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Proceedings V Japan —Germany Workshop on Bioremediation roceed i ng s R e s e a r c h Ins t i t u t e of Inn o v a t i v e T ec h n o I og y for the E a r t h December 4 (Mon) 5 (T u e),1995 Tokyo, Japan CONTENTS PAGE ■ Opening Items ...... 1 Program ...... 2 ■ Keynote Lecture “Environmental Preservation Using Biotechnology" -I. Karube (University of Tokyo, Japan) ...... 6 “Environmental Technology in Germany: Status,Achievements.and, Problems -R. D. Schmid (University of Stuttgart, Germany) ...... 7 ■ Abstracts (Oral Session) • Session 1 : “Microbiological Aspects of Bioremediation ” “Bacteria with Different Degradation Kinetics Predominate Depending on the Substrate Concentration in Biological Purification Process’ -Y. Urushigawa...... 8 “Development of Genetically Engineered Microorganisms for Biological Treatment of Chlorinated Enthylenes -K, Nakamura ...... 10 “Comparative Studies on Trichloroethylene(TCE) Degradation by Phenol Degrading Bacteria" M Fujita ...... 12 “Dehalogenating Bacteria, their Dehalogenating Enzymes and the Comparison of their activities in Liquid Culture and in Soil -R. Muller ...... 14 “Protein Engineering on a Polychlorinated Biphenyl Degradation Enzyme ” -M. Fukuda ...... 15 “The Application of Nitrate as an Alternative E1ectronacceptor for the Remediation of Contaminated Sites -P. Werner ...... 17 “ “Soft” Remediation Strategies: Examples for Activation of Microbiological Self Cleaning Potencies in Contaminated Water, Sediment and Soil -U. Stottmeister ...... 18 Session 2 : “Environmental Monitoring" - “Sensor Technology Applied to the Environment" -F. Spener ...... 19 “Recent Approaches to Immunochemical Analysis of Environmental Pollutants” -B. Hock ...... 20 “Construction of Highly-Sensitive Biosensors-for the Use of Enviromnetal Monitoring-” -F. Mizutani ...... 21 “Screening Assays for Environmental Pollutants -S. Neumann ...... 22 “Biosensors for Biomonitoring" -K. Ikebukuro ...... *... 23 “Methods to Chase Microorganisms in the Environment" -H. Oyaizu ...... 25 “Detection and Enumeration of' Methylotrophic Bacteria in Groundwater Samples by Molecular Probing” -T. Shimomura ...... 26 “Biomonitoring Systems in Drinking Water Plants-Peraanent Control of Quality Parameters" -H. H. Stabel ...... 28 “Highly Sensitive and Selective Analysis for Pesticides in the Environment -Y. Takimoto ...... ^ :...... :...... %...... 29 “Atmospheric Monitoring using an Eye Safe Lidar System -M. Kamata ...... '...... 30 Session 3 : “Engineering Aspects of Bioremediation “Industrial Aspects of Bioremediation in Germany ” -Y. Schulz-Berendt ...... 32 “Simulation of Biodegradation of Crude Oil on Beaches” -M. Ishihara ...... 33 “Bioremediation of Oil-Contaminated Soil in Kuwait -H. Chino ...... 34 “Application of Bioreactors for Bioremediation " -J. Klein ...... 36 “Reaction Engineering Aspects of the Degradation of Xenobiotics by Microbial Specialists -I. D. Deckwer ...... 37 “Model-Based Analysis of Coexistence in Mixed Culture Degradation of Xenobiotics -M. Reuss ...... 38 • Session 4 : Panel Discussion indluding Closing Summary “Perspective of Bioremediation and Environmental Monitoring” ...... 39 Chairpersons; -R. Kurane -I. D. Deckwer Panelers; -Y. Urushigawa -P. Werner -M. Fujita -S. Neumann -S. Harayama -V. Schulz-Berendt List of Participants 42 Japan—Germany Workshop on Bioremediation Date: December 4(Mon)-5(Tue), 1995 Venue: Kikai Shinko Kaikan (8-5, 3-chome Sibakouen, Minato-ku, Tokyo, Japan) Sponsored by: Bundesministerium fur Bi Idung, Wissenschaft, Forschung und Techno I ogie New Energy and Industrial Technology Development Organization Research Institute of Innovative Technology for the Earth Organization: Advisory Committee Program Committee Isao Karube Tomoho Yamada Tokyo University Ministry of International Noboru Yumoto Trade and Industry Ministry of International Yoshikuni Urushigawa Trade and Industry National Institute for Tamotsu Mukai Resources and Environment New Energy and Industrial Ryuichiro Kurane Technology Development Org. National Institute of Bioscience Tsutomu Yamaguchi and Human-Technology Research Institute of Innovative Kiichiro Yamagishi Technology for the Earth New Energy and Industrial Technology Development Org. Yasukatsu Miyoshi Research Institute of Innovative Technology for the Earth Masahiko Hachiya Project Center for C02 Fixation and UtiIization, RITE Secretariat Project Center for C02 Fixation and Utilization, Research Institute Innovative Technology for the Earth(RITE) 7 Toyokaiji-Bldg. 8F.8-11 Nshishinbashi 2-chome, Minato-ku, Tokyo, 105 JAPAN Tel +81-3-3503-5666 Fax +81-3-3503-4533 —1— Japan-Gerroany Work Shop on Bioremediation RROGRAM DEC. 4 (MON) Registration 9:00 — 9:30 Opening Greetings 9:00 — 9:45 Lecture 9:45 —10:15 “Environmental Preservation Using Biotechnology ” — Isao Karube(Japan, University of Tokyo) 10:15—10:45 "Environmental Technology in Germany: Pol icy, Achievements,Perspectives ” — Rolf D. Schmid(Germany, University of Stuttgart) Coffee break 10:45—11:00' Session 1 : “Microbiological Aspects of Bioremed±ationn Chairpersons : Peter Werner and Yoshikuni Urushigawa 11:00—11:25 “Bacteria with Different Degradation Kinetics Predominate Depending on the Substrate Concentration in Biological Purification System.” — Yoshikuni Urushigawa (Japan, National Institute for Resources and Environment) 11:25—11:50 “Development of Genetically Engineered Microorganisms for Biological Treatment of Chlorinated Ethylenes” — Kanji Nakamura(Japan, Kurita Water Industries Ltd.) 11:50—12:15 “Comparative Studies on TCE Degradation by Phenol Degrading Bacteria — Masanori Fujita (Japan, Osaka University) Lunch break 12:15-13:15 Session 1(Continued) 13:15—13:40 “Dehalogenating Bacteria, their Dehalogenating Enzymes and the Comparison of their activities in Liquid Culture and in Soil — Rudolf Muller (Germany, Technical University of Humburg-Harburg) 13:40—14:05 “Protein Engineering on a Polychlorinated Biphenyl Degradation Enzyme” — Masao Fukuda (Japan, Nagaoka University of Technology) -2- 14:05-14:30 “The Application of Nitrate as an Alternative Electronacceptor for the Remediation of Contaminants" — Peter Werner(Germany, University of Dresden) 14:30-14:55 'Soft' - Remediation Strategies: Examples for the Activation of Microbiological Self Cleaning Potencies in Contaminated Water, Sediment and Soil ” — Ulrich Stottmeister (Germany, Environmental Research Centre Leipzig) Coffee break 14:55-15:15 Session 2 “ Environmental Monitoring” Chairpersons : Siegfried Neumann and Masanori Fujita 15:15-15:40 “Biosensor Technology Applied to the Environment ” — Friedrich Spener (Germany, Institute for Chemo-and Biosensors, Munster) 15:40-16:05 “Recent Approaches to Immunochemical Analysis of Environmental Pollutants" — Berthold Hock(Germany, Technical University of Munchen) 16:05-16:30 “Construction of Highly-Sensitive Biosensors-For the Application of EnviromentaI Monitoring" — Fumio Mizutani (Japan, National Institute of Bioscience and Human Technology) 16:30-16:55 “Screenig Assays for High-Priority Pollutants in Soil and Water" — Siegfried Neumann(Germany, Merck AG) 16:55-17:20 “Biosensors for Biomonitoring" — Kazunori Ikebukuro (Japan, University of Tokyo) 17:20-17:45 “Methods to Chase Microorganisms in The Environment ” — Hiroshi Oyaizu(Japan, University of Tokyo) Move to Reception Room 17:45—18:30 Reception 18:30-20:00 —3— DEC. 5 (TUE) Session 2(Continued) 9:00 — 9:25 “Detection and Enumeration of Methylotrophic Bacteria in Groundwater Samples by Molecular Probing ” — Tatsuo Shimomura (Japan, Ebara Reserch Co. , Ltd.) 9 :25— 9:50 • “Biomonitoring Systems in Drinking Water Plants-Permanent Control of Quality Parameters” — Hans-Henning StabeI(Germany, Bodenseewasserversorgung,SiissenmuhIe ) 9:50 —10:15 “Highly Sensitive and Selective Analysis for Pesticides in the Environment ” — Yoshiyuki Takimoto (Japan, Sumitomo Chemical Co. ,Ltd.) 10:15—10:40 “Atmospheric Monitoring using an Eye Safe Lidar System” — Masao Kamata (Japan, Ishikawajima-Harima Heavy industries Co. ,Ltd.) Coffee break 10:40-11:00 Session 3 “ Engineering Aspects of Bioremediation” Chairpersons : Shigeaki Harayama and Schulz-Berendt 11:00-11:25 “Industrial Aspects of Bioremediation in Germany” — Schulz-Berendt (Gremany, Umweltschutz Nord GmbH) 11:25-11:50 “Simulation of Biodegradation of Crude Oil on Beaches” — Masami Ishihara (Japan, Marine Biotechnology Institute Co. ,Ltd.) 11:50-12:15 “Bioremediation of Oil-Contaminated Soil in Kuwait” — Hiroyuki Chino (Japan, Obayashi Corp. ) Lunch break 12:15-13:15 Session 3(Continued) 13:15—13:40 “Application of Bioreactors for Bioremediation ” — Jurgen Klein(Germany, DMT Institute) 13:40-14:05 “Reaction Engineering Aspects of the Degradation of Xenobiotics by Microbial Specialists ” — Wolf-Dieter Deckwer(Germany, GBF, Braunschweig) 14:05—14:30 “Model-Based Analysis of Coexistence in Mixed-Culture Degradation Xenobiotics" — Matthias ReuB (Germany, University of Stuttgart) —4— Coffee break 14:30—14:50 Session 4 Panel Discussion Including Closing Summery “Perspective of.Bioremediation and Environmental Monitoring" 14:50—16:50 Chairpersons : — Ryuichiro Kurane (Japan, National Institute of Bioscience and Human Technology) — Wolf-Dieter Deckwer(Germany, GBF, Braunschweig) Panelers : — Yoshikuni Urushigawa (Japan, National Institute for Resources and Environment) — Peter Werner(Germany, University of Dresden) — Masanori" Fujita (Japan, Osaka University) — Siegfried Neumann(Germany, Merck AG) — Shigeaki HarayamaCJapan, Marine Biotechnology Institute Co. ,Ltd.) — Schulz-Berendt (Gremany, Umweltschutz Nord GmbH) Closing Greetings 16:50-17:00 -5- Environmental preservation using biotechnology Isao Karube Research Center for Advanced Science and Technology, 4-6-1 Komaba, Meguro-ku, Tokyo 153, Japan Now we have" reached a period when the subject of biotechnology is very much recognizable. Japanese industries have been changed their phase gradually from learning and beginning to harvesting the products of their research in this field. The technology market, mainly concerned with pharmaceuticals, is said to be currently as high as eight hundred billion yen and is expected to reach three trillion yen by the turn of the century as new medicines are developed and technology is applied to agriculture, food processing and other industries. Above all, its application to the environment is quite important. Vigorous efforts are being made in the research and application of "bio-remediation" with the purpose of removing environmentally burdensome materials by biotechnological processes. In the case of the United States, the demand for bio-remediation is expected to be more than 35 billion dollars, which is twice the value in 1992. Environmental problems are now one of the worldwide concern, and the need to develop technologies to solve these problems is claimed everywhere. Biotechnology is thought to be one way to solve one of the problems, for example, such as the global warming caused by the CO2 increase in the atmosphere. One trial of CO2 fixation by a biochemical means and reused as a source is now going on in our group. This project is now sponsored both by the New Energy Development Organization (NEDO) and the Research Institute of Innovative Technology for the Earth (RITE), and is performed by the cooperation of 16 private companies. One of the most effective method to convert CO2 into useful materials is the one using sunlight and biological processes. The amount of CO2 converted is limited because the fixation depends on the biological process. Our work for five years has revealed that the system required an extremely expensive light condenser. On the other hand, a strenuous efforts are being made in research into a method of generating useful organic resources from CO2. This is a critical project that may in the future solve the food shortage problem. This research is aiming at the development of an ideal bioreactor, and is planned to last next five years. Micro algae that have a tolerance of high CO2 concentrations are mainly used. Some of them have a property of producing useful substances from CO2. The fruitful results obtained from this project will contribute a noble way for the reuse of CO2. A process that uses microorganisms to decompose chlorinated organic materials such as trichloroethylene and tetrachloroethylene is now the focus of much attention. Semiconductor devices and the dry-cleaning of clothes require such substances. The technology for removing materials not readily decomposed from the environment, such as heavy metals and organic materials, are now being used widely, and the market for this technology is expected to grow much further. Biological processes are also considered to be useful for the conversion of the sludge in lakes and the ocean into some organic fertilizer. These examples illustrate that