KINETICS OF P-NITROPHENOL DEGRADATION BY PSEUDOMONAS SP - AN EXPERIMENT ILLUSTRATING BIOREMEDIATION

Toxic chemical pollutants such as trichloroethylene (TCE), polychlorin ated biphenyls (PCBs), and petroleum hydrocarbons have been released i nto the environment for years asa result of industrial, agricultural, and other commercial activities. Among the many efforts directed at re mediating contaminated sites, bioremediation is a growing technology t hat uses microorganisms to break down environmental pollutants to nont oxic by-products. According to the Organization for Economic Cooperati on & Development, by the year 2000 the worldwide market demand for bio remediation technologies will reach an estimated $75 billion (1). Beca use of the multidisciplinary nature of bioremediation and its potentia l societal importance, the subject should be of interest to a broad sp ectrum of engineering and science students. In this paper, we describe an experiment developed for a senior-level undergraduate class in env ironmental engineering where students studied the biodegradation of an important environmental contaminant, p-nitrophenol or ''PNP''. PNP is a toxic derivative of the insecticide parathion (O,O-diethyl-o-p-nitr ophenyl phosphorothioate), which has been used in a number of countrie s for controlling agricultural pests (2) and mosquito populations (3). A proposed biochemical pathway for the natural breakdown of parathion is shown in Figure 1. After parathion is released, bacteria naturally present in soil and water hydrolyze the insecticide to PNP and an org anic acid (diethylthiophosphoric acid). Bacterial strains have been is olated from PNP-contaminated sites, and several of these strains can c atalyze the ring hydroxylation of PNP to 4-nitrocatechol (4-NC) (see F ig. 1) (4). In a series of steps, 4-NC is broken down by naturally occ urring microbial consortia to CO2 and nitrate (4, 5). This laboratory exercise investigated the stoichiometry and kinetics of the hydroxylat ion of PNP to 4-NC in a batch system containing the bacterial strain P seudomonas sp. ATCC 29354. Several features of this system make it uni quely suited for undergraduate study: (i) both the substrate (PNP) and product (4-NC) can be assayed with a spectrophotometer; (ii) the stoi chiometry of the reaction can be quantified over time because 4-NC rem ains in solution as a stable intermediate; (iii) the reaction is relat ively rapid, so that six to eight hours of laboratory time should be s ufficient for most students to carry out the experiment; (iv) in the a bsence of microbial attack, PNP is not lost to wall adsorption, volati lization, or other nonconservative processes; and (v) the bacterial st rain used in this study is easy to grow easy to assay, and nonpathogen ic.