Isolation And Characterization Of A P-Nitrophenol Degrading Bacterium And Its Degrading Ability

Nitroaromatic compounds are common pollutants produced in developed countries by the industrial manufacture of dyes, explosives, pesticides, herbicides and drugs. The use of such materials has resulted in the release of nitrophenols into the environment. Among them, p-nitrophenol (PNP) is one of the main hydrolysis products of parathion and methyl parathion and it shows enhanced toxicity towards soil microflora. It is also generated during formulation, distribution, and field application of pesticides or photodegradation of pesticides that contain the nitrophenol moiety. Therefore, PNP poses significant health and environmental risks, because it is toxic to many living organisms and it may accumulate in the food chain.Many researchers have focused on the biodegradation of PNP. Most of them reported the PNP degradation at relative low concentrations. And the nitrite, which was toxic to many of the organisms, was rarely removed during PNP biodegradation, except the Rhodococcus Wratislaviensis by which detoxified PNP with an additional anoxic process. Also, there had been reports of cloning degrading gene clusters using the method of genome library construction.In this study, an effective PNP-mineralizing strain Y1 was isolated from the activated sludge by the enrichment technique. Its degrading abilities, mineralizing-mechanism degrading genes and enzymes were investigated. The main results are as follows:1. Several degrading bacteria were isolated from the activated sludge. And one of them Y1, was selected because of its effective degrading ability. Based on the results of phenotypic features, physiological-biochemical properties, and phylogenetic similarity of 16S rRNA gene sequences, strain Y1 was designated as Arthrobacter sp. Y1. Its 16S rRNA partial sequence was deposited in the GenBank under accession No. EU282772.2. The growth and degrading abilities were investigated under pure-culturing conditions by UV-Vis and HPLC. Strain Y1 could degrade 50 mg/L PNP within 6 h under the condition of pH 7 and 30℃. The effect of degrading abilities on PNP degradation under different inocula biomass amount, additional carbon source, temperature, initial pH and PNP concentrations were also investigated to determine the optical conditions. Results showed that the isolate could effectively degrade PNP with inocula 0.10～0.3 g/L, at 15～40℃, pH 6～11 and the PNP concentration was in 100～400 mg/L. Y1 could tolerant concentrations of PNP up to 600 mg/L, which was the highest among reported. Degradation could be effectively improved with the existing additional carbon sources (0~30 g/L). If the glucose concentration exceeded 30 g/L, however, degradation was repressed.3. 4-Nitrocathechol (4-NC) was detected as the main intermediate product by HPLC, which implied that degradation was mainly processed through the 4-NC pathway, the typical degrading pathway in gram-positive bacteria. Degradation of 4-NC using the isolate was investigated, which showed that the Y1 could also live on 4-NC as the sole carbon, nitrogen and energy source.4. Degradation of PNP by the supernatant of the inoculated cultures induced by PNP was investigated. Results revealed that the supernatant could degrade 50 mg/L PNP within 24 h. It is hypothesized that the degrading enzymes responsible for PNP degradation was secreted from bacterial cells.5. There had been publications of degrading gene cluster, which reported that a gene cluster responsible for the PNP conversion into 4-NC, was cloned by the method of constructing genome library. So a pair of primers was designed according to the consensus sequences coding for the PNP hydroxylase and phenol hydroxylase. A 1.5 kb fragment was obtained by PCR and was ligated into the expression vector to form into the recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21. Screening of the recombinant strains was achieved by the PNP conversion in the plates containing PNP. Investigation of the PNP conversion by the recombinant strain showed that the fragment we obtained was functionally expressed. Sequence analysis of the fragment showed that it encoded the oxygenase component of PNP hydroxylase which was similar to the PNP hydroxylase reported. Its sequence was deposited in into the GenBank under accession No. EU282773.6. SDS-PAGE analysis of the total proteins of strain under induced and noninduced cultures indicated that the degrading enzymes in Arthrobacter sp. Y1 could be inducible. Enzymetic degradation assays indicated that crude enzyme extracts could rapidly degrade PNP under induced and noninduced conditions. But the degradation rate in induced conditions were 7.2 times faster than in the noninduced ones, which implied that degrading enzymes in Arthrobacter sp. Y1 could be constitutively expressed.