Aerobic Biodegradation Of Nitrobenzene By R.mucilaginosa, S.albidoflavus And M.luteus

This dissertation reports three newly isolated strains, which are able to utilize nitrobenzene as the sole source of carbon, nitrogen and energy under aerobic condition. The physiological biochemical properties of the three strains were studied and the rDNA molecular identifications were carried out. The strain growth and nitrobenzene degradation characteristics of the free cells and irmnobilized cells were investigated. Metabolic pathway of the nitrobenzene degradation was proposed. The purpose of this dissertaion is to provide an academic basis for the treatment of practical nitrobenzene-containing wastewaters.According to the morphological characteristics, physiological biochemical properties and rDNA sequences, the three strains are identified as Rhodotorula mucilaginosaZ1, Streptomyces albidoflavusZ2 and Micrococcus luteusZ3, respectively. The rDNA sequences of Z1, Z2. and Z3 have been submitted to GenBank with the accession numbers DQ778627, DQ855477 and DQ855476, respectively. Z1, Z2 and Z3 are deposited as patent strains in China General Microorganism Culture Center with the accession numbers CGMCC 1758, CGMCC 1759 and CGMCC 1760, respectively.The optimal conditions for both strain Z1 growth and nitrobenzene degradation are: temperature 30℃, pH=7.0, shaking velocity 180r/min and inoculation size 9mg/L (dry weight); the optimal conditions for both Z2 growth and nitrobenzene degradation are: temperature 30℃, pH=7.0, shaking velocity 150r/min and inoculation size 12mg/L (dry weight); the optimal conditions for both Z3 growth and nitrobenzene degradation are: 25℃, pH=7.0, 150r/min, inoculation size 9mg/L (dry weight). When nitrobenzene serves as the sole source of carbon, nitrogen and energy and the initial concentration is 200mg/L, strain Z1 degrades nitrobenzene completely after 60h under the optimized conditions, Z2 72h and Z3 120h. TOC removing rates are over 98% for all the three strains, which demonstrate that nitrobenzene is mineralized to CO₂ and H₂O. Strain Z1 can tolerate nitrobenzene up to a concentration of 450mg/L, Z2 400mg/L and Z3 250mg/L. Under the optimized conditions, the degradation kinetics of nitrobenzene by the three stains can be expressed by Andrew equation. The kinetic parameters of strain Zl are: q_max=4.11(1/h), K_s=151.42mg/L, K_i=32.569mg/L; the kinetic parameters of Z2 are: q_max=3.19(1/h), K_s=106.73mg/L, K_i=38.03mg/L; the kinetic parameters of Z3 are: q_max=2.32(1/h), K_s=67.13mg/L, K_i=40.29mg/L.The nitrobenzene degradation by the three strains in high salinity was studied. When nitrobenzene serves as the sole source of carbon, nitrogen and energy and the initial concentration is 200mg/L, the three strains still degrade nitrobenzene effectively in 5% salinity (NaCl, mass concentration) under the optimized conditions. Even 150mg/L phenol or 75mg/L aniline mixed into 200mg/L nitrobenzene, strain Z1 degrades nitrobenzene effectively under the optimized conditions. 200mg/L phenol or 50mg/L aniline mixed into 200mg/L nitrobenzene, Z2 degrades nitrobenzene effectively under the optimized conditions. However, Z3 degrades nitrobenzene little when 25mg/L phenol or 25mg/L aniline is mixed into 200mg/L nitrobenzene under the optimized conditions.The surface-adsorption immobilization method was adopted. Macro-pore carrier of DW-22 was selected as the immobilized material. The optimal conditons for the growth of strain Z1 on DW-22 and nitrobenzene degradation are: temperature 30℃, pH=7.0, shaking velocity 160r/min and inoculation size 6mg/L (dry weight); the optimal conditons for the growth of Z2 on DW-22 and nitrobenzene degradation are: temperature 30℃, pH=7.0, shaking velocity 130r/min and inoculation size 10mg/L (dry weight); the optimal conditons for the growth of Z3 on DW-22 and nitrobenzene degradation are: temperature 25℃, pH=7.0, shaking velocity 130r/min and inoculation size 6mg/L (dry weight). When nitrobenzene serves as the sole source of carbon, nitrogen and energy and the initial concentration is 200mg/L, immobilized Z1 degrades nitrobenzene completely after 30h under the optimized conditions, Z2 40h and Z3 96h. Under the optimized conditions, the degradation kinetics of nitrobenzene by immobilized Zl, Z2 and Z3 can be expressed by Andrew equation. The kinetic parameters of immobilized Zl are: q_max=8.38(1/h), K_s=185.59mg/L, K_i=132.39mg/L; the kinetic parameters of immobilized Z2 are: q_max=6.34(1/h), K_s=153.63mg/L, K_i=126.73mg/L; the kinetic parameters of immobilized Z3 are: q_max=4.31(1/h), K_s=110.39mg/L, K_i=54.95mg/L. Compared to free cells, immobilized cells exhibit strong capacity to endure more heat, salinity and toxicity. Immobilized Z1 is selected for further study on nitrobenzene degradation under semi-continuous flowing modes and the experimental results indicate that immobilized cells can tolerate higher nitrobenzene shock-load than free cells.The metabolic pathway of nitrobenzene degradation by strain Z1 was investigated. The proposed pathway is: nitrobenzene undergoes partially reduction to form 2-aminophenol, and then 2-aminophenol is cleaved and mineralized. Meantime, in the cleavage of 2-aminophenol, picolinic acid emerges as a byproduct. Z1 also degrades picolinic acid. The proposed pathway is: picolinic acid undergoes hydroxylation to form 6-hydroxy picolinic acid and 6-hydroxy picolinic acid is cleaved and mineralized.In conclusion, Rhodotorula mucilaginosaZ1, Streptomyces albidoflavusZ2 and Micrococcus luteusZ3 have higher potential for being applied to nitrobenzene industrial wastewater treatment.