Phenol-degrading Moderately Halophilic Bacterial Community Structure And Molecular Mechanism Of Metabilism And Salt Tolerance

This study targeted to phenol degradation in high salinity conditions, moderately halophilic bacterial enrichment and strains which could degrade phenol effectively were gained through screening and acclimation from habitats with salinity. The community structure, strains identification, biodegradation characteristics of phenol, and osmoprotection mechanism were studied. The resultes provided further information on the understanding of phenol-degradation over a wide range of salinity in the environment.Firstly, salt and phenol with various concentration were added as the selection pressure to enrich and acclimated bacteria from the sediments of Qarhan Salt Lake. The enrichment was able to degraded phenol as the sole carbon source at a wide range of salt concentrations from 1 to 3 M NaCl. The ability of the enrichment to degrade phenol decreased with the increase of salinity. The degradation rate and the tolerance of phenol by the enrichments were notably improved by continuous acclimation. The optimal degradation was achieved at 1.5 M of NaCl and 350 mg/L of phenol. The log-phase of the enrichment growth started after a lag period of about 12 h, and the rate of degradation increased linearly as the biomass increases during the log-phase. Then the stationary phase was reached at about 52 h after inoculation. The influence of inoculation scale and additional nutrients on the growth of the flora and the biodegradation of phenol was investigated. The result showed that the degradation effect was best when the inoculation scale was 10%. The addition of yeast extract could significantly promoted the growth of the enrichment and the degradation ratios of phenol.Secondly, the community structure and the phenol-degrading pathway of the enrichment were studied. PCR-DGGE profile of the enrichment showed that the Acidobacterium sp. and Chloroflexus sp. dominated the community, and they might be the major functional populations of phenol degradation in the bacterial communities. These two species existed almost at all the salinity conditions indicated that the concentration of NaCl had no significant effects on the dominant species distribution. PCR detection of the key functional genes in the degrading pathway revealed that the enrichment contained all the known pathways for phenol degradation, including phenol hydroxylase gene, catechol 1,2-dioxygenase gene and catechol 2,3-dioxygenase gene, indicating the diversity of the phenol biodegradation by this enrichment.Thirdly, a high-throughput-microplate spectrophotometer system to screen halophilic bacteria for phenol degradation was developed. Two isolates were screened from the soil samples from Shanghai Laogang landfill, and showed the high phenol degradation effect. Comparative analysis of 16S rRNA gene sequences and phylogenetic tree suggested that the two isolates PDB-F2 and PDB-G1 belonged to genus Virgibacillus and Brachybacterium. Strain PDB-G1 and PDB-F2 were able to remove 500 mg/L phenol at the salinity ranging from 5%-15%, and they were able to tolerate up to 1200 mg/L and 1400 mg/L phenol, respectively. The culture temperature and pH had much effect on growth and phenol degradation by strain PDB-G1 and PDB-F2, and the optimum environmental conditions for phenol degradation was 30℃, pH 7.0-7.5 and pH 6.5-7.5. The addition of Ca2+could improve the removal efficiency of phenol, and the SO42- showed its stronger inhibition on the strains growth and phenol degradation than Cl-. When the volume ratio of bacterial suspension mixture of PDB-G1 and PDB-F2 was 1:2, the highest removal efficiency of phenol was 84.7%.Finally, Nuclear magnetic resonance (NMR) spectroscopy profiles showed that ectoine and hydroxyectoine were the main compatible solutes to adjust the bacterial osmotic pressure. The results showed that under hyperosmotic stress, PDB-F2 accumulated ectoine up to 73.12 mg/g·cdw in the LB medium containing 12% NaCl. The intracellular ectoine could released outwards in 15 min under hyperosmotic shock, and return to the original concentration by more than 75% in 12 hours. PDB-F2 could maintained the osmotic equilibrium in response to the changes in the external salt concentration, and be versatile in its adaptation to salinity.