The biodegradation of phenol, chlorophenols and recalcitrant biopolymer

• Main Authors: Chu-Fang Yang, 楊茱芳
• Other Authors: 李季眉
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/51136716347956087161

博士 === 國立中興大學 === 環境工程學系所 === 96 === Chlorophenolic compounds are common environmental contaminants and they are hazardous to human being. Especially pentachlorophenol (PCP) is toxic to all forms of life since it is an oxidative phosphorylation inhibitor. Thus, removing them from environment and decreasing their toxicity is an important issue. Biological treatment is harmless environmental friendly and relative economical treated procedure. Therefore, one part of this research focused on phenol and chlorophenols (CPs) biodegradation. In this field, pure phenol and CPs degrading pure strains were enriched, isolated and characterized. Moreover, PCP-degrading strain Sphingobium chlorophenolicum PCP-1 isolated previously was applied for PCP-contaminated groundwater bioremediation, and its genes involving in PCP degradation were also identified, sequencing and expressed. From phenol and CPs acclimated mixed cultures, we isolated and identified two phenol-degrading strains (Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6), one 4-CP-degrading strain (Rhizobium sp. 4-CP-20) and one 2,4-dichlorophenol (2,4-DCP) strain (Burkholderia cepacia strain 1-3b). After investigation, the optimum growth temperatures for strain P-1 and P-6 were 31°C and 39°C respectively. Strain P-1 could degrade 600 mg/l phenol completely within 57.5 hr, and the maximum degraded phenol concentration of stain P-6 was 200 mg/l within 93.1 hr. Strain P-1 showed higher applied potential than strain P-6. The optimum growth temperature and 4-CP degrading pH for strain 4-CP-20 were 36°C and 7.59, respectively. Strain 4-CP-20 could degrade 100 mg/l 4-CP completely within 3.95 days, but the bacterial growth and the 4-CP degrading activity were both inhibited while the initial 4-CP concentration was higher than 240 mg/l. The maximum 2,4-DCP degrading concentration of strain 1-3b was 75 mg/l. Strain 1-3b not only utilized 2,4-DCP but also phenol, 2-CP and 4-CP as sole carbon source. After inoculating immobilized Sphingobium cells to bioremediate PCP-contaminated groundwater with a biological treatment system, the results indicated the immobilized cells could be inoculated into PCP-contaminated groundwater without adding other supplementary nitrogen, phosphate and carbon sources. The optimum HRT in the bioreactor system was 12.6 hr. PCP removal in the bioreactor remained stable and PCP removal efficiency was higher than 92% at this phase, and PCP concentration in the biotreatment system effluent remained undetectable. It is possible to bioremediate PCP-contaminated groundwater using immobilized Sphingobium cells in a bioreactor system. The proposed biological treatment system could be maintained for at least for two months. Besides, PCP metabolic pathway of strain Sphingomonas chlorophenolica PCP-1 was the same with previous study, and its PCP-degrading genes pcpA, pcpB, pcpC, pcpD and pcpE were sequenced. Gene pcpB was successfully expressed with pET-system, but the expressed protein was aggregated into inclusion body form. Another part of this study was concerned with recalcitrant biopolymer biodegradation. Polythioesters (PTEs) are the newest eighth class of biopolymers, and the first PTEs member was identified in 2001. Among PTEs, homopolymer poly(3-mercaptobutyrate) (PMP) synthesized using non-natural pathway displays good potential for application. However, before its extensive utilization, the PMP biodegradability has to be addressed more throughoutly. In this field, PHA depolymerase genes (phaZ1, phaZ5 and phaZ7) from strain Paucimonas lemoignei were identified, sequenced, expressed and mutated. Our result demonstrated that gene phaZ1 and phaZ5 of strain P. lemoignei could be expressed by strain E. coli and pET-23a system and the expressed proteins contained activities of degrading dPHB granules. After mutation with XL1-red competent cells system and UV light, no mutant containing PMP degrading ability was successfully screened. The third gene phaZ7 also could be expressed, but its nPHB degrading ability was not detectable. According to all results mentioned above, we can summarize that functional strains could be applied successfully for bioremediation after appropriate enrichment and detailed characterization. Moreover, basing on the role of molecular biotechnology on environmental engineering becomes gradually important. To approach recalcitrant compounds biodegradation, sometimes genomic engineering methods could be powerful tools and the background of functional genes in functional strain also must be investigated carefully. We tried to change the substrate specificity of gene phaZ using molecular biotechnological methods. However, the results were not so successful.