biotechnology may become a key for environmental preservation. —6— Environmental Technology in Germany: Status, Achievements and Problems Rolf D. Schmid Institut fur Technische Biochemie, Universitat Stuttgart Over the past 30 years, development and implementation of environmental technologies have become a major issues in Germany, and a comprehensive legal framework has been passed in order to protect the environment Whereas air pollution technology has mainly been developed and financed by private enterprise, water sanitation and waste treatment were, and still are, predominantly in the public domain. Germany is a continental nation surrounded by populous neighbor countries. To most of ist neighbors, it is linked by the treaties of the European Community. Thus, environmental problems in Germany rarely are of national concern only, but rather relate to international challenges such as cross-border air pollution abatement, cleaning up of the major rivers, and protection of the Northern oceans. As a result, Germany is involved in many international networks and projects concerned with the development and implementation of pertinent technology. A special challenge resided in German unification, which took place in 1990. As the former German Democratic Republic shared the poor environmental conditions of all Central and Eastern European Nations, large-scale programmes were initiated to establish EU environmental standards in the new Eastern states of Germany. While considerable progress has been made, much remains to be done to equilibrate environmental standards in both parts of Germany. Environmental technology has become a major industry and an item for exports. Germany is doing well in this business with the developed countries, but it is clear that a successful entrance into emerging markets such as Russia or China will require specific technological solutions. Bacteria with different degradation kinetics predominate depending on the substrate concentration in biological purification process Yoshikuni Urushigawa and Yuichi Suwa National Institute for Resources and Environment, Onogawa 16-3, Tsukuba, Ibaraki 305, Japan In biological wastewater purification process, variety of microbial populations play different functional roles. The engineers, however, have not really paid attention to functional roles of the individual microbial populations in the consortia but have handled them as a whole. This could.be true for most of the biological purification processes targeting commonly occurring BOD-type natural organic matters. A relevant microbial community is established by given environmental conditions that are not really controllable. Engineers are primarily concerned with retaining biomass, however undifined, for accomplishment of BOD removal. Additionally , most microbiologists have not been interested in physiology of BOD utilization and therefore also have not studied the microbial communities involved. Is it also the case in purification of xenobiotic compounds? No, because a more detailed understanding of xenobiotic catabolism is required in treating those particular compounds. Cooperative actions among interdisciplinary research workers, such as civil engineers, mathematicians, microbial physiologists, ecologists, and even molecular biologists and geneticists have resulted in significant accomplishments in biodegradation of xenobiotic compounds . The recognition of the complexity and urgency of the problem has unified scientists of differing expertise . Such intensive and immediate attention is required to prevent further environmental contamination and to clean up existing problematic sites. We need practical knowledge of how - to retain microbial populations which effectively degrade the xenobiotic compounds of interest. This requires analysis of degradation mechanisms and ecophysiological features of microbes responsible for degradation. Here, we summarize substrate- microbe relationships to consider what types of organisms are preferable in xenobiotic degradation and how we could retain them in a treatment system. 1. Microorganisms capable of degrading xenobiotics may not always form the predominant population in the xenobiotic degradation consortia so that biomass itself is not always representing the desired population. This is quite different from the interpretation of whole biomass in BOD treatment. 2. Some xenobiotic compounds do not support growth of microorganisms but organisms require a co substrate for their degradation, which is known as co-metabolism. If this is the case, the most appropriate co-substrate has to be chosen, and this may select for degraders so that they become dominate. 3. Xenobiotics themselves are often toxic to microorganisms (including the degraders themselves) so that, beyond a critical concentration, degradation will not occur. A population that is tolerant to the compounds, which may not always be degraders, will then predominate and be maintained. 4. Toxic metabolites are sometimes produced, and they are usually very reactive. For example, it has been reported that meta-cleavage product from 3-or 4-chlorocatechol and degradation intermediates from TCE oxidation may be lethal to microorganisms involved in the process. Thus, a metabolic route that does not produce toxic products is very preferable for successful biodegradation. 5. Some microbes can degrade just a few structurally related compounds. If a variety of compounds is present in wastewater, a different microbial population may be required to degrade each group of compounds. If a coexisting compound is toxic to microbes responsible for degradation of another compound, it may be difficult to maintain all populations necessary for treatment of the wastewater. 6. Microbes display a variety of degradation kinetics. Kinetic parameters are necessary to predict both final concentration in treated water and also the rate and performance of degradation processes. Knowledge of these parameters is important for optimizing and maximizing degradation of xenobiotics, especially because of their toxic and hazardous nature. -8- In this presentation, we will illustrate the principle mentioned above in item six. We have studied two p-nitrophenol degrading strains showing different degradation kinetics. These strains degraded the compound with the identical mechanism. One of the two demonstrated conventional substrate saturation kinetics for degradation, and the other demonstrated substrate inhibition kinetics. The former had a higher degradation rate at low substrate concentration than the latter, which could degrade a much higher concentration of the substrate. A comparative study of these two degraders would be a good demonstration of the effect of substrate concentration in determining which type of organism will predominate. We acclimated an activated sludge to p-nitrophenol (PNP) in both batch and continuous modes and found that the operational mode of the PNP acclimation of activated sludge strongly affected the kinetic pattern of predominant microorganisms responsible for PNP degradation. The sludge of batch mode (Sludge B) had lower PNP affinity and was insensitive to PNP concentration. That of the continuous mode (Sludge C), on the other hand, had very high PNP affinity and was sensitive to PNP. MPN enumeration of PNP degraders in sludge B and C using media with different PNP concentrations (0.05, 0.5, 1.0 and 2.0 mM) supported the results of the kinetic studies. The ratios of MPN enumeration at 0.05 mM to 2.0 mM, an index of the predominancy of the sensitive group, were 1.3 for sludge B and 1.4x1 O'* for sludge C, showing that the each predominant population has different sensitivity to PNP concentration. When the operational modes for the two sludges were reversed, undegraded PNP appeared at the beginning but disappeared after ten days in both sludges. After twenty-eight days, the MPN ratio of sludge B increased to 2.9x10", showing that the predominant population shifted to the sensitive group. In sludge C, on the other hand, the MPN ratio was 1.7x10", indicating that the insensitive group did not fully recover. These data suggest that the sensitive group was tolerant to the given loading of PNP and that the insensitive group was not be a good competitor when the loading of the substrate was changed. Predominant PNP degraders were isolated from each activated sludge to confirm if the above observation was caused by different microorganisms. Among the isolates obtained, Pseudomonas spp. MSL1 and MSH1 did and did not show substrate inhibition, respectively. Based on carbon source utilization patterns and the 16SrRNA sequences, the two strains were different. Further, PNP degradation kinetics of the two strains was examined using cell free extract preparations. PNP degradation activity of MSL1 was inhibited by PNP at higher than 0.02 mM and only one-third of the maximum rate was found at 0.05 mM. On the other hand, PNP degradation by MSH1 fit the Monod equation up to 0.2 mM or higher. These results indicate that a kinetic pattern of PNP degradation shown by activated sludge was directly attributable to that of an enzyme responsible for PNP catabolism by a predominant degrader. It may not be unusual that bacteria with different degradation kinetics would predominate depending on substrate concentration in biological purification processes. However, the importance of this observation has not been recognized or applied in practice so far. Many efforts have been exerted to isolate degraders, and it may also be true that many of them were obtained with a high selective pressure using high concentration of xenobiotics as a substrate. One could argue that most of them are not good strains to explain many commonly found phenomena in treatment plants, though they are useful for , studying degradation mechanism qualitatively. Further, one could explain why direct application of kinetic data obtained from those strains is not always useful in practice. One could point out the fact that most of the purification processes are operated by continuous mode, including the conventional activated sludge process. In a well-treated process, the concentration of xenobiotics should reach to a low level and would be maintained. In such a case, organisms having degradation kinetics analogous to that shown by MSL1 may predominate. Further, one could say the environmental concentration of xenobiotics should be very low. To remediate and protect the environment from toxic substances, we should pay much more attention to organisms showing high affinity to the compounds and engineering systems that retain them. effectively would be superior. -9- DEVELOPMENT OF GENETICALLY ENGINEERED. MICROORGANISMS FOR BIOLOGICAL TREATMENT OF CHLORINATED ETHYLENES Kanji Nakamura Kurita Water Industries Ltd., 7-1, Wakamiya, Morinosato, Atsugi-city, Kanagawa, 243-01, Japan Introduction Trichloroethylene (TCE) is one of the most common soil and ground water contaminants in Japan. Although there is no microorganism that can use TCE as a sole carbon source, several bacteria are known to decompose TCE by co-metabolism only in the existence of their growth substrate such as methane, toluene or phenol Therefore, in order to utilize those microorganisms, it is necessary to add the above-mentioned inducer substrates. The development of a genetically engineered microorganism (GEM) that carries a gene responsible for TCE degradation would be a good way to promote TCE decomposition without inducer substrates. We report herein on the degradation of TCE and other chlorinated ethylenes by OEMs containing phenol hydroxylase genes. Development of a GEM for TCE degradation Phenol hydroxylase (PH) genes, which are responsible for TCE decomposition, and a catechol- 2,3-dioxygenase (C230) gene were cloned from the genomic DNA of a phenol-utilizing bacterium, Pseudomonas putidaKNl. These genes were subcloned into pRCLIOO having the replicon of RSF1010 and the recombinant plasmid was named pNEM 101 (Fig. 1). The plasmid of pRCLIOO carries lac Iq and the trc promoter, which means that cloned PH and C230 genes could only be expressed in the presence of isopropyl-B-D-thiogalactoside (IPTG). The plasmid of pNEMlOl was transferred to Ps. putida KN1 Degradation of chlorinated ethylenes by Ps. putida KN1 (pNEMlOl) Ps. putida KN1 (pNEMlOl) was cultivated in LB medium and induced by 5 mMof IPTG for two hours. The cells were harvested and washed, then resuspended in a basal salt medium (BSM) with an optical density of 2.0 at 600 nm. Ten milliliters of the culture were transferred Phenol Hydroxylase 6.0 kb BamHI site +IPTG pRCLIOO 8855 bp Time (hrs) Fig.l Recombinant plasmid pNEMlOl Fig.2 TCE degradation by Ps. putida KN1 (pNEMlOl) —10— into a 155ml vial, and TCE was added to a final concentration of 10 mg/1. The vial was capped and incubated with shaking at 30°C. The time course of TCE degradation