Dept. of Environmental Science and Engineering
Oregon Graduate Institute of Science & Technology
Reduction reactions contribute to a significant portion of the chemical transformations that take place in anaerobic sediments. While these processes are known to be strongly linked to microbial activity, little is known about the specific reducing agents responsible for contaminant reduction. One approach to identifying these reductants is to use molecular probes that give reaction products (or kinetics) that are diagnostic for particular reaction mechanisms. A method using 2-chloroacetophenone (2-CAP) has been developed for this purpose. 2-CAP is reduced by electron transfer to form acetophenone (AcPh) and by hydride transfer to form 2-chloro-1-phenylethanol (2-CPE). AcPh can be further reduced by hydride transfer to form 1-phenylethanol (1-PE). Using an appropriate extraction technique and high-performance liquid chromatography (HPLC), 2-CAP and its reduction products can be detected and quantified in anaerobic sediments. The results of numerous batch experiments consistently showed that 2-CPE formation is favored over AcPh production in sediment collected from a tributary of Rock Creek in Beaverton, Oregon. AcPh reduction to 1-PE was rarely observed and control experiments showed that the kinetics of this reaction are very slow in comparison to 2-CAP reduction. 2-CAP reduction and product formation appear to follow first-order reaction kinetics. However, further analysis revealed that the observed kinetics actually represent the combined effects of adsorption and reduction processes. Detailed kinetic modeling will be necessary to fit both adsorption and reduction data simultaneously. Model systems allow the 2-CAP probe response to be calibrated in ways that may eventually allow the probe to be used to identify particular reductants. In the presence of NADPH and Thermoanaerobium brockii alcohol dehydrogenase, 2-CAP has been shown to react by hydride transfer to form 2-CPE. In the absence of the enzyme, 2-CAP was not reduced. Previous studies in sediments have shown that production of 2-CPE is temperature dependent and distribution of the 2-CPE stereoisomers suggests that the reducing agents are enantioselective. Both phenomenon are indicative of an enzyme-mediated reaction. To further validate the notion of an enzyme-mediated transformation, the effects of enzyme inhibitors (p-chloromercuribenzoate, 1-formylpiperidne, and pyrazole) on 2-CAP reduction were studied in an NADPH/dehydrogenase model system. The results showed small, but consistent, decreases in the rate of 2-CAP reduction with increasing inhibitor concentration. When applied to sediment, all three inhibitors caused a decrease in both the rate of 2-CPE formation and the amount of 2-CPE formed, but had only minor effects on 2-CAP reduction. In contrast, product inhibition, employing the addition of 2-CPE, successfully inhibited 2-CAP reduction, causing a decrease in the rate of reduction with increasing inhibitor concentration. The results obtained with these inhibitors are further evidence that reduction of 2-CAP in sediment is an enzyme-mediated process. Additional studies were performed using 2-bromoacetophenone (2-BAP) and 2, 2- dichloroacetophenone (2,2-CAP) as complimentary probes to 2-CAP. Both compounds were reduced by hydride and electron transfer in Rock Creek tributary sediment. Preferential production of AcPh from 2-BAP, in a system where hydride activity is apparently high (based on observed 2-CAP reduction trends), may provide evidence for the presence of NAD(P)H which is capable of reducing 2-BAP external to a dehydrogenase enzyme.
Reilkoff, Thea E., "2-Chloroacetophenone as a probe compound for studying reduction reactions in anaerobic sediments" (2000). Scholar Archive. 172.