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Degradation of petroleum in soil by biological methods
250
Abstracts
Isolation of a Gram-Positive p-CB Degrading Bacterial Strain M. C. SPPgua”, M. H. Paveia”, M. G. Vieira” and A. M. Anselmo” “Department o[Biol. Veg., Faculdade de Ciencias de Lisboa, Portugal, and Inst. Nat. Eng. e Techn. Ind., Portugal The high physical and chemical inertness of halogenated biphenyls places these compounds among the most resistant pollutants. However microbial degradation of polychlorinated biphenyls (PCBs) has been reported repeatedly. In bacteria the major catabolic aerobic pathway of PCBs is an extradiol cleavage of the aromatic ring with production of chlorobenzoate. The key enzymes involved in this pathway are encoded by several genes usually designated bph. We isolated a mixed microbial population (7 bacteria and 1 fungus) from a soil sample of an unexposed natural environment which after enrichment and establishment in a defined medium containing p-CB (p-Chlorobiphenyl) as the sole carbon source, was able to utilize this synthetic organic compound. The growth of the pure cultures of each microorganism on p-CB medium showed that only one of the bacterial strain could transform p-CB to chlorobenzoate indicating a metacleavage pathway. This gram-positive strain had the ability to degrade approximately 50% of p-CB (300 mg/l) within 3 days of incubation. The cloning of the p-CB degrading genes is in progress.
Ecological Interactions Between Specially Selected Strains and Natural Microorganisms A. Vu. Fedorov and E. V. Volchenko Institute qfGenetics and Selection of Industrial Microorganisms (GNII Genetics), Saratov Branch. Tulaykova str. 1650, Saratov. 4 10020, Russia In contrast to genetically engineered microorganisms, strains -destroyers and -transformers of xenobiotics (SD) obtained from the environment are not alien to nature. However. selection and adaptation processes in vitro are often carried out taking into account only degradative activity without regard for other properties. It is known that the content of destroyers in biocenosises, even in polluted sites, is comparatively low and that ecosystems can balance or remove small inoculum; but when natural microorganisms altered by selection are returned into the environment in massive amounts during the process of bioremediation or together with purified effluents, it may cause ‘ecological aggression’. Studies on ecological interactions between SD from our collection and natural microorganisms from various aqueous ecosystems have shown that the most of SD would have little chance to survive under natural conditions. Clear river bicenosis possessed higher ecological protection whereas microflora from polluted river manifested lower activity. At the same time, several SD repressed growth of the majority of the natural microorganisms and could influence model river ecosystems.
Biodegradation
of Surfactant and Mineral Oils
0. Turkovskava”, L. Panchenkos, A. MuratovaB, E. Duhrovskaya” and G. Shuhb UInstitute of Genetic and Selection of Industrial Microorganisms, Saratov Branch, Tulaykova st.. p/b 1650, Saratov 4 10020, Russia, and hSaratov Medical University, B. Kazachja 112, Saratov 4 10020, Russia The collection of strains primary degrading a wide spectrum
from the genus Pseudomonas of anionic and nonionic
surfactants was created. Pseudomonas putida TP- 19 is the most active strain which utilized alkylphenol ethoxylate along with alcohol and fatty acid ethoxylates. It was determined that destruction proceeds and follows the path of gradual PEGchain reduction and carboxylation of hydrocarbon radical. In the case with alkylphenol ethoxylate in cultural liquid, the alkylphenol was collected. The microbial consortium formed on the basis of TP-19 under continuous cultivation was capable of destroying alkylphenol ethoxylate in full. In strains utilizing alkylsulfate alkyladdition. and benzensulfonate were selected. Often industrial sewage contains a composition of surfactants and oil products (mineral oils in particular). We succeeded in screening for corresponding strains and microbial associations. Depending on the structure and heterogeneity of substances 70-100% of oils and surfactants were degraded in 24 h in their concentrations 1 g/l. The obtained microorganisms may be used in microbial treatment of sewage and for introduction into environmental objects.
