemical fertilizer (50, 100, and 200 kg/ha super phosphate 15.5% P2O5). The results showed that inoculating the corn with yeast PSY-4 or B. polymyxa caused significant increases in the shoot and root dry weights and P-uptake in the shoots and roots. The P-fertilization level also had a significant influence on the shoot and root dry weights and P-uptake in the shoots and roots when increasing the P-level from 50 up to 200 kg/ha. Dual inoculation with yeast strain PSY-4 and B. polymyxa at a P-fertilization level of 200 kg/ha gave higher values for the shoot and root dry weights and P-uptake in the shoots and roots, yet these increases were nonsignificant when compared with dual inoculation with yeast strain PSY-4 and B. polymyxa at a P-fertilization level of 100 kg/ha. The best increases were obtained from dual inoculation with yeast strain PSY-4 and B. polymyxa at a P-fertilization level of 100 kg/ha, which induced the following percentage increases in the shoot and root dry weights, and P-uptake in the shoots and roots; 16.22%, 46.92%, 10.09%, and 31.07%, respectively, when compared with the uninoculated control (fertilized with 100 kg/ha).

Abstract Forty yeast strains isolated from soils taken from different locations in Egypt were tested for their P-solubilizing activities on the basis of analyzing the clear zone around colonies growing on a tricalcium phosphate medium after incubation for 5 days at 25degreesC, denoted as the solubilization index (SI). Nine isolates that exhibited P-solubilization potential with an SI ranging from 1.19 to 2.76 were genetically characterized as five yeasts belonging to the genus Saccharomyces cerevisiae and four non-Saccharomyces, based on a PCR analysis of the ITS1-26S region amplied by SC1/SC2 species-specific primers. The highest Psolubilization efficiency was demonstrated by isolate PSY- 4, which was identified as Saccharomyces cerevisiae by a sequence analysis of the variable D1/D2 domain of the 26S rDNA. The effects of single and mixed inoculations with yeast PSY-4 and Bacillus polymyxa on the P-uptake and growth of corn were tested in a greenhouse experiment using different levels of a phosphorus chemical fertilizer (50, 100, and 200 kg/ha super phosphate 15.5% P2O5). The results showed that inoculating the corn with yeast PSY-4 or B. polymyxa caused significant increases in the shoot and root dry weights and P-uptake in the shoots and roots. The P-fertilization level also had a significant influence on the shoot and root dry weights and P-uptake in the shoots and roots when increasing the P-level from 50 up to 200 kg/ha. Dual inoculation with yeast strain PSY-4 and B. polymyxa at a P-fertilization level of 200 kg/ha gave higher values for the shoot and root dry weights and P-uptake in the shoots and roots, yet these increases were nonsignificant when compared with dual inoculation with yeast strain PSY-4 and B. polymyxa at a P-fertilization level of 100 kg/ha. The best increases were obtained from dual inoculation with yeast strain PSY-4 and B. polymyxa at a P-fertilization level of 100 kg/ha, which induced the following percentage increases in the shoot and root dry weights, and P-uptake in the shoots and roots; 16.22%, 46.92%, 10.09%, and 31.07%, respectively, when compared with the uninoculated control (fertilized with 100 kg/ha).

Abstract Ten yeast strains acquired from different sources and capable of utilizing vegetable oil or related compounds (fatty acid or oleic acid) as sole carbon sources were inoculated into a sequencing batch reactor (SBR) for the treatment of high-strength vegetable oil-containing wastewater. The SBR system stably removed >89% of chemical oxygen demand (COD) and >99% of oil when fed with wastewater containing 15 g/L COD and 10 g/L oil in average. Denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 26S rRNA genes showed that among the ten yeast strains, only Candida lipolytica, Candida tropicalis, and Candida halophila were dominant in the system. To elucidate the major factors affecting the selection of yeast strains in the SBR system, the three dominant strains were compared with two non-dominant strains in terms of COD removal performance, biomass yield, cell settleability, cell flocculation ability, cell emulsification ability, and surface hydrophobicity. Results showed that hydrophobicity and emulsification ability of yeast cells were the two most important factors determining the selection of yeast strains in the treatment of high-strength oil-containing wastewater.

Abstract The combination of PCR amplification of 16S rRNA genes with denaturing gradient gel electrophoresis (DGGE) analysis was used to reveal the compositions and dynamics of bacterial communities in a sewage treatment plant with two systems, i.e., an anoxic-anaerobic-aerobic system (inverted A2O) and an anaerobic-anoxic-aerobic one (conventional A2O) over a period from February to July 2009, during which both systems experienced serious sludge bulking problems. The DGGE patterns showed that there were many common bands in both systems, suggesting the high similarity of bacterial communities of the two systems. Meanwhile, the moving window correlation analysis showed that the two systems experienced different microbial community structure changes during the period, which might be related with the different situations of the occurrence and disappearance of sludge bulking, as being reflected by sludge volume index (SVI) values. Major bands of DGGE patterns of sludge samples were further sequenced. Phylogenetic affiliation indicated that the majority of the sequences obtained were affiliated with Actinobacteria, Firmicutes, Bacteroidetes/Chlorobi group and α- and β-Proteobacteria. Two sequences showed high similarities to typical filamentous bacteria Microthrix parvicella and Nostocoida limicola I, indicating that these bacterial species have been involved in the sludge bulking problems.

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