Aerobic utilization of selected pharmaceutical and personal care product by estuarine heterotrophic bacteria.

Abstract

Pharmaceutical and personal care products (PPCPs) constitute a broad class of organic compounds, some of which belong to the list of the OECD high production volume (HPV) chemicals. These compounds have emerged as environmental contaminants with potentially detrimental effects. They have been detected in various environmental compartments typically in a nano- to microgram range and sewage treatment plants represent the major point source for the aquatic environment. Salicylic acid, a monohydroxybenzoic acid, is widely used in cosmetic and therapeutic products and is listed as HPV chemical. Benzyl salicylate and phenyl salicylate are diaryl ester compounds commonly used in pharmaceutical formulations, fragrances and household detergents. Benzyl salicylate is listed as HPV chemical. The fate of salicylic acid in the environment has been reported previously while those of benzyl salicylate and phenyl salicylate are unknown. Although studies are available on the microbial degradation of aromatic compounds, studies exclusive to the catabolism of PPCPs by marine heterotrophic bacterial isolates are rather limited. Therefore, the aim of this thesis was to characterize PPCPs (salicylic acid, benzyl salicylate and phenyl salicylate) utilizing bacteria from an estuarine environment (Durban Harbour, KwaZulu-Natal, South Africa). Selective enrichments were employed using artificial seawater medium typically supplemented with 2 mM of the target compounds (salicylic, benzyl salicylate or phenyl salicylate). After successive subculturing, bacteria capable of utilizing target compounds as sole carbon and energy source were characterized by morphological and physiological features, 16S rRNA gene sequence and MALDI-TOF MS analysis. Growth kinetics were assessed by monitoring the optical density, cell count and protein formation over time. The utilization of salicylic acid and phenyl salicylate was verified using UV spectroscopy and HPLC and the key reactions involved were verified by determining the specific oxygen uptake rates using resting cells and specific activities of representative enzymes. A Gram-negative coccus shaped bacterium belonging to the genus Acinetobacter degrading salicylic acid and phenyl salicylate, a Gram-negative rod shaped marine bacterium belonging to the genus Oceanimonas degrading salicylic acid and phenyl salicylate and a Gram-negative rod shaped bacterium belonging to the genus Pseudomonas utilizing benzyl salicylate in the presence and absence of synthetic surfactants (Tween 80) were isolated. The growth of Acinetobacter and Oceanimonas species was dependent on salicylic acid and phenyl salicylate as carbon source as growth was only observed when the carbon source was present and the compound was degraded almost to completion. Growth of Pseudomonas with benzyl salicylate was enhanced in the presence of surfactant. All three strains did not have an obligate requirement for NaCl. Acinetobacter and Oceanimonas strains were tolerant to high concentrations of salicylic acid and were inhibited at a concentration above 20 mM while phenyl salicylate did not show toxic effects on the strains; instead growth increased with the increase in concentration. Salicylic acid was utilized via catechol by both strains as they showed high specific oxygen uptake rates and catechol-1, 2-dioxygenase activity for this chemical. Phenyl salicylate was hydrolyzed at the ester bond to phenol and salicylic acid, as these were the metabolites that accumulated during growth with phenyl salicylate. The mono-aromatic metabolites resulting from the hydrolysis of diaryl substrate were further metabolized via catechol. Microbial catabolic activities were solely responsible for the loss of contaminant in the medium as confirmed by abiotic controls. Heterotrophic bacteria can therefore play an important role in the removal of contaminants from marine environments.