was examined by measuring TCE concentrations in the gas phase. Ps. putida KN1 (pNEMlOl) degraded TCE very efficiently with the IPTG induction as shown in Fig. 2. The inefficient TCE decomposition without the IPTG induction verified that the PH genes under the control of the trc promoter were responsible for TCE degradation. Degradations of other chlorinated ethylenes, tetrachloroethylene (PCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-dichloroethylene (c-DCE), trans-1,2-dichloroethylene (t-DCE) and vinyl chloride (VC) by Ps. putida KN1 (pNEMlOl) were examined with the IPTG induction. Fig. 3 shows the time courses of the degradation of all chlorinated ethylenes including TCE. The decomposition of c-DCE occurred at a maximum rate among the chlorinated ethylenes tested. The degradation rates of t-DCE and VC were slightly lower than that of TCE. 1,1-DCE was decomposed very slowly, and PCE was not degraded at all. In TCE or PCE contaminated soil, reductive dehalogenation proceeds gradually and those compounds are mainly transformed to c- DCE, then to VC. Although 1,1-DCE and t-DCE are also produced, usually the amount of those compounds is not so significant in contaminated soils. Therefore, the application of Ps. putida KN1- (pNEMlOl) capable of decomposing TCE, c-DCE, and VC is very practical. . Development of a stable GEM by homologous recombination Plasmids have been used to develop GEMs, however, recombinant plasmids are not maintained in host strains such as Pseudomonas sp. without the selection pressure of an antibiotic. Instead of using a plasmid as a carrier, homologous recombination was examined to insert a target gene into a genomic DNA to construct a GEM that would be stable even without an antibiotic. After determining the location of the PH genes in the chromosome of Ps. putida KN1, the tac promoter and the tetracycline resistant gene were inserted in front of the PH genes through homologous recombination as shown in Fig. 4. The constructed GEM, Ps. piitida KNl-lOA, constitutive!/ degraded TCE without an inducer. Although the TCE degradation rate of Ps. putida KNl-lOA was approximately one fifth of that of Ps. putida KN l (pNEMlOl), the trait of the new GEM was stable even in the absence of an antibiotic. Ps. putida KN1 K ■■ , ii l77Z///7///>7Z^> .* \PH genes ^ / \ Inserted by\homoIogous recombination t-DCE / Ps. putida Kljtl-lOA \ Time (hrs) Fig.4 Development of Ps. putida KNl-lOA Fig.3 Degradation of chlorinated ethylenes by homologous recombination by Ps. putida KN1 (pNEMlOl) A part of this work was conducted in a project on safety analysis related to bioremediation. The project was organized and supported by the Ministry of International Trade and Industry (MITI) of Japan. -11- COMPARATIVE STUDIES ON TRICHLOROETHYLENE(TCE) DEGRADATION BY PHENOL DEGRADING BACTERIA Masanori Fujita*, Michihiko Ike*, Koji Kataoka* and Masahiro Takeo** * Department of Environmental Engineering, Osaka University. ** Department of Applied Chemistry, Himeji Institute of Technology. Introduction. TCE is one of the most hazardous soil/groundwater contaminants, which is known to persist for a long period in natural environments and to be toxic for living things. It is of urgent necessity to remove TCE from polluted sites so as to protect our drinking water supplies from its contamination. As TCE in soils and aquifers are not fully mobilized by flowing water, physico-chemical treatment such as pump-and-treat strategy is not successful. Another alternative, digging up of the contaminated soils and their treatment or disposal, is practically impossible for large area contamination (Rittmann et al., 1994). Biological degradation of TCE at the targeted sites seems to be the best way for complete and cost- effective removal of TCE, and this approach is called in situ bioremediation. Until now, three major microbial groups, methanotrophic microbes, aromatic compound (phenol, toluene etc.) degraders, and ammonia oxidizers, have been known to be capable of degrading TCE by co-metabolic reaction (reviewed by Chaudhry and Chapalamadugu, 1991). Hopkins et al. demonstrated that high potential for in situ TCE bioremediation using phenol utilizing populations from the field studies (1993). However, this study was a case study and the microbial aspects of the remediation have not been fully elucidated. For successful in situ bioremediation, the quantitative and qualitative characterization of degrading microorganisms which are present in or are introduced into the soils or aquifers is required as to establish rational strategies for effectively enhancing their population size and/or catabolic activities. We carried out comparative studies on the biodegradation of TCE by a variety of phenol degrading bacteria in order to evaluate the potential of utilizing phenol degrading populations for in situ bioremediation of TCE contaminated sites. Phenol degrading bacteria in soils. In a preliminary experiment, we enumerated the phenol degrading bacterial population in soils. Several soil samples were collected and numbers of phenol degrading bacteria in them were counted by using plate count and replica plate techniques with a minimal salt medium containing 125 or 500 mg/1 of phenol. Total heterotrophic bacteria were enumerated with CGY agar containing 5 g of casitone, 5 g of glycerol, 1 g of yeast-extract and 1.5 g of agar in 1L of deionizedwater. Phenol degrading bacteria accounted for more than 5 % of total heterotrophic bacteria in all the tested soil samples, suggesting their common and abundant presence in natural soil environments. In a soil sample, the viable count of phenol degrading bacteria on 125 mg/1- phenol containing medium was 6.4 x 105 cfu/g, that on 500mg/l-phenol containing medium was 2.1 x 105 cfu/g, and that of total heterotrophic bacteria was 7.0 x 106 cfu/ml. Judging from the observation of colony morphology, each soil sample contained a few to several different types of phenol degrading bacteria, suggesting that there exists a wide variety of phenol degrading bacteria in soil environments. If all the phenol degrading bacteria present in soils have potential for TCE degradation, what we have to do is only that we propagate and induce TCE degrading activity of the indigenous phenol degrading populations. These results suggested that effective in situ TCE degradation will occur if we can successfully propagate and TCE degrading activity of indigenous populations. -12- TCE degradation bv a variety of phenol degrading bacteria. Further, TCE degrading properties of various types of phenol degrading bacteria were compared. Examined bacterial strains included authentic ones and soil/water isolates. Phenol degrading bacteria were cultivated in a minimal salt medium containing phenol as a sole carbon and energy source to late-log to stationary growth phase, washed with phosphate- potassium buffer (pH, 7.5), and suspended at an approximately turbidity of 2.0 at 600 nm (resting-cell). TCE degradation (1 mg/1) by the resting-cells were monitored during incubation at 25 °C with reciprocal shaking at 100 rpm in sealed vials. TCE was measured by head-space analysis with an electron capture detector-equipped gaschromatograph. Among more than ten distinct types of phenol degrading bacteria examined, all the strain with only one exception showed TCE degrading capability to a certain extent (ultimate TCE removal was between ca. 20 % and 100 %) when grown on phenol (when grown without phenol, no strain showed significant TCE biodegradation). The only strain which could not degrade TCE, even when grown on phenol, is different from the other strains in that it can grow on /7-nitrophenol as a sole carbon source. It was suggested that most of phenol degrading bacteria can exhibit TCE degrading capability, if only grown on or induced with phenol. Although the general trend was observed that bacterial strains with higher phenol degrading activities showed higher initial TCE degradation rates, the extents (ultimate removal) of TCE degradation had no relationship with the phenol degrading activity. TCE degradation by some strains apparently ceased in a few hours and that by others proceeded extensively for more than 10 hours. In other words, some strains could not completely remove 1 mg/1 of TCE, but others could reduce TCE to under a detectable limit. This indicates that the stability of TCE degrading activity depends on the strain, probably due to the differences in the stability of the phenol catabolic enzyme(s) of each strain. Here, the stability refers not only to the stability of the enzyme itself (as protein) but also to that against TCE or its metabolitefe) toxicity and that of the induction system (regulatory system for the enzyme production). TCE bioremediation with phenol degrading bacteria seems to lead to secondary contamination by phenol which is added to the soil/groundwater. In order to assess this possible hazard, phenol degradation by some strains were investigated. All the strain investigated lowered the phenol concentration in the medium below detectable limits (by high pressure liquid chromatograph), suggesting no possibility of secondary soil/groundwater pollution by phenol. Conclusions. Experimental results suggested that the addition of phenol (stimulation of indigenous soil bacteria) may be an effective way for removing TCE from contaminated sites. However, TCE removal efficiency seems to considerably depend on the TCE degrading properties of phenol degrading bacteria present in the targeted site. If there is no effective indigenous TCE degraders in the site, we have to introduce effective ones which are screened in the laboratory (bioaugmentation). Based on this study, system design or operation strategies for effective and safe in situ TCE bioremediation using phenol degrading bacteria will be developed in future. References Rittmann, B.E., Seagren, E., Wrenn, B.A., Valocchi, AJ„ Ray, C. and Raskin, L. 1994. "In situ bioremediation, 2nd. ed." Noyes Publ., Park Ridge, New Jersey, U.SA. Chaudhry, G.R. and Chapalamadugu, S. 1991. "Biodegradation of halogenated organic compounds. Microbiol. Rev., 55(1), 59-79. Hopkins, G.D., Semprini, L. and McCarty, P.L. 1993. "Microcosm and in situ field studies of enhanced biotransformation of trichloroethylene by phenol-utilizing microorganisms. Appl. Environ. Microbiol., 59(7), 2277-2285. —13— Dehalogenating Bacteria, their dehalogenating enzymes and the comparison of their activities in liquid culture and in soil Rudolf Muller ' < - Technical University Hamburg-Harburg, Biotechnology IT, Technical Biochemistry , DenickestraBe 15, D-21071 Hamburg, Germany ' /, . l - :' . V ''' ' : . " . - ' : ^ Chlorinated hydrocarbons represent one of the major classes of environmental pollutants. The stability of the carbon-chloride bond makes them difficult, to degrade in the environment. Nevertheless bacteria have devdqpped several mechanisms to cleave this bond. Under anaerobic conditions the chlorine is usually replaced by hydrogen in a reductive dehalogenation. For some molecules the replac^neit of chloride by water in a hydrolytic dehalogenation is possible, and finally under aerobic conditions an oxidative dehalogenation requiring molecular oxygen can be observed. The properties of a few new enzymes,:which were purified and characterized in oiir laboratory shall be described. I1 , ’ i : For thetechnical use Of bacteria containing such enzymes in bioremediation it is neccessary, that these bacteria develop their abilities in soil under environmental conditions. We therefore „ . incubated these bacteria in soil under different conditions and checked, which parameters influence their dehalogenating abilites in soil and compared the results with.those obtained in liquid-cultures. Amongst the parameters tested were soil type, organic carbon content, humidity, oxygen content, pH, temperature^ ionic strength, additional carbon sources, substeate concentration, other pollutants, heavy metals etc. From our results we conclude, that, there is no difference in the behaviour of bacteria in liquid culture and in soil, and that it is possible to predict from liquid culture experiments, under which conditions bioaugmentation with a special bacterium is a valuable option for bioremediation and under winch conditions it is not. 1 —14— Protein engineering on a polychlorinated biphenyl degradation enzyme Masao Fukuda Department of Bioengineering, Nagaoka University of Technology Nagaoka Niigata 940-21, Japan Polychlorinated biphenyls (PCBs) are serious environmental pollutants. Microbial degradation can be a practically efficient tool for the removal of PCBs from the environment and also for the treatment of PCBs in stock. In a PCB degrading aerobic microorganism, PCBs are cometabolize with biphenyl through a ring cleavage after a hydroxylation (figure below), and the ring cleavage enzyme, 2,3-dihydroxybiphenyl dioxygenase (BphC) plays a key role in a PCB degradation pathway. To design an efficient PCB-degradation pathway enzymes, we started the new approach to expand the substrate specificity of BphC enzyme. 