Isolation Of PAHs-degrading Halophiles And Study On The Mechanism Of Pyrene Degradation

Saline environments such as oil fields, deltaic coast and ocean are particularly vulnerable to petroleum pollution due to their close connection with oil industries. Among various petroleum hydrocarbons, polycyclic aromatic hydrocarbons(PAHs) are the main pollutants, and they could persist in the environment for a long period due to the stable chemical structure as well as high hydrophobicity. Biodegradation is considered the most effective way to remove PAHs in the natural environments. However, non-halophilic microorganisms may be not suitable for the PAH degradation in saline environments owing to their osmotic pressure imbalance and loss of metabolic activity. Therefore, it is vital to isolate more halophilic PAH degraders and further to study their degradation characteristics and degradation mechanisms for guiding the PAH-contaminated bioremediation in saline sites.In our study, four new pyrene degraders were isolated from the petroleum-polluted soil and identified as Thalassospira sp. strain TSL5-1, Thalassospira sp. strain TSL5-2, Baciallus sp. strain TSL5-3, alcanivorax sp. strain TSL5-4. TSL5-1 and TSL5-2 were selected to study their characteristics of PAHs degradation and the influence of environmental factors on their PAHs degradation. It showed that both TSL5-1 and TSL5-2 could also degrade phenanthrene, pyrene, fluoranthene, benzanthracene. Particularly, TSL5-1 could utilize benzopyrene. The pyrene degradation by them could occur at salt concentrations ranging from 0.5% to 19.5%, which indicated their outstanding salt-tolerance. pH fluctuation could significantly affected the pyrene elimination and neutral culture solution was more favorable for their degradation of pyrene. Yeast could efficiently promote pyrene and benzopyrene removal by TSL5-1 while peptone had the opposite effect. Yeast had little effects on pyrene and benzopyrene removal by TSL5-2, but peptone could stimulate TSL5-2 to degrade more pyrene and benzopyrene. Actually, TSL5-2 could hardly utilize benzopyrene as the sole carbon source. After adding peptone, the degradation rate of benzopyrene was 38.2% in 25 days.A pyrene metabolic pathway for Thalassospira sp. was first proposed based on the detection of eight intermediate products by GC-MS, which showed that TSL5-1 degraded pyrene beginning with the initial deoxygenation at C-4 and C-5 positions and via two parallel downstream pathways: phthalate and salicylic acid routes. The genome of TSL5-1 was sequenced and it was 4.98 Mb. Some genes related to the downstream pathway were localized in a region and this gene cluster worked together to complete the process of degrading protocatechuate to succinyl CoA and acetyl CoA. It is worth mentioning that the presence of two genes encoding protocatechuate 3, 4-dioxygenase(existing as alpha and beta subunit pair) and gentisate 1, 2-dioxygenase in the cluster also demonstrated that two downstream routes above were contained in the pathway. In addition, twenty genes responsible for detoxification of PAHs were contained in the genome, and they could encode glutathione S-transferases, O-methyltransferase, Quinone reductase, et al. These enzymes might take part in the generation of less reactive methoxy-derivatives, the reduction of quinones substances and the uncoupling reaction of aromatic to reduce the toxicity of PAHs.