Bacterial Degradation of PAHs Containing More than Three Rings - The State of the Art K. Rehmann and A. Kettrup GSF-Institut fur d’kologische Chemie, Neuherberg. D-85764 OberschleiJheim, Germany Relevant statements on the formation of environmentally hazardous microbial metabolites during the bioremediation of soils or water contaminated with polycyclic aromatic hydrocarbons (PAHs) require detailed information about their microbial degradation pathways. A short survey of the state of our knowledge concerning metabolite formation is given, focusing on PAH degradation by pure bacterial strains and emphasizing results currently obtained with pyrene and fluoranthene. A complete metabolic pathway for the degradation of pyrene by Mycobacterium and Rhodococcus spp. is presented. Furthermore, some reasons for the failure so far to isolate pure strains, which are able to utilize PAHs containing more than four rings, are discussed in brief. It is concluded that further progress in metabolite research may be achieved at least in three ways: (a) by exploring the genetic background of PAH degradation, (b) by improving the enrichment and screening techniques used to isolate bacteria1 PAH degraders for example by using PAH solubilizing agents, and (c) by investigating the metabolic potential of defined mixed cultures and model ecosystems to grasp the influence of cometabolic processes.
Degradation of Petroleum in Soil by Biological Methods T. Jamroz Department Bioprocess Engineering, Faculty of Process and Environmental Engineering, Technical University of &id?, ul. Wolczariska 175, Poland Biodegradation of a mode1 solution containing n-hexadecane, isooctane and benzene has been studied. The biological degradation of hydrocarbons in soil has been carried on; -indigenous microflora, -indigenous microflora activated by biogenous nutrients and indigenous microflora enriched with the biopreparate. Dynamics of hydrocarbon degradation and number of microorganisms included in this process were examined. The decontamination took place in different ways depending on microflora which was founded in soil. The best reduction of model petroleum solution (about 97%), was obtained using the biopreparate and the smallest one (about 32%) using only the indigenous microflora. The content of
251
Abstracts
Degradation of Polychlorinated Biphenyls (PCB) in Different Soils by Inoculated Alcaligenes xylosoxidans
types. The sorption of the majority of dyes tested could be described using Langmuir isotherms. Maximum sorption capacities were calculated. The prepared sorbents could be also used in analytical chemistry for the isolations of planar xenobiotics from biological and environmental samples prior to their analysis using standard analytical procedures.
K. Dercovi, L’. HaluBka and s. BaE Department of Biochemical Technology, Faculty of Chemical Technology. Slovak Technical University. Radlinskeho 9, 812 37 Bratislava, Slovakia
Induction of Alcohol Dehydrogenases Pseudomonas C 12B
microbiological cells in investigated soil persisted level both for contaminated and control samples.
at a similar
The degradation of polychlorinated biphenyls in soils by biphenyl-utilizing strain Alcaligenes xylosoxidans was studied in different soil types. In addition to the congener specificity, significant differences in the degradation of PCB by the strain in the different soil types were observed. Efficiency of degradation was generally better in sterilized soils, but the differences were not as significant as the differences observed among the different soil types. These results indicate that the degradation of PCB is probably related not only to the capabilities of the strain employed and quality and amount of competitive species inhabiting the soils, but also to the soil sorption of the PCB congeners: the degradation is faster in the soils containing an intermediate amount of organic carbon with a high portion of total and aromatic carbon in humic acids
Isolation of Planar Organic Compounds Using Copper Phthalocyanine Dye Immobilized on Magnetic Carriers 1. Safaiik and M. 6afaiikovB Laboratory of Biochemistry and Biotechnology, Institute of Landscape Ecology, Academy of Sciences. Branisbvska 31, 370 OS Ceske BudeYjovice. Czech Republic Magnetic separations represent a very promising procedure for the isolation and removal of environmental contaminants. Organic compounds with a planar structure form complexes dyes; magnetic phthalocyanine with phthalocyanine derivatives were tested as a possible specific sorbents of these compounds. Reactive copper phthalocyanine dye (Ostazin turquoise V-G; C.I. Reactive Blue 21) was immobilized on magnetic chitosan and on silanized magnetite particles. The prepared sorbents were used for the studies of sorptions of organic compounds with a planar molecular structure, namely water soluble organic dyes of heteropolyaromatic and triphenylmethane