2,3-dihydroxy- mefa-cieavage Cl dihydrodiol biphenyl biphenyl compound dioxygenase dehydrogenase If dioxygenase hydrolase c p H oh ^ OH C OOH OH benzoic acid biphenyl dihydrodiol 2,3-dihydroxybiphenyI meta-cleavage compound A series of chimeric enzymes between a BphC and a 3-methylcatechol dioxygenase (BnzC) of benzene degradation pathway were created to address the region responsible for the substrate specificity. The BphC enzyme gene (bphC) of PCB-degrader, Pseudomonas sp. KKS102 and the BnzC gene (bnzC) from benzene-degrader, Pseudomonas putida BE81 were employed, because they have a good homology to each other to construct chimeric enzyme genes by the aid of in-vivo recombination in E. coli. The chimeric enzymes obtained indicated dominant involvement of the carboxyl terminal region in the substrate preference. An additional series of mutant chimeric enzymes generated by site-directed mutagenesis revealed an key role of the residue 280 in the substrate preference. X-ray crystallographic analysis supported the conformational interaction between the side chain of the residue 280 and —15— substrates. A mutant enzyme was created to reduce the conformational interference of the residue 280 with substrates and showed improved activity on both 2,3-dihydroxybiphenyl and catechol. Although the activity of the mutant enzyme on chlorinated substrates should be examined, the new mutant enzymes obtained and those will be created in this approach are expected to take part in generating an efficient PCB degradation pathway. We recently isolated a significantly strong PCB degrader, Rhodococcus sp. RHA1 and cloned the enzyme genes for the first three PCB degradation steps. Introduction of the improved enzyme gene described above into such strong PCB degrader may intensify the PCB degradation activity. —16— THE APPLICATION OF NITRATE AS AN ALTERNATIVE ELECTRONACCEPTOR FOR THE REMEDIATION OF CONTAMINATED SITES PETER WERNER INSTITUTE FOR WASTEMANAGEMENT AND CONTAMINATED SITES 01062 DRESDEN TECHNICAL UNIVERSITY OF DRESDEN The biodegradation of contaminants in the subsurface is often limited by the lack of electronacceptors. Oxxygen can be dissolved at groundwatertemperature (10°C) up to 10 mg/1 water, which means that only about 3 mg/1 of organic compounds can be mineralized with this amount of oxygen. But in the groundwater of refinereries, military sites, airports etc. aromatic and aliphatic hydrocarbons can be found up to concentrations of 50 m/1. Therefore the supply with oxygen is insufficient. Nitrate could be an exellent additional electron acceptor, because it can be dissolved in high concentrations and even the temperature can be raised which means an increase of bioactivity. Experiences with the application of nitrate will be presented and problems existing will be discussed. One dicisive problem is, that not all contaminants are biodegradable by dinitrification pathway exclusively. Another problem is the formation of nitrogen gas as the final degradation product of denitrification. The gas might result in a clogging of the groundwater flow. Best experiences could be obtained with combined application of oxygen prior to nitrate. Solutions to overcome the problems will be presented and disenssed. —17 — "Soft"remediation strategies: Examples for activation of microbiological self cleaning potencies in contaminated water, sediment and soil. Stottmeister, Ulrich; WeiSbrodt, Erika; Seidel, Heinz; Kuschk, Peter; Ondruschka, Jelka* UFZ Centre for Environmental Research Leipzig-Halle, Section for Remediation Research, *Leipzig University, Laboratory for Biotechnology The careless exploitation of the nature by open cast lignite mining and lignite carbochemistry for decades in the former East-Gemany resulted in the destruction and contamination of landscapes in large territories. An economical and ecological remediation of these sites needs new approaches. In laboratory- and pilot-scale experiments are developed fundamentals of "soft" remediation principles: bioremediation and bioattenuation for high concentrated contaminants. The idea is the activation of autochthonic microorganisms or to eliminate inhibitions processes by simple technical methods. The following case studies are described in detail: 1. Pyrolysis water deposit: The highly toxic, black colored phenolic water of a deposit (2 Mio. m3, 9 ha) was decontaminated in a first step ~ by precipitation of humic-substances macromolecules with iron salts and activation of the autochthonic bacteria in the clear water for degradation of dissolved low molecular fractions of organic substances (in situ field experiments). 2. Constructed wetlands: Nitrification of pyrolysis deposit water proceeds in the rhizosphere of Fragmites and Thypha sp. in a subsurface flow wetland up to 68 % at a retention time of 7 days in continiuous flow pilot scale experiments. 3. Thiobacilli leaching: Heavy metals are leached from dregged river sediments by activation of autochthonic thiobacilli by addition of sulfur or other electron donators (field experiments). 4. Soil decontamination: PAHs in soil were decontaminated in a combination of a solid state - liquid bioreactor. The full-scale plant for off-site bioremediation is described. -18 Sensor Technology Applied to the Environment Friedrich Spener InstitutfOr Chemo- und Biosensorik, Mendelstr. 7, D-48149 Munster? Germany The evolution of biosensor research corresponds with the increasing need for fast, sensitive and selective measuring devices. After a short review of state of the art technology in environmental monitoring the lecture will focus on recent developments that have been advanced in our Institute. Microbial sensor systems for the detection of polycyclic aromatic hydrocarbons (PAH), representing the largest group of cancerogenic compounds present in the environment were developed. Biosensors based on microorganisms specialized in degradation of PAH can be a useful tool for rapid screening of high sample numbers. The complex parameter biochemical oxygen demand (BOD) can be estimated also by using a microbial sensor. Commercial sensor BOD analysers using normal microorganisms are influenced by heavy metal ions present in same contaminated waste water. These interference problems in the BOD estimation were avoided by using heavy metal resistant strains. The multireceptoral behavior of microorganisms is a suitable prerequisite for their use as a multichannel sensor. In our Institute for the first time a system for the simultaneous quantitative multicomponent analysis was developed by using only one microbial sensor in combination with a partial least squares regression method. Immunosensing of low molecular mass analytes is demonstrated by a flow injection system for the determination of herbicides in drinking water, such as 2,4-D. The immunoreactor applied in the system is manufactured in silica chip technology. The system is highly sensitive, fully automated and needs no sample pretreatment. The detection limit of 0.1 pg/l matches the requirements of EU guidelines for drinking water. Another already industrially applicable development of our Institute is an NIR - spectroscopical installation for the remote and fast recognition of polymers. A pilot plant is already in operation sorting German packaging waste. For this technology we seek production- and marketing partners for the Asian market. -19- RECENT APPROACHES TO IMMUNOCHEMICAL ANALYSIS OF ENVIRONMENTAL POLLUTANTS BerloldHock Technical University of Mttnchen at Weihenstephan Department of Botany, D-85350 Freising, GERMANY Immunochemical techniques supplement traditional analytical methods as screening tools because they are fast, extremely sensitive, simple, and inexpensive. Major progress is experienced in the fields of immunosensors and immuno- chromatography. The quality of these technologies primarily depends upon the employed antibodies, which represent the limiting factor of all immunochemical methods. Standardized methods require homogeneous antibody preparations with defined selectivities and high affinities to the analyte. Although the hyhridoma technology fulfills these requirements by providing monoclonal antibodies, ab development has entered an entirely new domain because of the potential of recombinant antibodies. Examples are given for the generation of recombinant single chain fragments (scFv) for the s-lriazines terbutiyn, terbuthylazine and atrazine. Immunomagnelic screening of phage particles displaying s-triazine-selective scFv with s-triazine- coated paramagnetic beads provides distinct advantages over classical panning for the selection. Furthermore soluble scFv were expressed, which can be used in a similar manner as the original monoclonal Abs. The next step is directed toward the generation of recombinant Ab libraries, which will provide a huge repertoire, generated at the DNA level, for screening new Ab types. —20— CONSTRUCTION OF fflGHLY-SENSmVE BIOSENSORS - FOR THE USE OF ENVIROMENTAL MONITORING - Fumio Mizutani National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan A biosensor is based on the combination of an electrochemical or electrical sensing device with a layer containing biomolecules. Much interest in biosensors has been existing in the field of analytical chemistry in the last two decades, since they provide rapid and selective measurement of species of biological and clinical importance. However, the improvement of the sensitivity of biosensors has still been required for applying them to environmental monitoring: the concentration of analyte is usually very low in the field of environmental anlysis. Here we describe approaches to obtaining highly-sensitive biosensors. Chemical amplification in enzymatic substrate determination is an excellent way of increasing sensitivity. We have prepared enzyme electrodes that gave chemically-amplified responses [1-3]. For example, an analyte, RH2, could be determined with high sensitivity by using a layer containing RH2-oxidase and RH2-dehydrogenase [1]: RHi-oxidase RHi-dehydrogenase RH2 + 02------s- R+H202;R+NADH + H+------^ RH2+NAD+ The regeneration of the analyte by the dehydrogenase reaction brings about the consumption of oxygen beyond stoichiometric limitation, which results in an enhanced response at an oxygen electrode, and, accordingly, in an increased sensitivity (detection limit, lO^-KT9 M). The preparation of a thin enzyme layer with high activity, in which the difiusion of the analyte (enzyme substrate) and the enzymatic reaction proceed rapidly, is a key technology for obtaining a highly-sensitive biosensor. For this purpose, the use of modified enzymes, instead of native ones, is considered to be useful: the modifiers on protein surface would enhance the affinity of the enzyme on the sensing device. For example, a biosensor using lipid-modified enzyme, which could easily be prepared by dip-coating of a glassy carbon electrode from the benzene solution of the modified enzyme, showed a high sensitivity (detection limit, lO'MO"8 M) [4]. The use of genetic engineering techniques provides an opportunity to introduce single immobilization sites onto protein surfaces which allow interfaces to be formed with high regularity. The introduction of accessible cystein residue has been demonstrated to be a viable method to immobilize proteins on gold surfaces [5]. Such a technique for preparing regular protein layers would be a potent way for obtaining highly-sensitive biosensors including immunosensors and receptor-based sensors. [1] F. Mizutani, T. Yamanaka, Y. Tanabe andK. Tsuda, Anal. Chim. Acta, 177,153 (1985). [2] F. Mizutani, S. Yabuki and M. Asai, Biosensors Bioelectron., 6,305 (1991). [3] F. Mizutani and S. Yabuki, Chem. Lett., 1569 (1994). [4] F. Mizutani, S. Yabuki and T. Katsura, Anal. Chim. Acta, 274,201 (1993). [5] S. J. Vigmond, M Iwaskura, F. Mizutani and T. Katsura, Langmuir, 10,2860 (1994). -21- SCREENING ASSAYS FOR ENVIRONMENTAL POLLUTANTS Dr. Siegfried Neumann Division Laboratory Products, Merck KGaA Frankfurter Str. 2S0, D-64271 Darmstadt (Germany) One of the global trends in environmental analytics is the growing need for on-site measure ment Whereas classical laboratory methods use quantitative standardized analytical tech niques on costly instruments alternate test procedures (ATP) are based on semiquantitative screening devices or sentiquantitatiye/quantitative tests for visual-colorimetric or photometric evaluation. We present results on alternate test procedures for industrial waste water analysis performed on test strips for nitrate, ammonium, phosphate, nitrite, iron, chlorine, copper, cyanide and sulphate ions on the band-hold RQ flex 6 reflectometer. In a second