Proposal of biostimulation for hexachlorocyclohexane (HCH)-decontamination and characterization of culturable bacterial community from high-dose point HCH-contaminated soils

Aims:  To locate a high-dose point hexachlorocyclohexane (HCH)-contaminated site, to identify HCH-degrading bacteria in it and assay HCH-decontamination by biostimulation.
Methods and Results:  Bacteria were isolated by serial dilution method from HCH-contaminated soil samples collected from areas near an HCH-manufacturing unit and its dumpsite in North India. After confirming the presence of indigenous HCH-degraders (seven of 24 strains), an ex situ biostimulation experiment was conducted. For this, residue levels in soil were diluted by mixing with pristine garden soil and aeration, moisture and nutrients were provided intermittently. This soil was monitored for reduction in Σ-HCH (sum of α-, β-, γ- and δ-HCH) levels and stimulation of HCH-degraders. Experiments were conducted twice, in March–April ( c.  75  μ g Σ-HCH g⁻¹ soil) and October–November 2006 ( c.  280  μ g Σ-HCH g⁻¹ soil) at 26–30°C. Σ-HCH levels were reduced to <30% of the original in 24 days and <3% in 240 days in the experimental pits. Terminal restriction fragment length polymorphism analysis reflected changes in microbial community structure during the course of experiment.
Conclusions:  Our results show presence of HCH-degrading sphingomonads at a high-dose point HCH-contaminated site and presents biostimulation as an effective approach for its decontamination via aeration, addition of nutrients and moisture, of the indigenous population.
Significance and Impact of the Study:  The study demonstrates that biostimulation of indigenous HCH-degrading microbial population can be used for decontamination of chronically HCH-contaminated sites.     

Dermal toxicity of hexachlorocyclohexane (HCH) in rabbit

Application of HCH (25 mg/kg) on dorsal, ventral and thigh regions of the skin of male rabbits resulted in poisoning and mortality of animals. Morphological changes in skin, liver, kidney, testes and cerebellum together with highly significant alterations in serum and liver enzymatic activity and residue in blood suggested that absorption of HCH and its toxicity could be severe when the pesticide comes in contact with the skin of thigh region of body.     

Biological cyanide destruction mediated by microorganisms

Many microorganisms have an inherent capacity to degrade the toxic organic compounds that enter the environment as a result of pollution and natural activities. Significant degradation of these compounds may take many years and it is frequently necessary to consider methods that can accelerate this process. There have been several demonstrations of enhanced biological degradation of toxic wastes, both in the laboratory and under field conditions. The prospects for enhanced biological cyanide degradation are reviewed. Compared with bench-scale processes, there are very few reports of field-scale processes for cyanide bioremediation. The implementation of such field-scale degradation requires inputs from biology, hydrology, geology, chemistry and civil engineering. A conceptual framework is emerging that can be adapted to develop new processes for bioremediation of toxic organic wastes. In terms of cyanide biodegradation, this framework incorporates identification of microbes, determination of the optimal conditions for degradation, establishment of the metabolic pathways involved in cyanide degradation, identification and localization of the genes involved, identification of suitable microbial strains for practical application and development of practical engineering processes. The present review addresses the progress that has been made in each of these aspects of cyanide biodegradation. It also examines the existing field applications of biological cyanide degradation and makes recommendations for future research.Dr S.K. Dubey is and Dr D.S. Holmes was with the Department of Biology, Clarkson University, Potsdam, NY 13699, USA. Dr D.S. Holmes is now affiliated with Centro de Estudios Cientifigos de Santiago, Av. Presedente Errazuriz 3132, Casilla 16443, Santiago 9, Chile.     

Biodegradation of trinitrotoluene (TNT) by indigenous microorganisms from TNT-contaminated soil,and their application in TNT bioremediation

Soil and groundwater contaminated by munitions compounds is a crucial issue in environmental protection. Trinitrotoluene (TNT) is highly toxic and carcinogenic; therefore, the control and remediation of TNT contamination is a critical environmental issue. In this study, the authors characterized the indigenous microbial isolates from a TNT-contaminated site and evaluated their activity in TNT biodegradation. The bacteria Achromobacter sp. BC09 and Citrobacter sp. YC4 isolated from TNT-contaminated soil by enrichment culture with TNT as the sole carbon and nitrogen source (strain BC09) and as the sole nitrogen but not carbon source (strain YC4) were studied for their use in TNT bioremediation. The efficacy of degradation of TNT by indigenous microorganisms in contaminated soil without any modification was insufficient in the laboratory-scale pilot experiments. The addition of strains BC09 and YC4 to the contaminated soil did not significantly accelerate the degradation rate. However, the addition of an additional carbon source (e.g., 0.25% sucrose) could significantly increase the bioremediation efficiency (ca. decrease of 200 ppm for 10 days). Overall, the results suggested that biostimulation was more efficient as compared with bioaugmentation. Nevertheless, the combination of biostimulation and bioaugmentation using these indigenous isolates is still a feasible approach for the development of bioremediation of TNT pollution.     

