Abd El-Latif Hesham

Soil enzymatic activities and microbial community structure with different application rates of Cd and Pb

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.

Comparison of bacterial community structures in two systems of a sewage treatment plant using PCR-DGGE analysis

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 A20) and an anaerobic-anoxic-aerobic one (conventional A20) 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 alpha- and beta-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.

Application of PCR-DGGE to analyse the yeast population dynamics in slurry reactors during degradation of polycyclic aromatic hydrocarbons in weathered oil

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. Copyright

Isolation and identification of a yeast strain capable of degrading four and five ring aromatic hydrocarbons

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.

Biodegradation Ability and Catabolic Genes of Petroleum-Degrading Sphingomonas koreensis Strain ASU-06 Isolated from Egyptian Oily Soil

Polycyclic aromatic hydrocarbons (PAHs) are serious pollutants and health hazards. In this study, 15 PAHs-degrading bacteria were isolated from Egyptian oily soil. Among them, one Gram-negative strain (ASU-06) was selected and biodegradation ability and initial catabolic genes of petroleum compounds were investigated. Comparison of 16S rRNA gene sequence of strain ASU-06 to published sequences in GenBank database as well as phylogenetic analysis identified ASU-06 as Sphingomonas koreensis. Strain ASU-06 degraded 100, 99, 98, and 92.7% of 100 mg/L naphthalene, phenanthrene, anthracene, and pyrene within 15 days, respectively. When these PAHs present in a mixed form, the enhancement phenomenon appeared, particularly in the degradation of pyrene, whereas the degradation rate was 98.6% within the period. This is the first report showing the degradation of different PAHs by this species. PCR experiments with specific primers for catabolic genes alkB, alkB1, nahAc, C12O, and C23O suggested that ASU-06 might possess genes for aliphatic and PAHs degradation, while PAH-RHDαGP gene was not detected. Production of biosurfactants and increasing cell-surface hydrophobicity were investigated. GC/MS analysis of intermediate metabolites of studied PAHs concluded that this strain utilized these compounds via two main pathways, and phthalate was the major constant product that appeared in each day of the degradation period.

Cell surface properties of five polycyclic aromatic compound-degrading yeast strains

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.

Study of enhancement and inhibition phenomena and genes relating to degradation of petroleum polycyclic aromatic hydrocarbons in isolated bacteria

Polycyclic aromatic hydrocarbons (PAHs) are xenobiotic compounds, which being degraded by chemical, physical or biological methods. The latter is the safest and the cheapest one. Two bacterial strains ASU-01 and ASU-016 were isolated from different Egyptian petroleum contaminated sites. They were genetically identified based on the analysis of the nucleotide sequences of the 16S ribosomal PNA gene and the phylogenetic tree as Enterobacter hormaechei and Pseudomonas pseudoalcaligenes respectively. When pyrene as high molecular weight (HMW)-PAH was added as a sole carbon source, both strains could degrade it with efficiency 77.7 and 83.7% within 15 days of incubation, respectively. However, when it was mixed with low molecular weight (LMW)-PAHs, two opposite phenomena appeared. The first one was enhancement, which occurred with ASU-01. This strain shifted pyrene efficiency to 98.5%. The second phenomenon was inhibition occurred with ASU-016 which completely retarded pyrene degradation. Naphthalene dioxygenase (nahAc), and catechol dioxygenases (C12O and C23O) genes were detected in the two strains based on PCR. The detected genes were confirmed by determining the different specific activities of their translated protein (enzymes) on different PAHs. The maximum values of biosurfacatant production activity and cell-surface and percentage of cell-surface hydrophobicity (CSH) were detected during the exponential phase. These latter factors increased the bioavailability and consequently, the assimilation of PAHs.

Effects of different sewage sludge applications on heavy metal accumulation, growth and yield of spinach (Spinacia oleracea L.).