part the evaluation of immunoassays of D-Tech * series for high priority pollutants in water or soil is reported. The detection of explosives (TNT, RDX) or polychlorinated biphenyls (PCB) is shown and compared with standard methods. BIOSENSORS FOR BIOMONITORING Kazunori IKEBUKURG and Isao KARUBE Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153 Japan In recent years, preservation of global environment gets much attention and has become recognized world wide as a problem. Monitoring of environmental pollution is therefore one of the most important controlling options. In our laboratory, a number of biosensors for environmental monitoring have been developed and reported. Here we would like to introduce several examples of those biosensors. Biochemical oxygen demand(BOD) sensor, phosphate sensor, cyanide sensor, detergent sensor, pesticide sensor and plankton sensor will be presented. BOD sensor The BOD test is a standard pollution monitoring tool since 1936(Mullis and Schroeder, 1971). In practice, the BOD test requires a 5 day incubation period at 20°O. Therefore, a number of papers concerning methods for rapid estimation of 5-day BOD have been published in recent years. However, these methods are neither rapid nor simple. Four essential characteristics must be included in a good measurement procedure. The method must have theoretical validity and, the procedure must be simple and inexpensive, the determination time must be short and the result must be precise and reproducible. Methods for rapid estimation of BOD which satisfy these criteria are still required. A microbial BOD sensor would be one of the powerful tools for rapid estimation of BOD. Since we first reported a microbial BOD sensor(Karube et al. 1977), we have been developing many kinds of biosensors for rapid estimation of BOD. Those BOD sensors will be presented. Phosphate sensor Phosphate concentration is commonly used as an index for the estimation of wafer enrichment. When water is enriched with phosphate, the amount of phytoplankton in the water increases (Krebs, 1972). Currently, there are very few simple sensing systems available for this purpose and thus the development of sensitive method for the determination of phosphate ions is of great importance for river water monitoring. A flow injection phosphate analysis system based on an enzymic reaction and a subsequent luminol chemiluminescence reaction has been developed. The system consists of an immobilized pyruvate oxidase column, mixing chamber for the chemiluminescent reaction and a photomultiplier. The H202 generated by the reaction of phosphate and pyruvate oxidase is detected using a luminol chemiluminescence. Cyanide sensor Cyanide discharged from the electroplating plants, for example, is very toxic to many life forms. Since we use river water as drinking water, cyanide contamination of river water is sometimes lethal. There have been several accidents involving cyanide contamination of rivers, so it is therefore necessary to monitor cyanide contamination of river water. Cyanide is commonly detected using spectrometric methods based on the Konig reaction(Epstein, 1946). Nevertheless these methods require laborious and time consuming procedures. Therefore there is an urgent need to develop a simple, rapid and inexpensive sensing system for monitoring cyanide in river water. -23- Cyanide is very toxic to life because it inhibits respiration by binding to the cytochrome oxidase complexes. By using this respiration inhibition, the cyanide sensing system can be constructed. When cyanide is added to the microorganism its respiration is inhibited and the decrease in oxygen consumption can consequently be observed resulting in a current increase from the oxygen, electrode. We have constructed a batch type microbial sensor for cyanide detection, which has a yeast immobilized membrane on the oxygen electrode. Detergent sensor . Detergent is also one of the serious pollutants of river waters in Japan. We have developed a microbial sensor for linear alkylebenzene sulfonate(LAS) using a LAS degrading bacteria(Nomura et al. 1994). The sensor consists of a.bacteria immobilized reactor and an oxygen electrode which can measure respiratory activity of immobilized bacteria. Pesticide sensor Pesticide remaining in soil and natural waters is a world wide problem as an environmental pollution. There has been very few rapid and easy methods for .a pesticide determination, but there is increasing interests inimmunoassays for rapid and easy detection of a pesticide. We have developed a sensor using antibodies for pesticide and quartz crystal as a transducer of siganl from antibodies. Using a competitive method, highly sensitive detection has been achieved. Plankton sensor Plankton concentration is also an index for the estimation of water enrichment. When the amount of phytoplankton in the water increases, it spoils a scenary and bad smells sometimes occur. Therefore it is very important to monitor plankton cocentration in a river water, but there are very few simple sensing systems available for this purpose. Thus the development of simple and sensitive method for the determination of plankton is of great importance for river water monitoring. A plankton sensor based on fluorometric measurement will be presented. References Epstein, J. E., Anal. Chem., 19,272 (1947) Karube, I., Mitsuda, S., Matsunaga, T. and Suzuki, S., J. Ferment Technol., 55, 243(1977) Krebs, CJ. Ecology, The experimental analysis of distribution and abundance. Harper & Row Publishers, New York, 454 (1972) Mullis, M.K. and Schroeder, E.D., J. Water Poll. Control Fed., 43,209 (1971) Nomura, T., Dcebukuro, K., Yokoyama, K. Takeuchi, T., Arikawa, Y„ Ohno S. and Karube, I., Anal. Lett., 27, 3095 (1994) -24- METHODS TO CHASE MICROORGANISMS IN THE ENVIRONMENT Hiroshi Oyaizu Graduate School of Life Science and Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Various kinds of microorganisms with special abilities are used for the bioremediation. For successful bioremediation it is very important to chase the applied strain in the applied environment. The chase methods include two steps, isolation in a selective medium and identification of the isolated strain. Identification at strain level can be done by several kinds of analyses of cellular constituents. Cellular protein profile on PAGE is powerful to discriminate a strain from related organisms. However, analysis of cellular protein profile is laborious and this method is difficult to be applied to the enumeration. A strain can be identified by an antibody labeled by fluorescent pigment, and the method is called immunofluorescence. However, usually many indigenous strains in the same species share the same antigen. DNA fragments specific to a strain can be used for strain identification. The DNA fragment is very easily detected by PCR. The enumeration based on a specific DNA fragment is also very easy. We were able to chase a fluorescent pseudomonad with antifungal activity in field soil. The fluorescent pseudomonad (strain HP72) was isolated from turf grass roots of golf course, and identified as Pseudomonas fluorescens. The strain showed activities to inhibit the growth of plant pathogens, Rhizoctonia solani, Sclerotima homoeocarpa, Pythium aphanidermatum and Gaeunnomyces graminis. The strain also showed an activity to promote the growth of turf grass in the field. To enumerate the pseudomonad a DNA fragment which was strictly specific to the strain was selected by RAPD PCR. The DNA fragment was cloned into pTTBlue vector and sequenced. Based on the sequence very specific primers were synthesized. Finally, the strain HP72 was enumerated in the field by MPN-PCR method, and survival conditions of the strain were elucidated. The number of the strain on turf grass roots was correlated with turf grass growth. Therefore, if the number of the strain on the plant roots is kept very high, the strain can be used as a biological pathogen controlling agent. The enumeration method by a specific DNA fragment was useful to assess the plant growth promoting bacteria. The MPN- PCR method can also be used for the assessment of a microorganism which is applied to bioremediation. Since 1993 we had some experiences for bioremediation of Kuwait oil contaminated soils under the PEC-KISR joint program. From the bioremediation experiments we would like to present the following questions and our answers. 1) Are the genetically modified microorganisms necessary for bioremediation? — Our answer is " No." 2) What is the most important factor of the soil bioremediation? — Our answer is "Application of materials to enhance biodegradation by indigenous microorganisms is the most important factor. " 3) What is important for the application of microorganisms? — Our answer is " The most important thing is to use the most suitable microorganism for the applied environment and to establish the most suitable application method." -25- DETECTION AND ENUMERATION OF METHYLOTROPHIC BACTERIA IN GROUNDWATER SAMPLES BY MOLECULAR PROBING T. SEDMOMURA1, H. UCHIYAMA2 and O. YAGI2 1 Ebara Research Co., Ltd., 2-1 Honfujisawa 4-chome, Fujisawa-shi 251, Japan 2 National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305, Japan 1.Introduction Indigenous methylotrophic bacteria (methylotrophs) have been studied for practical restoration of trichloroethylene(TCE)-contaminated soil and groundwater by in-situ biostimuIa.tion technology . The key point of success of biostimulation technology largely depends on the accuracy and speed of a monitoring method for responsible microbes. MPN (Most Probable Number method0 ) which consists of enumerating methylotrophs in the microbial population of a test sample after selective proliferation has been employed for continuous monitoring of soil and groundwater with least satisfaction, as it takes 3-4 weeks for cultivation; and all methylotrophs including TCE-degraders as types II and as well as non- TCE-degraders such as type I are nonselectively counted. This paper describes a rapid and selective detection and enumeration method specific for all TCE-degrading methylotrophs by a molecular probing technique using the segment of the soluble methane monooxygenase gene X (mmo X). 2.Experimental Base sequence data of the mmoX genes were supplied from Murrell ’ and Uchiyama (unpublished data). Specific DNA sequences which were perfectly conserved in three mmoX genes carried by type’ll and X methylotrophs, such as Methylocystis sp. M, Methylosinus trichosporium OB3b and Methylococcus capsulatiis Bath, were searched and detected, utilizing computer software GENETYX. These sequences were designed for DNA primer for subse quent Polymerase Chain Reaction 51 (PCR) experiments. Two types of DNA primers (forward and reverse segments) coding each of these sequences were obtained from TaKaRa. Groundwater samples were periodically collected from a TCE-contaminated aquifer in Chiba Prefecture which was subjected to biostimulation by supply of mineral salts, methane and oxygen. Microorganisms in fresh groundwater samples were captured on Millipore membrane filters. DNA was recovered from the filters by sonication and hot water extraction,and em ployed as template DNA for PCR. Taq DNA polymerase kit was obtained from TaKaRa. The Perkin Elmer DNA Thermal Cycler, type 480 was used for PCR amplification. The populations of TCE-degrading methanotrophs in the samples were estimated by comparing the dilution limits of the samples for detection by the PCR, with that of known 10- -26- fold cell dilution series of Methylocystis sp. M (type II methanotroph). .For comparison, the same lots of the groundwater samples were simultaneously analyzed by MPN. 3. Results a) Search of conserved sequences among the mmoX genes Two types of DNA sequences which are conserved among the three mmoX genes carried in methylotrophs described above were detected and resynthesized as DNA primers. b) Analysis of TCE-degrading methylotrophs by the PCR method All TCE-degarading methanotrophs which had been isolated from various TCE contami nated sites by the authors could be detected by this PCR detection method. Non TCE-degrad ing methanotrophs and heterotrophs; such as Mettiylobacterium extorquens, Bacillus subtilis, Pseudomonas cepacia and Pseudomonas putida, could not be detected by this method. c) Comparison of MPN and this PCR method for methylotroph enumeration The data of TCE-degrading methanotrophs ’ populations in the site detected by the PCR method were propotional to that of MPN method. The data detected by the PCR method are constantly higher than that of MPN at 10-50 times. 