Biodegradation of xanthan by salt-tolerant aerobic microorganisms

Summary Three salt-tolerant bacteria which degraded xanthan were isolated from various water and soil samples collected from New Jersey, Illinois, and Louisiana. The mixed culture, HD1, contained aBacillus sp. which produced an inducible enzyme(s) having the highest extracellular xanthan-degrading activity found. Xanthan alone induced the observed xanthan-degrading activity. The optimum pH and temperature for cell growth were 5–7 and 30–35°C, respectively. The optimum temperature for activity of the xanthan-degrading enzyme(s) was 35–45°C, slightly higher than the optimum growth temperature. With a cell-free enzyme preparation, the optimum pH for the reduction of solution viscosity and for the release of reducing sugar groups were different (5 and 6, respectively), suggesting the involvement of more than one enzyme for these two reactions. Products of enzymatic xanthan degradation were identified as glucose, glucuronic acid, mannose, pyruvated mannose, acetylated mannose and unidentified oligo- and polysaccharides. The weight average molecular weight of xanthan samples shifted from 6.5·10⁶ down to 6.0·10⁴ during 18 h of incubation with the cell-free crude enzymes. The activity of the xanthan-degrading enzyme(s) was not influenced by the presence or absence of air or by the presence of Na₂S₂O₄ and low levels of biocides such as formaldehyde (25 ppm) and 2,2-dibromo-3-nitrilopropionamide (10 ppm). Formaldehyde at 50 ppm effectively inhibited growth of the xanthan degraders.     

海洋石油污染物的微生物降解与生物修复

石油是海洋环境的主要污染物 ,已经对海洋及近岸环境造成了严重的危害。微生物降解是海洋石油污染去除的主要途径。海洋石油污染物的微生物降解受石油组分与理化性质、环境条件以及微生物群落组成等多方面因素的制约 ,N和P营养的缺乏是海洋石油污染物生物降解的主要限制因子。在生物降解研究基础上发展起来的生物修复技术在海洋石油污染治理中发展潜力巨大 ,并且取得了一系列成果。介绍了海洋中石油污染物的来源、转化过程、降解机理、影响生物降解因素及生物修复技术等方面内容 ,强调了生物修复技术在治理海洋石油污染环境中的优势和重要性 ,指出目前生物修复技术存在的问题。     

Isolation of polyacrylamide-degrading microorganisms from soil

Two polyacrylamide-degrading bacterial strains, No. 2 and No. 11, were isolated from soil, and identified asBacillus sphaericus No. 2 andAcinetobacter sp. No. 11, respectively. Both strains grew on medium containing polyacrylamide as sole carbon and nitrogen sources.B. sphaericus No. 2 andA. sp. No. 11 reduced by 16% and 19% of the initial polyacrylamide concentration, respectively. Optimal pH and temperature in growth ofAcinetobacter sp. No. 11 were 8.0 and 37°C, respectively. After 14-day cultivation ofA. sp. No. 11, the average molecular weight of polyacrylamide has been shifted from 2.3×10⁶ to 0.5×10⁶.     

Genetic diversity of soil microorganisms assessed by analysis ofhsp70 (dnaK) sequences

The genetic diversity of a soil microbial community was assessed by analysis of clonedhsp70 sequences. A clone library was generated by polymerase chain reaction-mediated amplification of a 650-base pair fragment of thehsp70 gene, using DNA extracted from soil, without culturing the microorganisms. Fifty-five random clones were sequenced and their amino acid sequences deduced. Analysis of the amino acid sequence of the clones revealed the presence of signature sequences in common with known prokaryotic and lower eukaryotic HSP70 homologs. None of the 55 analyzed sequences were identical to each other or to a published sequence. These results confirm the presence of considerable genetic diversity within soil microbial communities, the major proportion of which remains uncharacterized.     

Reductive dechlorination of hexachlorocyclohexane (HCH) isomers in soil under anaerobic conditions

The biological anaerobic reductive dechlorination of beta-hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of beta-hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of beta-hexachlorocyclohexane within 4 months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (alpha-, beta-, gamma-, and delta-), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30 degrees C. Dechlorination of beta-hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10 mM sulphate in the influent led to simultaneous dechlorination of beta-hexachlorocyclohexane and sulphate reduction. When the column was fed with 10 mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1 month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of beta-HCH. The significance of these experiments for in situ bioremediation of polluted soils is discussed.     