ABSTRACT In this study, we present the response of spinach to different amendment rates of sewage sludge (0, 10, 20, 30, 40 and 50 g kg−1) in a greenhouse pot experiment, where plant growth, biomass and heavy metal uptake were measured. The results showed that sewage sludge application increased soil electric conductivity (EC), organic matter, chromium and zinc concentrations and decreased soil pH. All heavy metal concentrations of the sewage sludge were below the permissible limits for land application of sewage sludge recommended by the Council of the European Communities. Biomass and all growth parameters (except the shoot/root ratio) of spinach showed a positive response to sewage sludge applications up to 40 g kg−1 compared to the control soil. Increasing the sewage sludge amendment rate caused an increase in all heavy metal concentrations (except lead) in spinach root and shoot. However, all heavy metal concentrations (except chromium and iron) were in the normal range and did not reach the phytotoxic levels. The spinach was characterized by a bioaccumulation factor <1.0 for all heavy metals. The translocation factor (TF) varied among the heavy metals as well as among the sewage sludge amendment rates. Spinach translocation mechanisms clearly restricted heavy metal transport to the edible parts (shoot) because the TFs for all heavy metals (except zinc) were <1.0. In conclusion, sewage sludge used in the present study can be considered for use as a fertilizer in spinach production systems in Saudi Arabia, and the results can serve as a management method for sewage sludge.

Biocontrol of apple blue mould by new yeast strains: Cryptococcus albidus KKUY0017 and Wickerhamomyces anomalus KKUY0051 and their mode of action

Seeking new yeast strains having the ability to protect apple fruits against blue mould for a long time under different storage conditions was the main goal of this work. Based on the in vitro test, yeast strains KKUY0017 and KKUY0051 were selected as the most effective antagonists against Penicillium expansum. Sequencing of 26S rDNA of both yeasts confirmed that the identity of KKUY0017 and KKUY0051 was Cryptococcus albidus and Wickerhamomyces anomalus, respectively. The two strains protected the apple fruits from the blue mould disease under a wide range of temperature (5–30°C); however, W. anomalus KKUY0051 was more effective. At 25°C, W. anomalus KKUY0051 involved in the reduction of disease severity and disease incidence of blue mould by 56.49% and 57.78%, respectively. When either of the two yeasts was applied in concentration of 108 or 109 cells/mL, the maximum reduction in disease severity and disease incidence was achieved. Under cold storage (5°C), both yeast strains succeeded to protect the apple fruits free from the infection up to 24 days. Electron micrograph showed a fit attachment between the cells of C. albidus KKUY0017 and the fungal hyphae leading to the degrading of the hyphae; however, W. anomalus killed the fungal hyphae without direct attachment to them. Gas chromatography–mass spectrometry analysis of the cell-free extract of W. anomalus KKUY0051 revealed the presence of toxic compounds such as the nitrophenol derivatives. The results support the assumption that the main mode of action of this yeast is by killer toxins. We conclude that application of these yeasts under cold storage condition could keep the apple fruits free from blue mould infection for a long time.

PCR-DGGE and real-time PCR dsrB-based study of the impact of heavy metals on the diversity and abundance of sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) are widely used for heavy metal (HM) treatment in bioreactors but their growth and biological activity can be inhibited by such treatment. Here, bioreactor experiments were used to investigate changes in the SRB community and the copy number of the dissimilatory sulfite reductase β-subunit functional gene (dsrB) under high doses of sulfates and HMs. The SRB community was investigated using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing techniques, while the dsrB gene abundance was measured by quantitative real-time PCR (qRT-PCR). The sulfate reduction rate was initially much higher in reactors without HMs than in those containing HMs (p = 0.001). Sulfate levels were reduced by 50% within the first 3 days of operation. As a result, the HM removal rate was initially much lower in the reactors containing HMs. Most of the HMs reduced to safe limits within 9 ~ 12 days of operation. The SRB community mainly consisted of Desulfovibrio vulgaris, D. termitidis, D. desulfuricans, D. simplex and Desulfomicrobium baculatum, as determined by PCR-DGGE. qRT-PCR revealed a decreasing trend in the copy numbers of a functional gene (dsrB) after 6 days in samples lacking HMs; however, the opposite trend was observed in the HM-containing samples.