Development of xenobiotic-degrading bioaugmentation products

Xenobiotics in the environment are pollutants, substances foreign to an entire biological system. Pesticides widely used for crop protection are xenobiotics, they did not exist before their manufacturing. Contamination of ground or surface waters with pesticides may be caused by direct intentional application, by discharge of industrial wastewater or spray liquid remainders, by the incidental contamination or by percolation or leaching out. Pesticide contamination of water should be prevented both because of the possible direct toxicity to man, and because of its influence on water biocenosis. Beyond that, many agent molecules of pesticides or their degradation products are very toxic to non-target organisms and may have carcinogenic, mutagenic, teratogenic, immuno-modulating or endocrine disrupting properties. Pesticides also affects the soils: reduces biodiversity and results in lower soil quality. Pesticide residues in the soil are taken up by plants and get into humans consuming the crops directly or through the food chain, so quick microbiological degradation of pesticide residues in soil is also important. The pesticide active ingredients investigated in our work are the herbicides 2,4-D and diuron, and the fungicides carbendazim (systemic) and mancozeb (contact). Carbendazim is to be banned from 2010 in Hungary, but is being intensively used in Serbia. Mancozeb is widely used in both countries, first of all for protection of vegetables. Diuron, 2,4-D and carbendazim are endocrine disruptors and have a persistent character. Degradation of mancozeb results in a potent mutagenic persistent compound, ethylene thiourea. From diuron a mutagenic and persistent compound 3,4-dichloroaniline, from carbendazim 2-aminobenzimidazole and from 2,4-D 2,4- dichlorophenol are produced by the soil microorganisms. These compounds have higher water solubility than the original molecule, and can leach from treated agricultural land into surface waters. Diuron is highly toxic to aquatic invertebrates and algae. Our main aim is to develop an optimized bacterial mixture which is capable of quick and complete degradation of the harmful agents described above. Pesticide-degrading bacteria will be isolated from water and soil samples taken from natural environments, and especially from agricultural soils. For evaluation of the bioaugmentation potential of the xenobiotic-degrading strains, accurate and rapid molecular diversity methods (RISA and community-ARDRA) will be used for monitoring the microbial community structure and population sizes of the degraders. The efficient xenobiotic-degrading bacteria will be taxonomically identified by partial sequencing their 16S rDNA and rpoB genes. Their basic physiological parameters: pH, water activity and temperature tolerance will be determined. Copper, cadmium, manganese and zinc tolerance levels of the strains will also be investigated. Degradation kinetics and products will also be determined.

pesticide-degrading bacteria (>50) were isolated from natural sources with the ability to use carbendazim, mancozeb, diuron and 2,4-D as a single carbon/nitrogen source. Molecular diversity methods (RISA and community-ARDRA) were used for monitoring the microbial community structure and the sizes of the degrading population. The identification of the most efficient degraders was carried out by partial sequencing of their 16S rDNA and rpoB genes. A new colorimetric assay was invented to follow the decomposition of ethylenethiourea (ETU), the spontaneous degradation product of mancozeb. The best ETUdegraders proved to be Bacillus subtilis, B. amyloliquefaciens, B. mojavensis and Pseudomonas fluorescens. Isolation of diuron-degrading bacteria required special experimental approaches. Twenty four 2,4-D-degraders were found. Tasks in the second trimester were identification the isolates, co-culturing the pesticide degrading bacteria, and determination of the effect of environmental factors (pH, temperature, water activity) on the growth of the selected degraders. We identified the best 2,4 D degrading isolate as Variovorax sp. and the diuron hydrolyzing bacterium as Rhodococcus sp. Co-culturing in pesticide containing media was made by the combinations of Pseudomonas-Variovorax and Bacillus-Variovorax strains. The investigated strains were able to degrade 40-95% of the pesticides within 10-12 days at 25°C at pH 6.5. They were able to grow at various pH ranges from4.5 to 9.5. The minimal water activity required for growth varied between 0.95 and 0.98. Rhodococcus sp. ACD2 was unable to grow below 10 °C and above 35 °C, the other strains grew at 10-40 °C. In the third trimester the heavy metal tolerance (copper, cadmium, manganese and zinc) of these isolates were investigated. The carbendazim degrading Variovorax and the diuron degrading Rhodococcus strains were extremely sensitive to cadmium, it decreased their degrading activity even at low concentrations. Copper ions strongly inhibited the degradation process of ETU degradation of PS27. 2,4D-degradation by Variovorax was highly accelerated by copper ions. All of the strains grew well at 100 mg/l Mn concentration. Zinc, copper and manganese (20-50 mg/l) accelerated the carbendazim and diuron degradation. optimized the UPLC instrument parameters for the chromatographic separation of selected pesticides. A multi-residue method able to simultaneously determine a group of selected pesticides with a broad spectrum of chemical characteristics in environmental matrices was developed.