Surfactant mediated enhanced biodegradation of hexachlorocyclohexane (HCH) isomers by Sphingomonas sp. NM05

Environmental biodegradation of several chlorinated pesticides is limited by their low solubility and sorption to soil surfaces. To mitigate this problem we quantified the effect of three biosurfactant viz., rhamnolipid, sophorolipid and trehalose-containing lipid on the dissolution, bioavailability, and biodegradation of HCH-isomers in liquid culture and in contaminated soil. The effect of biosurfactants was evaluated through the critical micelle concentration (CMC) value as determined for each isomer. The surfactant increased the solubilization of HCH isomers by 3-9 folds with rhamnolipid and sophorolipid being more effective and showing maximum solubilization of HCH isomers at 40 μg/mL, compared to trehalose-containing lipid showing peak solubilization at 60 μg/mL. The degradation of HCH isomers by Sphingomonas sp. NM05 in surfactant-amended liquid mineral salts medium showed 30% enhancement in 2 days as compared to degradation in 10 days in the absence of surfactant. HCH-spiked soil slurry incubated with surfactant also showed around 30-50% enhanced degradation of HCH which was comparable to the corresponding batch culture experiments. Among the three surfactants, sophorolipid offered highest solubilization and enhanced degradation of HCH isomers both in liquid medium and soil culture. The results of this study suggest the effectiveness of surfactants in improving HCH degradation by increased bioaccessibility.     

Biodegradation of p-nitrophenol and 4-chlorophenol by Stenotrophomonas sp

A bacterium named LZ-1 capable of utilizing high concentrations of p-nitrophenol (PNP) (up to 500 mg L(-1)) as the sole source of carbon, nitrogen and energy was isolated from an activated sludge. Based on the results of phenotypic features and phylogenetic similarity of 16S rRNA gene sequences, strain LZ-1 was identified as a Stenotrophomonas sp. Other p-substituted phenols such as 4-chlorophenol (4-CP) were also degraded by strain LZ-1, and both PNP and 4-CP were degraded via the hydroquinone pathway exclusively. Strain LZ-1 could degrade PNP and 4-CP simultaneously and the degradation of PNP was greatly accelerated due to the increased biomass supported by 4-CP. An indigenous plasmid was found to be responsible for phenols degradation. In soil samples, 100 mg kg(-1) of PNP and 4-CP in mixtures were removed by strain LZ-1 (10(6) cells g(-1)) within 14 and 16 days respectively, and degradation activity was maintained over a wide range of temperatures (4-35 degrees C). Therefore, strain LZ-1 can potentially be used in bioremediation of phenolic compounds either individually or as a mixture in the environment.     

Degradation of 4,5-dichloroguaiacol by soil microorganisms

No microorganisms could be isolated from chemostats or from a soil column fed with 4,5-dichloroguaiacol as the only carbon source. If guaiacol was added to chemostats with 4,5-dichloroguaiacol, either soil microbial consortia or guaiacol-degrading bacteria could dechlorinate the 4,5-dichloroguaiacol provided it was <0.2mm. A microbial consortium from farm soil removed 4,5-dichloroguaiacol under aerobic or anoxic conditions, with or without chlorolignin. Dichlorocatechol was the only 4,5-dichloroguaiacol-derived metabolite detected. In aerobic incubations, 4,5-dichlorocatechol was further degraded whereas under anoxic conditions it accumulated.     

The degradation of arsenobetaine to inorganic arsenic by sedimentary microorganisms

Two growth media containing arsenobetaine [(CH₃)₃ As⁺ CH₂COO^–] were mixed with coastal marine sediments, the latter providing a source of microorganisms. The mixtures were kept at 25 °C in the dark and shaken for several weeks under an atmosphere of air. The disappearance of arsenobetaine and the appearance of two metabolites were followed by HPLC. The HPLC-retention time of the first metabolite agreed with that of trimethylarsine oxide [(CH₃)₃AsO]. The second metabolite was identified as arsenate (As(V)) using hydride generation/cold trap/GC MS analysis and thin layer chromatography. This is the first scientific evidence showing that arsenobetaine is degraded by microorganisms to inorganic arsenic via trimethylarsine oxide. The degradation of arsenobetaine to inorganic arsenic completes the marine arsenic cycle that begins with the methylation of inorganic arsenic on the way to arsenobetaine.     

Use of Genetically Engineered Microorganisms (GEMs) for the Bioremediation of Contaminants

ABSTRACT

This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.     