in
M. Suchinek”, J. N. T. Rindb and B. KrAlov$” ODepartment of Biochemistry and Microbiology. Faculty of Food and Biochemical Technology Institute of Chemical Technology, Praha, 166 28 Praha 6, Czech Republic. and hDepartment of Biochemistry, School of Molecular and Medical Biosciences, University of Wales, College of Cardiff, Cardiff CFI 3US, UK Pseudomonas C 12B is a detergent-degrading soil bacterium that was isolated for its ability to grow on sodium dodecyl sulphate. Grown in batch culture on nutrient broth, the organism produces four cytosolic alcohol dehydrogenases distinguished by non-denaturating PAGE. These enzymes require NAD’ as a cofactor of the reaction. Decane induces two membrane-bound enzymes that are not dependent on presence of NAD+ in assay mixture. The natural cofactor of membrane-bound alcohol dehydrogenases is not known, but phenazine methosulphate coupled with suitable redox dye can be used for both quantitative and qualitative assays. Pseudomonas C12B was grown in batch culture in 200ml portions of nutrient broth until onset of stationary phase. Cultures were pooled and aliquot portions were centrifuged in individual centrifuge pots. Then each pellet was washed in sterile basal salts medium and recentrifuged. For induction experiment each pellet was resuspended in sterile basal salts medium and some cultures were inoculated with a variable concentration of decane and incubated for a fixed time span. The remainder was inoculated with a specific concentration of decane and the time of incubation was varied. Controls lacking decane were also used. All cultures were lysed and the lysate was separated into a cytosolic and membranous fractions and these fractions were then assayed individually for protein content and alcohol dehydrogenase activity. The fractions were also subjected to PAGE-electrophoresis and Gradient PAGE-electrophoresis. Inducer concentration giving half-maximum induction and optimum time of induction were established from collective results.
Abstracts
Isolation of a Gram-Positive p-CB Degrading Bacterial Strain M. C. SPPgua”, M. H. Paveia”, M. G. Vieira” and A. M. Anselmo” “Department o[Biol. Veg., Faculdade de Ciencias de Lisboa, Portugal, and Inst. Nat. Eng. e Techn. Ind., Portugal The high physical and chemical inertness of halogenated biphenyls places these compounds among the most resistant pollutants. However microbial degradation of polychlorinated biphenyls (PCBs) has been reported repeatedly. In bacteria the major catabolic aerobic pathway of PCBs is an extradiol cleavage of the aromatic ring with production of chlorobenzoate. The key enzymes involved in this pathway are encoded by several genes usually designated bph. We isolated a mixed microbial population (7 bacteria and 1 fungus) from a soil sample of an unexposed natural environment which after enrichment and establishment in a defined medium containing p-CB (p-Chlorobiphenyl) as the sole carbon source, was able to utilize this synthetic organic compound. The growth of the pure cultures of each microorganism on p-CB medium showed that only one of the bacterial strain could transform p-CB to chlorobenzoate indicating a metacleavage pathway. This gram-positive strain had the ability to degrade approximately 50% of p-CB (300 mg/l) within 3 days of incubation. The cloning of the p-CB degrading genes is in progress.
Ecological Interactions Between Specially Selected Strains and Natural Microorganisms A. Vu. Fedorov and E. V. Volchenko Institute qfGenetics and Selection of Industrial Microorganisms (GNII Genetics), Saratov Branch. Tulaykova str. 1650, Saratov. 4 10020, Russia In contrast to genetically engineered microorganisms, strains -destroyers and -transformers of xenobiotics (SD) obtained from the environment are not alien to nature. However. selection and adaptation processes in vitro are often carried out taking into account only degradative activity without regard for other properties. It is known that the content of destroyers in biocenosises, even in polluted sites, is comparatively low and that ecosystems can balance or remove small inoculum; but when natural microorganisms altered by selection are returned into the environment in massive amounts during the process of bioremediation or together with purified effluents, it may cause ‘ecological aggression’. Studies on ecological interactions between SD from our collection and natural microorganisms from various aqueous ecosystems have shown that the most of SD would have little chance to survive under natural conditions. Clear river bicenosis possessed higher ecological protection whereas microflora from polluted river manifested lower activity. At the same time, several SD repressed growth of the majority of the natural microorganisms and could influence model river ecosystems.