4. Discussion For effective execution of in-situ biostimulation, it is essential to rapidly monitor the TCE-degrading methanotrophs in samples. As described above, it takes 3-4 weeks for MPN, which means the critically important methylotroph count is unavailable at the time of in-situ decision making for practical treatments. In contrast, this PCR method is finished in 10 hours after sample collection, which indicates that the methylotroph count can now be taken into consideration as one of the essential parameters for environmental monitoring. It is noteworthy that this PCR method described above doesn't give us the absolutiy accurate populations of the bacteria, because the detection limit varies depending on DNA extraction efficiency and PCR amplification efficiency. But it will be useful to detect the mag nitude of the populations, at least. As this method seems promising, the current authors are going to further improve the assay conditions and collect more field data for objective confir mation of the utility of this method. 5. References 1) Hazen, T.C. WSRC-RD-91-23. 1992, Revision 3,16. 2) Tsien, H.C. and Hanson, R.S. Appl. Environ. Microbiol. 1992, 58,953-960. 3) Stainthorpe, A.C., et. al., Gene 1990,91,27-34. 4) Cardy, D.L.N., et. ah, Mol. Microbiol., 1991,5, 335-342. 5) Saiki, R.K., et. ah, Science 1988,239,487-491. -27- BIOMONITORING SYSTEMS IN DRINKING WATER PLANTS - PERMANENT CONTROL OF QUALITY PARAMETERS Hans-Henning Stabel 2V Bodensee-Wasserversorgung, Labor, D-70354" Sipplingen Biotests are frequently used in drinking water treatment plants, either for. . screening the raw water for adverse chemical compounds, or for the control of the finished top water. These tests were designed to compensate for expensive and laborious chemical analytical methods, while higher detection limits sometimes have to be tolerated. Thus low concentrations of xenohiotic substances, such as herbicides, etc. can be measured rather simply,and rapidly. A quick response of the test organisms to changing chemical signals is mandatory for automatical control systems. Sudden chemioa! spills in the raw water, however, can be detected by the combined application of different continuously monitoring flow-through biotesi systems, equipped with defined thresholds for pollution alarms. These tests operate over longer time periods (from weeks to years) and their combined spectrum of resonance to specific toxic compounds may add to chemical control of the water quality. Such on-line systems are: various fishtests, musseltests, the dynamic daphnia tost, the fluoromelric algal test, and several bacterial tests. Here I present practical experience from routinely application of some of these tests under conditions prevailing in water treatment plants. -28- HIGHLY SENSITIVE AND SELECTIVE ANALYSIS FOR PESTICIDES IN THE ENVIRONMENT YoshiyuM Takhnoto Environmental Health Science Laboratory Sumitomo Chemical Co., Ltd. To monitor residues of pesticides in the environment, analytical methods should have high sensitivity and selectivity due to relatively low residue levels of pesticides. Some gas chromatographic detectors have such good characteristics and hence the analytical methods can be simplified; good examples are organophosphorus pestiddes-FPD (flame photometric detector)-GC combination. Residues of these pesticides can be often determined at levels as low as ppb without any dean-up procedures from soil and water extracts. NPD (nitrogen, phosphorus detector) is specifically highly sensitive to both phosphorus and nitrogen atoms, and hence is less selective than FPD. However, for the determination of nitrogen-containing pesticides such as carbamate insecticides, herbicides, fungicides and some pyrethroid insecticides, NPD-GC is a useful tool, accompanied with dean-up procedures. ECD (electron capture detector) is highly sensitive to halide- and nitro group- containing pesticides such as organochlorinated pesticides, but less selective, and usually requires dean-up steps to determine their residues. Pesticides without such key atoms in the molecule as described above are usually measured with GC-MS. Taking account of the selectivity and sensitivity of detectors for a target pesticide, dean-up procedures are selected among the following; liquid-liquid partition between hexane and acetonitrile or GPC (gel permeation chromatography) to remove oily substances and column chromatography using silica gel or Plorisil. Extraction is carried out by using polar solvents such as methanol or acetone and then non-polar pesticides are extracted by partition into non-polar solvents such as hexane, ethylacetate or dichloromethane from aqueous solutions. Pesticides in air are trapped by Tenax or silica gel column under suction and then extracted with polar solvents. Pesticides in water are either directly extracted by partition with non-polar solvents or trapped by XAD-resins or SPE (solid phase extraction) column, followed by polar solvent extraction. General principles for determination of pestiddes at sub ppm levels will be presented at the Workshop. -29- ATMOSPHERIC MONITORING USING AN EYE SAFE LIDAR SYSTEM Masao Kamata,Chief Engineer Staff Group,Research & Engeneering Division, Ishikawajima-Harima Heavy Industries Co.,Ltd.(IHI) 1.Introduction Comprehensive studies were conducted in recent years to proceed remote sensing technologies using laser radar systems (Lidar).These systems use the backscattered energy from illuminated aerosols to remotely measure pollution concentration in urban areas. This report presents a brief description of the eye-safe lidar system developed by IHI for an industrial use and results of demonstrations in the fields of monitoring weather,exhaust plume in power stations and dust scattering in ore stock yards. 2.Outline of the system Fig. 1 illustrates the system configuration. The laser is a Ho-YAG laser of wave length 2.lum with E/0 Q-switch. The system operates at a pulse energy of 5OmJ,repetition rate of 5Hz,pulse width 100ns. The lidar return signal is collected with a 30 cm telescope. The laser system and the telescope are accommodated at a rigid table. The table is controlled by a servo-mechanism and can be rotated. Received signals are processed in a computer and concentration level of aerosols is displayed in a display as a two-dimensional color graphic. Table 1 shows the system parameter of the system. 3.Demonstration in atmospheric monitoring (1) Weather monitoring ' Fig.2 shows an example of weather observation done in cooperation with Tsukuba University in August,'95.The right side of the figure shows the record of radiosonde observation,which shows the air temperature, dew-point temperature and relative humidity with the altitude. We can recognize the region of inverted temperature at the altitude 800m. Therefore we can -30- Table 1 System Parameter Aerosol Range 0.5~r5.0km Range Resolution Scan Rate <10deg/s TELESCOPE Display Scan Angle Personal -10~95° Pitch Computer Weight 300kg iecori Inlet Power <3.Okw Fig.1 System Blockdiagram assume the presence of the inversion layer at altitude 800m or below. This assumption is confirmed by a record of the lidar system operation. The concentration level is recognized to be much higher in the region of similar altitude. Laser radar observation Radiosonde observation Distance from radar (km) Relative humidity (%) Temperature ("C) Fig.2 Weather monitoring data (2) Monitoring of exhaust plume of chimney-stacks in power stations Smoke flow emitted from 300m-tall chimney-stacks were observed to track the smoke downstream. Details will be explained in oral presentation. (3) Monitoring of dust scattering at ore stock yards Careful control is required at ore stock yards in power stations or steelworks to prevent dust scattering under the current strict environmental regulations. Frequent spraying of water is generally implemented regardless of yard conditions. Monitoring using the Lidar system was recognized to be useful •for realtime analysis of yard conditions. 4.Conclusion An eye safe Lidar system developed for industrial use demonstrated effectiveness in realtime analysis for environmental and weather monitoring. -31- INDUSTRIAL ASPECTS OF BIOREMEDIATION IN GERMANY Dr. V. Schulz-Berendt and E. Poetzsch -' , Umweltschurz Nord GmbH & Co., Ganderkesee, Germany In Germany the biological treatment of contaminated soils has been developed during the last 10 years-and is today considered to be the better alternative to special waste land" disposal for volumes from some hundreds up to several thousand cubicmeters. The large-scale regeneration and recultivation of conta- _ minated solids by microorganisms is based on the following principles: selection, adaption and optimization of microorganism cultures;, selection of substrates used to enhance the microbial activities; optimization of nutrient sup ply; preparation of the soil and mixing of the process components; construction end operation of biological treatment plants; maintenance and control of the •process. Large-scale experience in soil bidremediation in Germany exists in the fields: T. On-sitc-remediatlon of soil contaminated with Petroleum-Hydrocarbons (TPH) and Polycyclic Aromatic Hydrocarbons (PAH) in amounts of some hundred thousends of tons. 2. Off-site-treatment of TPH- and PAH-contaminated soil as well as volatile compounds like aromatic (BTEX) and chlorinated pollutants (Perchloro- ethylene. Trichloroethylene, Dichloromethana). in Germany a capacity of about 750.000 t/a Is Installed today. 3. In-sltu-treatment of sites contaminated with TPH, BTEX and chlorinated - compounds at areas of some ten thousand square meters and a depth of about 10 to 15 m. Biological soil remediation has developed from simple land farming to a sophisti cated technology including the use of bioreactors. At the same time, the large scale use of biological breakdown processes expanded from simple applications, like the degradation of petroleum hydrocarbons, to more complicated substan ces like aromatic or chlorinated compounds. In the most cases. It Is not the lack of enzymatic potential for degradation of the contaminant that limits the biologi cal breakdown but other properties like solubility or volatility. Therefore, tech nical efforts are necessary to make the pollutants available for microorganisms or their extracellular enzymes. There are .technical solutions to these problems but their realization eliminates the economical advantages of bioremediation as compared with other technolo gies. Therefore we have to keep in mind that bioremediation can be a simple and cheap way to improve the condition of nature damaged by human acti vities, provided space and time are available. -32- SIMULATION OF BIODEGRADATION OF CRUDE OIL ON BEACHES Masami Ishihara Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi-city, Iwate 026, JAPAN To develop bioremediation technologies for the treatment of an oil-contaminated beach, we investigated the biodegradation of crude oil in various cultural systems: in batch cultures, rolled cultures, flow columns and mesoscale beach simulator units (BSUs). Batch cultures in test tubes and flasks modeled the oil degradation in seawater. Rolled culture system mimicked the oil degradation at an intertidal gravel layer. Flow column system was the cylindrical model of gravel layer beneath the water table. Mesoscale BSUs simulated the biodegradation in the intertidal shore. The BSU consists of a plastic tank, a reservoir, and a level-controlling device. Seawater in the reservoir was aerated and temperature-controlled at 20°C. The internal volume of each tank was 1.5 m3 (1 m x 1.5 m x 1 m), filled with approximately 1 m3 of gravel (2-8 mm in diameter). The level-controlling device moved between 20 and 80 cm high two cycles per day, thus realizing a tidal cycle of seawater in these tanks. At the beginning of the experiments, 1.5 kg of crude oil (artificially weathered by distillation up to 230 °C) was poured into each tank. A culture (3L, approximately 108 cells/mL) of the mixed population called SMS (isolated from a sediment in SMzugawa Bay, Japan) and two types of fertilizers, namely a slow-release granular nitrogen, fertilizer and a granular phosphorus fertilizer, were added in one tank but not in the other (control) tank. When the dissolved oxygen concentration at 5 cm below the surface of high-tide seawater was measured in the amended tank, it was at 30% the saturated level in the first two days after the inoculation, then went up to 70% of the saturated level in 10 days. The dissolved oxygen concentration subsequently decreased again to 20% the saturated level for next 70 days. The dissolved oxygen concentration of seawater in the control tank remained unchanged. The numbers of total heterotrophic bacteria were high immediately after the inoculation, then drifted between 105 and 106 cfu/mL. In the control tank, the viable counts of indigenous bacteria were around 2 x 104 cfu/mL. In the amended tank, gas-chromatography-resolvable crude oil associated with gravel disappeared within 80 days, while no significant biodegradation was observed in the control tank. This observation