Laboratory and field scale bioremediation of hexachlorocyclohexane (HCH) contaminated soils by means of bioaugmentation and biostimulation

Hexachlorocyclohexane (HCH) contaminated soils were treated for a period of up to 64 days in situ (HCH dumpsite, Lucknow) and ex situ (University of Delhi) in line with three bioremediation approaches. The first approach, biostimulation, involved addition of ammonium phosphate and molasses, while the second approach, bioaugmentation, involved addition of a microbial consortium consisting of a group of HCH-degrading sphingomonads that were isolated from HCH contaminated sites. The third approach involved a combination of biostimulation and bioaugmentation. The efficiency of the consortium was investigated in laboratory scale experiments, in a pot scale study, and in a full-scale field trial. It turned out that the approach of combining biostimulation and bioaugmentation was most effective in achieving reduction in the levels of α- and β-HCH and that the application of a bacterial consortium as compared to the action of a single HCH-degrading bacterial strain was more successful. Although further degradation of β- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of β- and δ-HCH, respectively, did not occur by the strains comprising the consortium, these metabolites turned out to be less toxic than the parental HCH isomers.     

Characterization of phosphoinositide hydrolysis products induced by hexachlorocyclohexane isomers in rat brain cortex

Water-soluble inositol metabolites were separated by anion-exchange chromatogrphy in order to determine whether or not -hexachlorocyclohexane (-HCH, lindane) and related compounds affect phosphatidylinositol hydrolysis in rat brain cortex slices. Hydrolysis was increased by -and -HCH, while - and -HCH were inactive. Muscarinic receptor stimulation of rat cortical slices with carbachol increases inositol phosphates formation. The combined effect of carbachol and the hexachlorocyclohexane isomers together were approximately equal to the sum of the effect of each one separately. The results suggest that lindane stimulates phosphoinositide phospholipase C and/or inhibits the phosphases implicated in dephosphorylation of inositol phosphates.     

微生物降解多环芳烃的研究进展

多环芳烃是一类长久存在于环境中,具有毒性、致突变与致癌等特性的环境优先污染物。本文对降解多环芳烃的微生物类群进行了阐述,介绍了在土壤与厌氧条件下细菌降解多环芳烃的研究情况,最后介绍了降解多环芳烃的相关酶类以及分子生物学的研究,并对消除环境中多环芳烃的相关生物技术提出展望。     

Utilization and degradation of lindane by soil microorganisms

Of 147 microorganisms isolated from a loamy sand, 71 showed good growth with lindane (-1,2,3,4,5,6-hexachlorocyclohexane) and produced chloride in an aqueous medium. Thirteen soil microorganisms were selected to study the utilization of lindane. Lindane was metabolized by the microbes to -2,3,4,5,6-pentachloro-1-cyclohexene (-PCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), and pentachlorobenzene (PCB). Cells of Pseudomonas sp. No. 62 grown on lindane simultaneously adapted to -PCCH, -TCCH, -TCCH, -TCCH, PCB, 1,2,3,4-tetrachlorobenzene (1,2,3,4-TCB) and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TCB). The bacteria degraded each of these chemicals at least partially as indicated by an increased rate of oxygen consumption.Abbreviations Lindane -1,2,3,4,5,6-hexachlorocyclohexane - -PCCH -2,3,4,5,6-pentachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - PCB pentachlorobenzene - 1,2,3,4-TCB 1,2,3,4-tetrachlorobenzene - 1,2,3,5-TCB 1,2,3,5-tetrachlorobenzene - 1,2,4,5-TCB 1,2,4,5-tetrachlorobenzene - 1,2,3-tCB 1,2,3-trichlorobenzene - 1,2,4-tCB 1,2,4-trichlorobenzene - 1,3,5-tCB 1,3,5-trichlorobenzene - 1,2-DCB 1,2-dichlorobenzene - 1,3-DCB 1,3-dichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene Contribution No. 631, Research Institute, Agriculture Canada, University Sub Post Office, London, Ontario N6A 5B7     

Biodegradation of 4-nitrophenol by indigenous microbial populations in Everglades soils

The Everglades in South Florida are a unique ecologicalsystem. As a result of the widespread use of pesticides andherbicides in agricultural areas upstream from these wetlands,there is a serious potential for pollution problems in theEverglades. The purpose of this study was to evaluate theability of indigenous microbial populations to degradexenobiotic organic compounds introduced by agricultural andother activities. Such biodegradation may facilitate theremediation of contaminated soils and water in the Everglades.The model compound selected in this study is 4-nitrophenol, achemical commonly used in the manufacture of pesticides. Themineralization of 4-nitrophenol at various concentrations wasstudied in soils collected from the Everglades. Atconcentrations of 10 and 100 µg/g soil, considerablemineralization occurred within a week. At a higherconcentration, i.e., 10 mg/g soil, however, no mineralizationof 4-nitrophenol occurred over a 4-month period; such a highconcentration apparently produced an inhibitory effect. Therate and extent of 4-nitrophenol mineralization was enhancedon inoculation with previously isolated nitrophenol-degradingmicroorganisms. The maximum mineralization extent measured,however, was less than 30% suggesting conversion to biomassand/or unidentified intermediate products. These resultsindicate the potential for natural mechanisms to mitigate theadverse effects of xenobiotic pollutants in a complex systemsuch as the Everglades.