Mutation Breeding Of Bacillus Studies On The Degradation Of Phenanthrene And Naphthalene By Pseudomonas Aeruginosa PAO1

It is more pressing to develop new antibiotics or the substitute of antibiotics with the high tendency of bacterial resistance to antibiotics.A strain which was isolated from the subcuticular of capsicumu was indentified to be in the genus of Bacillus. The ingredient extracted from the the culture was preliminarily proved to be a new antibacterial material. However, the strain produced low level of this antibacterial material and lots of polysaccharide in fermentation process. These are not conducive for the further industrial production and the subsequent purification.In order to screen the mutants with high yield of the antibacterial material and low polysaccharide production, the mutation breeding of the strain was carried out by ultraviolet mutagenesis. At first,30 mutants with higher yield of antibacterial material were obtained from1.0x105 mutants by testing drug sensitive analysis using Staphylococcus aureus. The mutant with the highest yield of antibacterial material was used for the second round mutation to get low polysaccharide yield strain. By analysing of the colony morphology and polysaccharide production, two mutants with higher yield of antibacterial material and lower polysaccharide production was gotten from 1.2×105 mutants. And the two mutants showed stable characters after subculture.Finally, the fermentation conditions of the mutant were optimized by the single factor experiment, and the optimum fermentation conditions were:fermentation time 60 h, initial pH value of 6.5, temperature 30℃, shaking revolution 200 r/min.This research provides a theoretical basis for the industrial production of this new antibacterial material. Polycyclic aromatic hydrocarbons(PAHs)that are classified as fused-ring aromatic compounds occur ubiquitously as environmental pollutants. The presence of these compounds have been seriously concerned for human health and the environment because most of the compounds are mutagenic, teratogenic, and potential carcinogenic. Microbial biotransformation has been found to be the major environmental process affecting the fate of PAHs. The majority of the PAHs-degrading strains isolated from environment were identified to be in the genus of Pseudomonas, but little is known about the degradation of phenanthrene and naphthalene in Pseudomonas aeruginosa PAO1.The degradation of phenanthrene and naphthalene by PAO1 stored in the lab has been investigated including degradation pathway, related and key degradation genes.The key intermediates of the phenanthrene and naphthalene degradation were used as the sole carbon and energy source to explore the degradation pathways of PAO1. TLC and HPLC were also used for futher confirmation. These results revealed that PAO1 degrade phenanthrene by distinct routes, via phthalate and salicylate, degrade naphthalene via salicylate.The effect of the related genes on the degradation of phenanthrene and naphthalene in PAO1 were investigated.20 related genes were obtained by screening the PAO1 random promoter library. Among them, PA2666 and PA4008 were selected for the further study as the expressions of their promoters were affected by phenanthrene or naphthalene effectively. Two mutants were constructed and their growth were analysed using phenanthrene or naphthalene as sole carbon and energy source with PAO1 as control. These results proved that they are related but not key to the degradation of phenanthrene or naphthalene.In order to find the key genes in PAO1 responsible for phenanthrene or naphthalene degradation, a transposon insertion library of PAO1 containing 5000 clones has been constructed.23 insertion mutants that can not grow on MM plate containing phenanthrene or naphthalene as the sole carbon and energy source were obtained by screening the transposon insertion library. The genes which may have the key role in phenanthrene or naphthalene degradation will be conformed by random PCR, sequencing and sequence alignment in the following experiments. The further library construction and other related works were still in progress. This research facilated the further understanding on the PAHs degradation pathway and promoted the PAHs biodegrading development on environmental bioremediation.