Microbiological remediation of chlorinated hydrocarbons in contaminated soils Széchenyi plan NKFP 3/007/2001

Identifying the main goal or goals of the project The aim of the project is to work out bioremediation technology for cleaning soils polluted by chlorinated hydrocarbons. Chlorinated hydrocarbons are the most frequent persistent pollutants in Hungary. For example huge amount of waste containing chlorophenols and chlorinated pesticide residues are deposit in Balatonfűzfő. After transporting the barrels containing polychlorinated benzene from Garé the soil remaining is polluted with these compounds. Furthermore, chlorobenzene polluted soils can be found in Hidas region in an industrial area of Budapest Chemical Company. Even today large quantity of chlorinated intermediers and solvents are used in chemical industry. In order to fit the concentration of chlorinated hydrocarbons and biodegradation technology in the firs part of the project we estimate the pollution of chlorinated hydrocarbon is known legal and illegal deposition sites. Prallel to this work we work out modern analytical methods (GC, GC-MS, HPLC, HPLC-MS, HPLC-MS-MS) for monitoring biodegradation process and for isolation and structural elucidation of metabolites formed during the procedure. Distribution of the pollutants (degradation products of biotic and abiotic process) is determined by the type of the soil. Experimental work in determination of sorption capacity of different soils will give data about it. Research in biodegradation of chlorinated hydrocarbons is the base of the selection of strains or composite of strains for field experiments. Last but not least a closed mobile plastic redemption house can be built. Summary of the aims of the project is the following:  Determination of condition of pollution  Developing analytical methods  Selection of bacteria and filamentous fungi strains for degradation of chlorinated hydrocarbons  Soil sorption experiments and working out distribution model  Application of biodegradation in the polluted field small and large scale up to 100 tones Building a closed and mobile house for bioremediation of polluted soils.

The results obtained to date TUB Department of Agricultural Chemistry and Technology For degradation of 2,4-dichlorophenol és 2,4,6-trichlorophenol branches was searched and found: bacteria: TUB B-645 TUB B-671 microspores::TUB F-1224 TUB F-1234, TUB F-1238 – TUB F-1239, TUB F-1260 – TUB F-1261 The strains were lyophilized and placed in the microbiological collection of the TUB. TUB Department of General and Analytical Chemistry Development of the proper analytical methods (GC, GC-MS, HPLC) for the following of the biodegradation are ready. The selection of the strains happened on the base of these analytical results. University of Debrecen, Medical and Health Science Center Department of Human Genetics During the research was isolated 18 strains. Morphological investigation was done with 13 strain and was developed methods for the storing of the strains. In the investigated period (5 months) with one of the storing method was possible the storing without decreasing of the population.

Konzorciumi partnerek Name of the consortium partner AddressPhoneFax TUB Department of General and Analytical Chemistry 1111, Budapest, Szt. Gellért tér 4. TUB Department of Agricultural Chemistry and Technology 1111, Budapest, Szt. Gellért tér 4. Szt.István University, Department of Soils Science and Agrochemistry 2103, Gödöllő, Páter Károly u / / 1817 University of Debrecen, Medical and Health Science Center Department of Human Genetics 4032, Debrecen Egyetem tér / / Biokör Technological and Environmental Ltd. 1089, Budapest, Bláthy O. u / Dekosta-Biokör Environmental Technological Ltd. 1089, Budapest, Bláthy O. u / PEMÜ Plastic Process Corp. 2083, Solymár, Terstyánszky u / /

Start of the project July

Role of the consortium members Szt.István University Department of Soils Science and Agrochemistry Investigation of the chlorinated hydrocarbons adsorption and desorption on different soils. Determination the chlorinated hydrocarbons buffering capacity of different soils. Soil investigation on chlorinated hydrocarbons polluted fields. Determination the soil parameters of experiments and semi industrial technology for chlorinated hydrocarbons polluted soil remediation Optimizing the soil properties of remediation technology.

Role of the consortium members TUB Department of General and Analytical Chemistry The analytical background of the project. Developing analytical methods for determining chlorophenols, chlorobenzenes and other chlorinated hydrocarbons and its degradation products in soil. Developing of quick, solvent saving sample preparation methods. Monitoring of the microbiological degradation with these methods during the whole project. Isolation and preparation of degradation product

Role of the consortium members TUB Department of Agricultural Chemistry and Technology Isolation, characterisation and freeze-drying of bacteria and filamentous fungi capable of partial degradation or total mineralization of chlorinated phenols and chlorinated pesticide residues (both pure cultures and consortia). Research on degradation spectra of different bacterial consortia. Influence of various additives (nutrients), physico- chemical parameters and soil characters on the biodegradation. Installing and operating two laboratory bioreactors (size: 5-20 liter) in controlled conditions for soil bioremediation studies. One bioreactor will be operated in anaerobic conditions, and the other will be used for aerobic biodegradation studies. Solid substrate fermentation experiments with the most promising filamentous fungi. Preparation of inocula for larger scale experiments. Strong collaboration with colleagues carrying out the pilot plant bioremediation experiments.

Role of the consortium members University of Debrecen, Medical and Health Science Center Department of Human Genetics Biological experiments for degradation of chlorobenzenes: Isolation, selection and cultivation of bacterial strains. Investigation of the spectrum of degradation of different chlorobenzene derivatives of the strains, determination of their requirements for media supplements and aeration. Investigation of the molecular biology of the degradation. Establishment and testing of bacterial consortia. Improvement of the bacterial consortia to achieve wider spectrum of degradation. Scale-up of the degradation up to 10 litre of volume. Setting up a controlled fermentation. Production of biomass for pilot plant experiments. Testing of the improved bacterial consortia.

Role of the consortium members Dekosta-Biokör Environmental Technological Ltd. Pilot tests with kg soil in bioreactors fabricated by PEMÜ and equipped with measuring apparatus for continuos detection of major biogenic elements. Pilot test with 5-10 m 3 soil placed in windrows and providing a sequential anaerobic/aerobic treatment. Application of the strains which were efficient in the base experiments in the big laboratory expeiments

Role of the consortium members Biokör Technological and Environmental Ltd. Site characterisation of a chlorinated hydrocarbon polluted area. Soil, groundwater and soil gas sampling. Assessment of the contaminants and its intermediates subsurface distribution. Examination of the major geochemical parameters that may enhance biodegradability. Installation of monitoring system. Degradation experiments continuously carried out with soils of various types and polluted in various concentrations, based on the achieved results of our partners in screening and in research of optimal additive supply. Research for potential modification of soil parameters to effectively degrade the target compound in a coupled anaerobic-aerobic process. Microbiological, chemical and ecological toxicity testing of degradation process. Evaluation of achievable pollutant reduction.

Role of the consortium members PEMÜ Plastic Process Corp. Investigation of polyolefin plates considering the pollutants in soil Design and manufacturing of unique devices for big laboratory experiments Investigation of the mobilization of soil handling technology