indicated that the addition of SMS and/or fertilizers was effective for the removal of contaminated crude oil. In the nonamended tank, oil slick remained at the surface for 80 days but disappeared within 15 days from the surface in the amended tank. Thus, the effectiveness of the bioremediation of crude oil was demonstrated in the BSUs both by visual inspection and by chemical and biological methods. The biodegradability of crude oil was strongly influenced by its physical state. When dry gravel was smeared with crude oil and subsequently submerged into seawater, the biodegradation of the crude oil adhered to gravel was less extensive than that of crude oil poured and dispersed in seawater. The similar phenomena were observed in the flow column system. This work was performed as a part of the Industrial Science and Technology Frontier Program supported by the New Energy and Industrial Technology Development Organization of Japan. -33- Bioremediation of Oil-Contaminated Soil in Kuwait Obayashi Corporation Technical Research Institute Hirokazu TSUJI, Hiroyuki CHINO Kuwait Institute for Scientific Research Nader AL-AWADHI, M. Talaat BALBA, Reyad AL-DAHER 1. Introduction During the Arabian Gulf War in 1991, Iraqi forces bombed and set fire to over 600 Kuwait oil wells, spilling enormous amounts of crude oil. The oil that flowed out above ground created over300 oil lakes, covering a combined area of over 49 km2area. To solve the urgent problem of treating the oil-contaminated soil, the Petroleum Energy Center (PEC; a foundation established under the auspices of MITI) progresses the collaboration with KISR (Kuwait Institute for Scientific Research) to facilitate work progress. The remediation of the oil lake beds is now actively considered so that oil contamination does not pose a critical health hazard to man and also to stimulate the restoration of the damaged ecosystem. Bioremediation technology was selected for the treatment of the contaminated soil and three different methods evaluated on die field: landfarming , windrow composting soil piles and static piles fitted with forced aeration. The project is primarily intended to lay the foundation for rehabilitating Kuwait's environment through the field-scale demonstration of biological to develop an "Action Plan" for the remediation of all-oil contaminated desert soil in Kuwait Content (%) 2. Distribution and properties of the 30 40 30. 10 12 contaminated soil The penetration depth of the oil contamination into the subsurface soil was investigatedby collecting soil samples at various depth from several test pits, according to a predetermined grid. The degree of nil .con tamination was examined visually, documented by photographs and by chemical analysis. The soil was analyzed for total extractable material (TEM) by gravimetric analyses, total petroleum hydrocarbons (TPH), using IR spectrophotometric technique. The oil distribution profile within the subsurface soil didnot follow any clear pattern, Fig.-2 Oil Content in Fig.-l Oil Content in Depth Depth (<50cm) possibly due to the variable geological nature of the Fig.3 GC/FTD-Cromatogram of Fig.4 GC/HD-Cromatogram of n-alkanes the Soil Extract in Surface in Kuwait Crude Oil —34— Burg an area and the presence of gatch lenses in the subsurface soil. In some areas, oil penetration was limited to the first 50 cm (Fig.l) and in other areas penetration was as deep as 2.5 meters. The concentrations of TEM in the analyzed samples, ranged between 20 to 60 % in the oil sludge and between 0.03 to 24.8 % (w/w) in the subsurface soil. In some spots excavation was extended an additional 140 cm down from the 50 cm below ground level (to a total depth of 190 cm). As clearly shown in the TEM data in Fig. 2 there was barely any contamination at a depth of 60 to 70 cm, but at 160 cm the contamination level surged to a TEM of over 10 %. In addition to the above analyses, selected samples were extracted with freon and analyzed by Gas chromatograph fitted with flame ionization detector (GC/FID), to determine the general characteristics of oil con tamination (Fig. 3). The results were compared with those of crude oil sample from the Burgan field (Fig. 4) and standard of authentic alkane compounds. The results indicated that the oil contamination has lost significant portion of the lower molecular- weight compounds (C8-C13), which is not surprising due to the exposure to harsh weather condition over an extended period. 3. Laboratory tests to enhance microbial biodegradation in oil contaminated soil In the bioremediation of oil contaminated soil, three of the most important factors affecting the activity of microorganisms are nutrition, temperature, and moisture. Accordingly, laboratory tests were conducted to ascertain fhe degree of these factor's effects. In the experiment 10 g of contaminated soil were placed in a 300 ml triangular flask, and to this were added nutrient (N:P:K=6.5:6:19), oil degrading microorganisms, and water. Properties of the soil is shown in Table 1. In the flask, small bottle with 5 ml of lN-NaOH was set and C02 production amount by microorganisms was measured once a week. Measurement of residual TPH was performed at the end of experiment. Fluctuations in COz levels due to differences in nutrient additive volume are shown in Fig. 5. Nutrients must be added in order to facilitate microbial degradation of oil contaminated soil. No variance was found in the volume of C02 generated during biodegradation or in the TPH biodegradation rate even when the volume of .additive was altered from 0.5 % to 1.5% of soil. From this it was concluded that 0.5% or more is an adequate nutrient additive volume. This amount is equivalent to 0.5 kg nitrogen per cubic meter soil. The microbial degradation f L T ... . ; Table-1 Properties of Soil of readily biodegradable substances, such as saturated hydrocarbon compounds, occurs rapidly at first, and this is followed by a more gradual biodegradation of less readily Oil Contaminated Soil Color - Dark Brown biodegradable hydrocarbons, such as aromatics. Oil components of residual TPH after Smell Oily Smell Touch Slightly Sticky 10 months incubation were measured. The best results of biodegradation testis shown Moisture (%) 43 Ig. Loss (%) 9.6 in Table 2. Most of saturated hydrocarbon had degraded and aromatic hydrocarbons pH 8.05 had degraded to half of the amount. However, resin and asphaltene group in the oil T-C(%) 5.20 T- N (%) 0.019 components remained. C/N 274 TPH (%) 5.47 4. Field experiment On the basis of laboratory tests and site investigation, a field demonstration experiment is now being carried out at the site near oil lake No. 102, located about 70 km south of the city of Kuwait. The lake covers an area of more than 50 hectares and an area of one hectare (75 m x 135 m) of this lake was —O—Nutrient 0% designated to the project Biological treatment of the contaminated soil, using —•—Nutrient 0.5% —1O—Nutrient 1.0% three techniques is being evaluated at a field scale. —♦— Nutrient 1.5% Biological treatment was selected for the treatment the soil with highest degree of contamination (the top 20 cm of soil following the sludge layer). The technology is also being assessed for the lightly contaminated soil. Three different types of bioremediation techniques are being used: landfarming Time (day) (1440 m3), windrow composting soil piles (480 m3) and static piles, fitted Fig.-5 Effect of Nutrient with enforced aeration system (240 m3). Composite soil samples are routinely Table-2 Oil Components in the de collected from all the soil plots and piles for the analyses of petroleum graded Soil hydrocarbon concentration and other key parameters. Certain landfarming Satorated Aromatic Resin Asphaltene Loss Group Group Group plots and soil piles will be inoculated with hydrocarbon-utilizing microbial Non-treatment 2.07 1.57 0.70 0.51 0.45 strains, which have been isolated in the laboratory. 4 Months 0.84 134 0.67 0.52 0.63 10 Months 0.27 0.70 1.00 0.49 0.94 Soil wt% -35- Application of Bioreactors for Bioremediation Jurgen Klein DMT- Gesellschaft fur Forschung und Prttfiing mbH, Essen The prerequisites for bioremediation of soil are: - degradability of contaminants -bioavailability of contaminants in soil matrix - sufficient supply of microorganisms with nutrients and oxygen during the whole remediation period. Especially for fine- grained soil the latter requirement can be met by application of bioreactors. As compared with the conventional techniques of land farming and bio- heaps the advantages of bioreactors can be summarized as follows: - treatment of soil with high ratio of fines - realization of optimum milieu conditions for degradation - shortening of remediation period for contaminants with low degradation rates - improved emission control. For the selection of the most suitable kind of reactor economic aspects, e. g. investment and operation costs, are of major importance. Taking the water content of the soil, the reactor techniques can be differentiated into dry- reactor and slurry-reactor processes. Dry- reactor systems treat the soil at a moisture content of 50 to 70 % of its maximum water retentioncapacity in rotary drums or static reactors with internal mixing arrangements and therefore correspond to the dynamic heap- process. In slurry- rector- systems the soil ratio amounts up to 30 weight-%. Here reactors of the pachuca-, air-lift- and fluid- bed- type are used. They ensure separation of the individual particles and ideal mixing, thereby producing a maximum of available surface for the contaminants mass transfer between soil particles and aqueous phase (microorganisms). Of great interest for soil decontamination are combinations of wet classification, e: g. flotation, and bioreactor- treatment as an economically and ecologically appropriate solution. -36- REACTION ENGINEERING ASPECTS OF THE DEGRADA TION OF XENOBIOTICS BY MICROBIAL SPECIALISTS W.-D. Deckwer, GBF - BVT, Braunschweig Despite the many biological systems proposed to degrade xenobiotics there are only few examples where microbial specialists are used goal-oriented in larger industrial scale for the detoxification of effluent streams and for the remediation of our environment. One of the reasons for this situation is certainly the insufficient understanding of microbial degradation processes in bioreactors and particularly in the environment. However, the development of safe technologies for microbial abatement systems requires detailed knowledge of the activity of the biochemical machinery and specifically its behavior under process relevant conditions. From the reaction engi neering point of view die recognition and evaluation of the rate limiting steps such as bioavailabi lity, transport phenomena (nutrients supply), fluid dynamics, microbial degradation kinetics etc., all considered under steady as well as transient conditions, may play an important role. The R+D work at GBF’s Biochemical Engineering Division focuses on these aspects with the aim to improve the theoretical knowledge and the experimental methodology to be "applied in environmental detoxification technologies. Presently, die subsequent selected processes are under investigation: • Biocatalytic treatment of gaseous effluents with volatile organic compounds (VOCs and halogenated hydrocarbons) - development of an adsorption supported laminar flow trickle bed and a TCA bubble column scrubber • Comparison of the performance of various strains (wild, differently genetically engineered) for degradating substituted aromates (alkylated, oxygenated, chlorinated) in controlled .bioreactors under various stress conditions • Degradation of heteroaromates (quinoline) - development of process strategies for strongly inhibited catabolic pathways by immobilization and cell recycle • Cultivation of pentachloiphenol degrading strains as starter cultures for remediation studies, investigation on induction and degradation mechanisms • Biotransfbrmation of mercury compounds (Hg(II)) by wild and genetically engineered strains • Microbial degradation of polymers - recognition of relationships between chemical structure - physical properties - biodegradability at environmentally relevant conditions, development of biodegradable plastics with predictable degradation and usage properties. The talk will particularly concentrate on die effects of inhibition kinetics on reactor performance and the advantages of immobilization to overcome shock loadings. -37- MODEL-BASED ANALYSIS OF COEXISTENCE IN MIXED CULTURE DEGRADATION OF XENOBIOTICS Matthias Reuss Institut fuer Bioverfahrenstechnik, Universitaet Stuttgart Allmandring 31, D - 70569 Stuttgart, Germany Tel: 49-711 - 685 4573 Fax: 49-711 - 685 5164 A considerable amount of work has been devoted to investigations of microbial degradation of various xenobiotics with the aid of mixed cultures. Most of the work has been