ISOLATION AND CHARACTERIZATION OF PHENOL-RESISTANT MICROORGANISMS

Abstract

A phenol-degrading Comamonas testosteroni strain P15 was isolated from a soil sample obtained from the DYNO industrial processing plant and could utilize phenol up to 15mM as the sole source of carbon and energy. Two mutant strains, designated as Y20 and E23 strains were further isolated by fed-batch cultivation and ethyl-methyl sulphonate (EMS) mutagenesis respectively. Mutants Y20 and E23 showed maximum tolerance to phenol concentrations of 17.5mM and 20mM respectively. Phenol at a concentration of up to 23mM could be completely degraded by mutant E23 after adaptation to 20mM phenol in the medium. Determination of the batch kinetic constants were carried out by cells which were grown previously in media containing 5mM of phenol. The maximal growth rate (µmax), saturation constant (Ks) and inhibition constant (Ki) of the wild type strain P15 were established at 0.52h-1, 0.066mM and 10.6mM respectively. Mutant Y20 showed similar values of µmax and Ks as those of the wild type strain P15, but with a higher Ki value of 13.6mM. Compared to the wild type strain P15 and mutant Y20, mutant E23 demonstrated a lower µmax value (0.3h-1) but higher values of Ks (0.177mM) and Ki (19.3mM). After adaptation to a high phenol concentration (20mM), the values of µmax, Ks and Ki of mutant E23 were elevated to 0.36h-1, 0.24mM and 20.3mM respectively. The mechanisms by which the mutant strains gained an increase in phenol tolerance were investigated. By measuring the intracellular phenol concentrations, it was established that neither the wild type P15 nor mutant strains Y20 and E23 was able to accumulate phenol in the cells. Compared to the wild type, mutant Y20 displayed a similar phenol hydroxylase activity but an average 1.6-fold higher activity of catechol 2,3-dioxygenase (C23O) in the presence of 5 to 15mM phenol concentrations. The activities of both enzymes from mutant E23 were generally lower than those of the other two strains. Investigations of changes in membrane lipids were carried out by studying the mechanisms of phenol tolerance in the Comamonas testosteroni strains. The major classes of phospholipids in the wild type strain P15 and mutant E23 were phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). Without phenol exposure, mutant E23 showed a 2-fold greater amount of CL than the wild type. Upon exposure to phenol, the wild type strain P15 demonstrated an increase in CL at the expense of PE. However, there was no significant difference in the major phospholipid contents between cells of mutant E23 grown in the presence and absence of phenol. The changes in fatty acid compositions of each phospholipid for the wild type strain P15 and mutant E23 were also studied. For both strains, a dramatic increase of C16:1 9trans unsaturated fatty acid upon exposure to phenol was observed in all fractions of phospholipids examined. It was noted that the ratio of trans/cis fatty acid of PE and CL in mutant E23 was 65-70% higher than that in the wild type strain P15. In the absence of phenol, the degree of saturation of CL in mutant E23 was 33% higher than that in the wild type strain P15. Analysis of the CL and PE fractions of the wild type strain P15 and CL fraction of mutant E23 revealed that the cells reacted to phenol with an increase in C16:1 9trans fatty acid and a simultaneous decrease in the corresponding C16:1 9cis. However, an increase in C16:1 9trans was accompanied by a reduction of C18:1 11cis instead of C16:1 9cis in the PG fraction of both strains and PE of mutant E23. Higher activity of cis-trans isomerase or a new enzymatic system might offer an explanation for this event.