Study On Isolation And Biodegrading Characteristics Of Chlorobenzoate-degrading Bacteria

Chlorobenzoates (CBAs), as important chlorinated aromatic compounds, were widely used in agriculture and industry. However, the residue and risk of CBA in environment can not be neglected. Study the degradation characteristics exhibit theoretical and application importance. The detailed work of this thesis is as follows:(1) The diversity of 2-, 3-, and 4-CBA degraders in two pristine soils and one contaminated sewage sludge was compared. Results showed that the ability of microbial populations in undisturbed soils to mineralize CBA was widespread. The result also indicated that the 3- and 4-CBA degraders were more diversity than the 2-CBA degraders.(2) Seven strains, using 3-CBA as sole carbon sources, were isolated from the sewage sludge. Three strains were Pseudomonas sp., the other four were Rhodococcus sp., Arthrobacter sp., Alcaligenes sp. and Burkholderia sp. respectively. One of the strains, named S-7, can degrade 3-CBA effectively, so it was chosen as study strain. S-7 was identified by morphological, biochemical and 16S rDNA sequence analysis as Rhodococcus erythropolis.(3) Strain S-7 can fully degrade 3-CBA in a range of temperatures from 10 to 30°C. Strain showed higher degradability at 20°C than at 30°C. The psychrotolerant ability is significant for bioremediation in low temperature regions.(4) Plasmid purification and elimination showed strain S- 7 do not contain plasmid, the degrading function might be controlled by chromosome DNA. Catechol and chlorocatechol 1, 2-dioxygenase activities were present in cell free extracts, but no (chloro)catechol 2,3-dioxygenase activity was detected. HPLC detected the chlorocatechol in the fermentation broth. Spectral conversion assays with extracts from R. erythropolis S-7 showed accumulation of a compound with a similar UV spectrum as chloro-cis,cis-muconate from 3-CBA. Based on the results, a pathway for degradation of 3-CBA was proposed: 3-CBA was converted initially to chlorocatechol, and then degraded by modified ortho-pathway. Products entered the TCA cycle.(5) When glucose was added as grow substrate, strain S - 7 can cometabolic degrade 2-CBA, but cannot biodegrade 4-CBA. Protein electrophoresis result showed that the unspecific enzymes for 2-CBA degradation were induced by 2-CBA itself when glucose was served as growth substrate.(6) The methods of immobilization were studied. Using alginate or polyvinyl-alcohol (PVA) alone, the immobilized cell can not form steady beads. When added PVA to the sodium alginate, both strength and tenacity were enhanced greatly, and the biodegradation rate was faster than the free bacteria. Using sodium alginate and chitosan as carrier, although the properties of immobilized ball was better than using sodium alginate alone, the degradation rate was a little slower than the free bacteria.