Degradation of phenol via ortho-pathway by Kocuria sp. strain TIBETAN4 isolated from the soils around Qinghai Lake in China

Based on the feature of high-altitude permafrost topography and the diverse microbial ecological communities of the Qinghai-Tibetan Plateau, soil samples from thirteen different collection points around Qinghai lake were collected to screen for extremophilic strains with the ability to degrade phenol, and one bacterial strain recorded as TIBETAN4 that showed effective biodegradation of phenol was isolated and identified. TIBETAN4 was closely related to Kocuria based on its observed morphological, molecular and biochemical characteristics. TIBETAN4 grew well in the LB medium at pH 7–9 and 0–4% NaCl showing alkalophilicity and halophilism. The isolate could also tolerate up to 12.5 mM phenol and could degrade 5 mM phenol within 3 days. It maintained a high phenol degradation rate at pH 7–9 and 0–3% NaCl in MSM with 5 mM phenol added as the sole carbon source. Moreover, TIBETAN4 could maintain efficient phenol degradation activity in MSM supplemented with both phenol and glucose and complex water environments, including co-culture Penicillium strains or selection of non-sterilized natural lake water as a culture. It was found that TIBETAN4 showed enzymatic activity of phenol hydroxylase and catechol 1,2-dioxygenase after induction by phenol and the corresponding genes of the two enzymes were detected in the genome of the isolate, while catechol 2,3-dioxygenase or its gene was not, which means there could be a degradation pathway of phenol through the ortho-pathway. The Q-PCR results showed that the transcripts of both the phenol hydroxylase gene and catechol 1,2-dioxygenase gene were up-regulated under the stimulation of phenol, demonstrating again that the strain degraded phenol via ortho-degradation pathway.