Biodegradation
of Surfactant and Mineral Oils
0. Turkovskava”, L. Panchenkos, A. MuratovaB, E. Duhrovskaya” and G. Shuhb UInstitute of Genetic and Selection of Industrial Microorganisms, Saratov Branch, Tulaykova st.. p/b 1650, Saratov 4 10020, Russia, and hSaratov Medical University, B. Kazachja 112, Saratov 4 10020, Russia The collection of strains primary degrading a wide spectrum
from the genus Pseudomonas of anionic and nonionic
surfactants was created. Pseudomonas putida TP- 19 is the most active strain which utilized alkylphenol ethoxylate along with alcohol and fatty acid ethoxylates. It was determined that destruction proceeds and follows the path of gradual PEGchain reduction and carboxylation of hydrocarbon radical. In the case with alkylphenol ethoxylate in cultural liquid, the alkylphenol was collected. The microbial consortium formed on the basis of TP-19 under continuous cultivation was capable of destroying alkylphenol ethoxylate in full. In strains utilizing alkylsulfate alkyladdition. and benzensulfonate were selected. Often industrial sewage contains a composition of surfactants and oil products (mineral oils in particular). We succeeded in screening for corresponding strains and microbial associations. Depending on the structure and heterogeneity of substances 70-100% of oils and surfactants were degraded in 24 h in their concentrations 1 g/l. The obtained microorganisms may be used in microbial treatment of sewage and for introduction into environmental objects.
Bacterial Degradation of PAHs Containing More than Three Rings - The State of the Art K. Rehmann and A. Kettrup GSF-Institut fur d’kologische Chemie, Neuherberg. D-85764 OberschleiJheim, Germany Relevant statements on the formation of environmentally hazardous microbial metabolites during the bioremediation of soils or water contaminated with polycyclic aromatic hydrocarbons (PAHs) require detailed information about their microbial degradation pathways. A short survey of the state of our knowledge concerning metabolite formation is given, focusing on PAH degradation by pure bacterial strains and emphasizing results currently obtained with pyrene and fluoranthene. A complete metabolic pathway for the degradation of pyrene by Mycobacterium and Rhodococcus spp. is presented. Furthermore, some reasons for the failure so far to isolate pure strains, which are able to utilize PAHs containing more than four rings, are discussed in brief. It is concluded that further progress in metabolite research may be achieved at least in three ways: (a) by exploring the genetic background of PAH degradation, (b) by improving the enrichment and screening techniques used to isolate bacteria1 PAH degraders for example by using PAH solubilizing agents, and (c) by investigating the metabolic potential of defined mixed cultures and model ecosystems to grasp the influence of cometabolic processes.
Degradation of Petroleum in Soil by Biological Methods T. Jamroz Department Bioprocess Engineering, Faculty of Process and Environmental Engineering, Technical University of &id?, ul. Wolczariska 175, Poland Biodegradation of a mode1 solution containing n-hexadecane, isooctane and benzene has been studied. The biological degradation of hydrocarbons in soil has been carried on; -indigenous microflora, -indigenous microflora activated by biogenous nutrients and indigenous microflora enriched with the biopreparate. Dynamics of hydrocarbon degradation and number of microorganisms included in this process were examined. The decontamination took place in different ways depending on microflora which was founded in soil. The best reduction of model petroleum solution (about 97%), was obtained using the biopreparate and the smallest one (about 32%) using only the indigenous microflora. The content of
251
Abstracts
Degradation of Polychlorinated Biphenyls (PCB) in Different Soils by Inoculated Alcaligenes xylosoxidans
types. The sorption of the majority of dyes tested could be described using Langmuir isotherms. Maximum sorption capacities were calculated. The prepared sorbents could be also used in analytical chemistry for the isolations of planar xenobiotics from biological and environmental samples prior to their analysis using standard analytical procedures.