performed, however, under laboratory conditions in shake flask cultures. These conditions are important and interesting for illustrating the microbial potential, they are, however, far from those conditions we are faced with in technical plants. The most difficult questions, which needs to be answered when trying to transfer the results of laboratory observations to a technical plant, are related to the problems of coexistence of the mixed culture under dynamic conditions during continuous operation. Well known and very potential tools for tackling this kind of problems are the application of reaction engineering principles of structured modeling and stability analysis. Essential prerequisites for this analysis, however, are reliable experimental observations aimed at understanding the kind of interaction between the species and afterwards finding reasonable mathematical descriptions. The present contribution is aimed to discuss this kind of problems (experimental observations and mathematical modeling) with the aid of two examples: (1) Microbial degradation of sulfanilic acid with the aid of a two species mixed culture (2) Horizontal gene transfer (TOL plasmid) via transconjugation within freely suspended and immobilized Pseudomonas putida. -38- Panel Discussion Including Closing Summery “Perspective of Bioremediation and Environmental Monitoring" Chairpersons: -Ryuichiro Kurane (Japan,National Institute of Bioscience and Human Technology) -Wolf-Dieter Deckwer(Germany,GBF,Braunschweig) Panelers: -Yoshikuni Drushigawa(Japan,National Institute for Resources and Environment) -Peter Werner(Germany,University of Dresden) -Masanori Fuijita(Japan,Osaka University) -Siegfried Neumann(Germany,Merck AG) -Shigeaki Harayama(Japan,Marine Biotechnology Institute Institute Co.,Ltd.) -Schulz-Berendt (Germany,Ummeltschutz Nord GmbH) Thank you very much indeed all panelars and all participants for your giving very useful and helpful discussion. At first, can I make three sessions' very brief summary record. ■ . " ' ‘ ' . " " 1) Bioremediation in its broadest sense has been shown to be an effective technology both in its traditional uses and in more recent applications. This important workshop addressed the use of biotechnology including biomonitoring in remediation of pollutants. 2) There is an increasing potential for the use of bioremediation in pollusion prevention and remediation because of several considerations including the flexibility of the technology, problems with alternative technology, the possible cost benefits and the social -39- acceptance of biological rather than conventional physical or chemical techniques. I had an opportunity to attend and present in OECD Amsterdom Workshop *95 on wider application and diffusion of bioremediation technology which was held last November 19-22, Amsterdom, The Netherlands, and also in EU/EUREKA Project which was held last November 23rd and 24th, Brussels, Belgium. OECD Amsterdam Workshop's brief conclusions are as follows; 1) Research activities in academic not always directed to appropriate environmental problems. And so we need for closer cooperation between academic study and practical application, and we need understanding of scale up. 2) It is important for us to remove bottlenecks, technological, economical, etc. 3) It is quite important for us to make R&D, especially in field test level to remove technological bottlenecks. 4) Hollistic approach is also quite important, e.g. co-operation between microbiologist, engineer and so on. 5) Facilitation of introduction in practice, this means that we need, at first, success story for wider application and diffussion of bioremediation technology. 6) Efforts to public acceptance should be continued. 7) Co-operation in OECD member countries and technology transfer to developing countries. Finaly, could I present state of the Art and make recommendation of this Japan-Germany Workshop on Bioremediation. State of the Art; 1)German's situation is quite different from Japan's —40— situation; German's practical remediation technologies and their engineering are advanced and Japan has a few experience in practical bioremediation. 2) Basic researches for bioremediation in both countries, Germany and Japan, are quite excellent and at the same level. 3) Japanese Biomonitoring researches related to practical applications are advanced more than German's ones. Recommendations of bilateral Workshop on Bioremedialion and Biomonitoring between Japan and Germany; 1) Basic & Practical knowleges should proceed in expanding the use of bioremediation to wider applications. 2) There is a need to increased both scientific and practical inputs in establishing methods for evaluating efficacy, and the use of this information to improve the predictability to foster rapid development of this technology. 3) We should make follow up of our successful workshop's activities by continuing an information exchange through bilateral and OECD Workshops. Thank you very much indeed all members for your nice cooperation. —41— LIST OF PARTICIPANTS GERMANY (Guest speaker) 1 ' Schmid, Rolf R - Prof. , Dr, Institut fur Technische Biochemie, Universitat Stuttgart TIB Tencbn. Biochemie Allmandring 31 70569 Stuttgart TEL NO. 0771/685-3193, FAX NO. 0771/685-3196 Declarer, Wolf-Dieter Prof., Dr., Gesellschaft fur Biotechnologische Forschung, Braunschweig Hock, Berthold Prof. , Dr., Lehrstuhl fur Botanik, Technische Universitat Munchen Klein, Jurgen Prof. , Dr., DMT-Institut fur Umwelt-und Verfahrenstechnik, Essen Muller, Rudolf . - . Prof. , Dr., Technische Universitat Hamburg-Harburg Neumann, Siegfried Dr., E. iferck/Forschungsreagenzien, Darmstadt Reuj3,_ Matthias Prof. , Dr., Institut fur Bioverfahrenstechnik, Universitat Stuttgart Schulz-Berendt Dr., Umweltschutz Nord GmbH, Ganderkesee Spener, Friedrich Prof. , Dr., Institut fur Chemomnd Biosensorik, Munster Stabel, HansTfenning Dr., Bodenseewasserversorgung, Sussenmuhle Stottmeister, Ulrich Prof. , Dr., Umwe1tforschungszentrum Leipzig Werner, Peter Prof. , Dr., Institut fur Grundwasserwirtschaft, Ihiversitat Dresden -42— J A PAN (Guest speaker) University of Tokyo Karube, Isao Professor, Doctor Ministry of International Trade and Industry (MITI). Urushigawa, Yoshikuni National Institute for Resources and Ihvironment Kurane, Ryuichirou Applied Microbiology Dept. Nat’ 1 Institute of Bioscience and Human-Technology Mizutani, Fumio Biomolecular Eng. Dept. , Nat' 1 Institute of Bioscience and Human-Technology Nagaoka University of Technology Fukuda, Masao Professor Osaka University Fujita, Masanori Professor University of Tokyo Oyaizu, Hiroshi Assistant Professor Ikebukuro, Kazunori Assistant Ebara Research Co. , Ltd. Shimomura, Tatsuo Investigator Ishikawa,iima-Harima Industries Co. , Ltd. Kamata, Masao Manager Kurita Water Industries Co. , Ltd. Nakamura, Kanji Manager, Kurita Central Laboratories Marine Biotechnology Institute Co. , Ltd. Harayama, Shigeaki Manager Ishihara, Masami Chief Researcher -43 Obayashi Corporation I _ ___ Chino, Hiroyuki _ . Chief Research Engineer, Bio-Environmental Engi. Dept. , Tech. Research Institute „ I _ i Sumitomo Chemical Industries, Ltd Takimoto, Yoshiyuki _i • Chief Investigator GERMAN EMBASSY Schroeter, Klaus First Counsellor Yako, Yumi Researcher JAPAN Ministry of International Trade and Industry ftflTI) Yumoto, Noboru Biochemical-Industry Div. Basic Industries Bureau Sasaki, Takafuni Global Environmental Affairs Office, Environmental Protection and Industrial Location Bureau Yamada, Tomoho Biochemical-Industry Div. Basic Industries Bureau I to, Hiroshi Biochemical-Industry Div. Basic Industries Bureau Mikami, Eiichi Applied Microbiology Dept. , Nat’ 1 Institute of Bioscience and Human-Technology New Energy and Industrial Technology Development Organization (NEDO) Okumura, Sato^ii Industrial Technology Dept. Minemura, Norishige Global Environment Technology Dept. Mizukoshi, Tatsuya Global Environment Technology Dept. J A PAN (Audi axe) | Aisin Cosmos R & D Co. , Ltd Yamamoto, Takekazu Deputy Research Director, Bio-Medical Field ■44- Asahi Glass Co. , Ltd Takeuchi, Jurrichi Canon Research Center Sakuranaga, Masanori Asst. General Manager, Bio-Engineering Div. Chiyoda Corporation Toyohara, Hircki Lead Engineer, Environmental Facility Engineering Dept. DKK Corporation Asano, Yasukazu Vice Director, Research & Development Headquarter Ebara Corporation Kato, Masuo General Manager, Environmental Restoration Dept. Ebara Research Ca, LTd. Kitagawa, Masani Manager, Biotechnology Lab., Center for Environmental Engineering Eniricerche Ciocca, Giuseppe Representative, Japan Liaison Office Fu ji Electric Corporate Research & Development Ltd. Shimizu, Yasuji Manager, Environment & Energy Lab. Hazama Corporation Yamauchi, Hiroshi Chief Engineer, Technical Research Institute Hitachi Ltd. Takagi, Masato Deputy Chief Engineer, Chemical Plant & Systems Dept. Mori, Toshikatsu Senior Researcher, The 2nd. Dept, of Energy System Research, Hitachi Research Lab. Hitachi Plant Engineering Construction Co. , Ltd. Iijima, Yoshinori Senior Chief Engineer Mori, Naomichi Deputy Senior Chief Engineer Hitachi Zosen Corporation Miyazawa, Masayoshi Chief Condinator, Technology & Development Headquarter —45— Sakaba, Masaru Manager, Environmental Research & Development Center Idemitsu Kousan Corporation Murakami, Nobro Ishikawaiima-Harima Heavy Industries Co., Ltd. Akashi, Shigebaru Senior Manager, Technical Planning & Control Dept Ikegami, Yuji Senior Manager, Technical Development Div. Ka.iima Technical Research Institute Arai, Akio Manager, 6th Dept. Kami, Tatsushi Research Engineer, Bio Environmental Engineering Dept. Kawasaki Heavy Industries, Ltd. Yoshida, Kohsuke • • Technology Development Headquarters Kawasaki Steel Co. , Ltd. Iizuka, Tokio Senior Researcher, Chemical Research Center Uehara, Ken-ichi Senior Research Scientist, Technical Research Lab. Kikkoman Corporation Matsuyama, Asahi Manager, Research & Development Div. Kubota Corporaticn Asao, Goro Manager, Research & Development Planning Dept. Kurita Water Industries Ltd. •. Morita, Akira • Chemicals Research Group, Research & Development Div. ^ 1 ' i i Kvowa Hakko Kogvo Co. , Ltd. Mori, Hideo Scientist, Tokyo Research Lab Kyushu Matsushita Electric Co. , Ltd. Motegi, Itsuro Engineer, Water Environment Lab . —46— Marine Biotechnology Institute Kawase, Taichiro General Ifenager, Research Planning Div. Mei.ii Milk Products Co. , Ltd Imada, Masaru Deputy Director, Meiji Institute of Health Science Mercian Co. Tsunekawa, Hiroshi Manager, Process Development Lab. Mitsubishi .Tuko Kankyo Engineering Ltd Fukuda, Yoshinori Manager, Water Treatment Div., Kobe Branch Mitsui Engineering & Shipbuilding Co., Ltd. Fukuju, Yoshibaru General Manager, Research & Development Dept. , Chiba Lab. Nakatani, Tatsuo Manager, Research & Development Dept. , Chiba Lab. Morinaga Nvugyo Go. , Ltd Takahashi Eiji Sec. Head., Biochemical Research Lab. NEC Nakayama, Noriyuki Researcher, Resources & Environment Protection Research Lab. Nippon Lever B. V. Snape, Jonathan Project Manager, Japan Technology Group Nippon Oil Co. , Ltd. Sakata, Ko Research Associate, Central Technical Research Lab. Nishihara Environmental Sanitation Research Coro. , Ltd. Tsuchiya, Yukinari Application Engineer, Hot Project Team NKK Corporation Nakahama, Akiko Researcher, Implied Technology Research Center NOK Co. . Ltd. Gotoh, Masao Chief Researcher, Research & Development Headquarter —47 — Obayashi Corporation i - Tsuji, Hirokazu General Manager . 1 Ishikawa, Yoji Deputy Chief Research Engineer Organo Corporation Eguchi, Masahiro Deputy Chief, Research & Development Plant Dept. Sasaki, Shouichi Manager, Global Environment Protection Dept. - Osaka Gas Co., Ltd. ; Tsubota, Jun Researcher, Advanced Technology Center- Shimizu Corporation Niwa, Chiaki Director, Technology Development Division Sumitomo Electric Industries Ltd. Masuda, Shigeo General kbnager Sumitomo Heavy Industries Ltd. Sugimoto, Takeo Principal Engineer, Corporate Technology Operations Group Sumitomo Metal Mining Co. , Ltd. Ushio, Ryozo Researcher, Central Research Lab. Taisei Corporation Hoaki, Toshihiro Chief, Biotechnology Section Takenaka Corporation Ohsawa, Takehiko , ■ , , , Chief Researcher, Research & Development Institute ~ * . , Tokyo Gas Co. , Ltd. Sato, Yoshiyuki Team Leader of Applied Microbiology, Frontier Technology Research Institute Toshiba Corporation Ishimori, Yoshio R & D Center —48 — Tovo Engineering Corporation Michiki, Hideyuki Senior Research Engineer Takada, Kazutoshi Manager, Technology Research & Development Center Research Institute of Innovative Technology for the Earth (RITE) Yamaguchi, Tsutomu Miyoshi, Yasukatsu Kotani, Yasutoyo Takamatsu, Hideaki Shigemori, Norihisa Suzuki, Kunio Hachiya, Masahiko Shinada, Tsuneo Takezawa, Kazuko Nakadai, Etsuko Ikenouchi, Masahiro Watanabe, Tatsurou Sannoumaru, Akiko —49— 03-3987-9368