| Phenols are a class of highly toxic substances protoplasm, serious harm to human health and survival. General strategies to eliminate phenol compounds are physical, chemical treatment and biodegradation. To get an economical, efficiently and no-secondly contaminated method, biodegradation attracts more intestests in recent years. In this work, two bacteria strains that growing on hydrolyzed p-tert-Butylcatechol (TBC) and phenol as sole energy and carbon source, were isolated from sludge of wastewater treatment workshops in a chemical plant. Degradation characteristics of these two strains were studied and so did their mechanisms. Main results are followed:(1) These two strains mentioned above were isolated by mediums added with TBC and phenol as single carbon source respectively. After enrichment and cultured by substrates, a TBC and phenol degrading strain obtained and named as YH1 and YH8 respectively. YH1 was classified as Pseudomonas corrugate by morphology, physiology, BIOLOG Auto Microbe System, sequence of 16S rDNA and gyrB. YH8 was classified as Acinetobacter guillouiae by the same method.(2) The degradation rate of TBC was higher than 82% with low sustrate concentration (below 500mg/L) when YH1 was cultured in a media added with TBC as single carbon and energy source at 24-36℃, pH 7.0-10.0. One-factor-at-a-time experiments demonstrate that optimal carbon and nitrogen source for TBC degradation are sucrose and tryptone respectively. Optimal temperature and pH are 30℃ and 7.0 with inoculation rate of 2%. In order to enhance TBC degradation rate, Plackett-Burman Design was conducted and sucrose, tryptone and initial pH were identified as main factors. These factors were approached to optimal region by steepest ascent design. Then optimal degradation condition (pH 8.12, TBC 400mg/L, sucrose 3%, inoculation rate of 2.97%, tryptone 1.44%,30℃,96h) was established by Box-Behnken design and response surface analysis. Degradation rate under this condition is 98.2%.(3) The degradation rate of phenol was higher than 70% with low sustrate concentration (below 1200mg/L) when YH8 was cultured in a media added with phenol as single carbon and energy source at 26-34℃, pH 7.0-10.0. One-factor-at-a-time experiments demonstrate that optimal carbon and nitrogen source for phenol degradation are sorbitol and NaNO3 respectively. Optimal temperature and pH are 30℃ and 9.0 with inoculation rate of 5%. In order to enhance phenol degradation rate, Plackett-Burman Design was conducted and initial pH, concentration of phenol and sorbitol were identified as main factors. These factors are approached to optimal region by steepest ascent design. Then optimal degradation condition (pH 9.26, phenol 1163.63mg/L, sorbitol 7.81%, inoculation rate of 5%, NaNO32%, 30℃,96h) was established by Box-Behnken design and response surface analysis. Degradation rate under this condition is 98.95%.(4) Localization experiments and TBC degrading activity showed that enzymes in relation to TBC degrading were intracellular proteins and synthesis of catechol 1, 2-dioxygenase (C12O) can be induced by TBC. Catechol 1,2-dioxygenase gene could be amplified from this strain by PCR. By detecting and eliminating plasmids, it was found that degrading genes located on plasmid.(5) Results showed that YH1 could use catechol, phenol, TBC, diphenylamine, tri-hydroxymethyl aminomethane, octaphenyl polyoxyethyiene, sodium dodecyl sulfate, quintozene as single carbon sources.And YH8 could use phenol, catechol, TBC, diphenylsemicarbazide, tri-hydroxymethyl aminomethane, octaphenyl polyoxyethyiene, sodium dodecyl sulfate, quintozene as single carbon sources. Growth rate of YH1 could be stimulated by Pb2+、Mn2+、Cu2+、Zn2+ and that of YH8 could be enhanced by Cr3+、Mn2+ Zn2+. Cd2+、Hg2+、Ag+ could inhibit both of them. Both strains could tolerant 5μg/mL of chloramphenicol, ampicillin, kanamycin, erythromycin, cefotaxime sodium and high concentration of NaCl. |
PDF Full Text Request