K. Dercovi, L’. HaluBka and s. BaE Department of Biochemical Technology, Faculty of Chemical Technology. Slovak Technical University. Radlinskeho 9, 812 37 Bratislava, Slovakia
Induction of Alcohol Dehydrogenases Pseudomonas C 12B
microbiological cells in investigated soil persisted level both for contaminated and control samples.
at a similar
The degradation of polychlorinated biphenyls in soils by biphenyl-utilizing strain Alcaligenes xylosoxidans was studied in different soil types. In addition to the congener specificity, significant differences in the degradation of PCB by the strain in the different soil types were observed. Efficiency of degradation was generally better in sterilized soils, but the differences were not as significant as the differences observed among the different soil types. These results indicate that the degradation of PCB is probably related not only to the capabilities of the strain employed and quality and amount of competitive species inhabiting the soils, but also to the soil sorption of the PCB congeners: the degradation is faster in the soils containing an intermediate amount of organic carbon with a high portion of total and aromatic carbon in humic acids
Isolation of Planar Organic Compounds Using Copper Phthalocyanine Dye Immobilized on Magnetic Carriers 1. Safaiik and M. 6afaiikovB Laboratory of Biochemistry and Biotechnology, Institute of Landscape Ecology, Academy of Sciences. Branisbvska 31, 370 OS Ceske BudeYjovice. Czech Republic Magnetic separations represent a very promising procedure for the isolation and removal of environmental contaminants. Organic compounds with a planar structure form complexes dyes; magnetic phthalocyanine with phthalocyanine derivatives were tested as a possible specific sorbents of these compounds. Reactive copper phthalocyanine dye (Ostazin turquoise V-G; C.I. Reactive Blue 21) was immobilized on magnetic chitosan and on silanized magnetite particles. The prepared sorbents were used for the studies of sorptions of organic compounds with a planar molecular structure, namely water soluble organic dyes of heteropolyaromatic and triphenylmethane
in
M. Suchinek”, J. N. T. Rindb and B. KrAlov$” ODepartment of Biochemistry and Microbiology. Faculty of Food and Biochemical Technology Institute of Chemical Technology, Praha, 166 28 Praha 6, Czech Republic. and hDepartment of Biochemistry, School of Molecular and Medical Biosciences, University of Wales, College of Cardiff, Cardiff CFI 3US, UK Pseudomonas C 12B is a detergent-degrading soil bacterium that was isolated for its ability to grow on sodium dodecyl sulphate. Grown in batch culture on nutrient broth, the organism produces four cytosolic alcohol dehydrogenases distinguished by non-denaturating PAGE. These enzymes require NAD’ as a cofactor of the reaction. Decane induces two membrane-bound enzymes that are not dependent on presence of NAD+ in assay mixture. The natural cofactor of membrane-bound alcohol dehydrogenases is not known, but phenazine methosulphate coupled with suitable redox dye can be used for both quantitative and qualitative assays. Pseudomonas C12B was grown in batch culture in 200ml portions of nutrient broth until onset of stationary phase. Cultures were pooled and aliquot portions were centrifuged in individual centrifuge pots. Then each pellet was washed in sterile basal salts medium and recentrifuged. For induction experiment each pellet was resuspended in sterile basal salts medium and some cultures were inoculated with a variable concentration of decane and incubated for a fixed time span. The remainder was inoculated with a specific concentration of decane and the time of incubation was varied. Controls lacking decane were also used. All cultures were lysed and the lysate was separated into a cytosolic and membranous fractions and these fractions were then assayed individually for protein content and alcohol dehydrogenase activity. The fractions were also subjected to PAGE-electrophoresis and Gradient PAGE-electrophoresis. Inducer concentration giving half-maximum induction and optimum time of induction were established from collective results.