APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 0099-2240/99/$04.00+0 June 1999, p. 2784–2788 Vol. 65, No. 6 Copyright © 1999, American Society for Microbiology. All Rights Reserved. Optimization of Simultaneous Chemical and Biological Mineralization of Perchloroethylene† FATI ˙ H BU ¨ YU ¨ KSO ¨ NMEZ, 1 THOMAS F. HESS, 1 * RONALD L. CRAWFORD, 1 ANDRZEJ PASZCZYNSKI, 1 AND RICHARD J. WATTS 2 Center for Hazardous Waste Remediation Research, University of Idaho, Moscow, Idaho 83844, 1 and Department of Civil and Environmental Engineering, Washington State University, Pullman, Washington 99164 2 Received 20 October 1998/Accepted 10 March 1999 Optimization of the simultaneous chemical and biological mineralization of perchloroethylene (PCE) by modified Fenton’s reagent and Xanthobacter flavus was investigated by using a central composite rotatable experimental design. Concentrations of PCE, hydrogen peroxide, and ferrous iron and the microbial cell number were set as variables. Percent mineralization of PCE to CO 2 was investigated as a response. A second-order, quadratic response surface model was generated and fit the data adequately, with a correlation coefficient of 0.72. Analysis of the results showed that the PCE concentration had no significant effect within the tested boundaries of the model, while the other variables, hydrogen peroxide and iron concentrations and cell number, were significant at  0.05 for the mineralization of PCE. The 14 C radiotracer studies showed that the simultaneous chemical and biological reactions increased the extent of mineralization of PCE by more than 10% over stand-alone Fenton reactions. Halogenated organic chemicals have been introduced into the environment from a variety of sources, including the im- proper disposal of degreasers and solvents (14). Many of the halogenated compounds, such as perchloroethylene (PCE) and carbon tetrachloride, are persistent in the environment due to their resistance to microbial degradation and their toxicity to microorganisms. For the treatment of these recalcitrant com- pounds, a number of chemical processes have been investi- gated, including oxidation by Fenton’s reagent. Compound degradation by Fenton’s reagent may yield (i) partial mineral- ization (15), (ii) lowered toxicity (1), and (iii) increased sus- ceptibility to biodegradation (4). Owing to their ability to lower the toxicity and increase the biodegradability of the parent compounds, chemical reactions have been coupled in sequence with biological reactions and have been the subject of much recent research. An extensive review is available elsewhere (19). However, the investigation of simultaneous chemical and biological transformation processes has received little atten- tion. Simultaneous processes could have both economic and process advantages if applied to industrial pollution or in situ hazardous waste treatment in the environment. Fenton (8) discovered the strong oxidizing power of mix- tures of hydrogen peroxide and ferrous iron solutions. Haber and Weiss (10) later identified the oxidizing element in Fen- ton’s reagent as a hydroxyl radical. The hydroxyl radical is a nonspecific, strong oxidant which reacts with most organic and biological molecules at near diffusion-controlled rates (10 9 M -1 s -1 ) (7, 12). The classic Fenton’s reaction involves the addition of dilute hydrogen peroxide to a degassed, acidic ferrous iron solution, which generates hydroxyl radicals (equa- tion 1). The degradation of organic chemicals by hydroxyl radicals then proceeds via hydroxylation, hydrogen atom ab- straction, or dimerization (21). H 2 O 2 + Fe 2+ 3 Fe 3+ + OH  + OH - (1) Some environmental applications of Fenton’s reagent involve reaction modifications, including the use of high concentra- tions of hydrogen peroxide, the substitution of different cata- lysts such as ferric iron and naturally occurring iron oxides, and the use of phosphate-buffered media and metal-chelating agents. These conditions, although not as stoichiometrically efficient as the standard Fenton’s reactions, are often necessary to treat industrial waste streams and contaminants in soils and groundwater (20). As a part of our overall simultaneous chemical and biolog- ical transformation study, PCE was chosen as a probe com- pound for experimental investigation. PCE was known to be degraded by Fenton’s reagent, yielding dichloroacetic acid (DCAA) (14, 18), a readily biodegradable compound. A major obstacle to combining the chemical and microbial reactions simultaneously was the toxicity of Fenton’s reagent to micro- organisms. We previously showed that the toxic effects of Fen- ton’s reagent were sufficiently reduced by preacclimation of microorganisms to high concentrations of hydrogen peroxide, thereby allowing a significant number of microorganisms to survive throughout the course of treatment (3). In a later study, we investigated coexisting chemical and biological reactions used for mineralization of PCE (2). The results of that study, with 14 C-labeled PCE, showed that the addition of microor- ganisms increased the extent of mineralization of PCE by more than 10% over that of a noninoculated control. This finding suggested that chemical and biological reactions could coexist and might be a viable alternative for the treatment of waste- waters containing PCE. In the present study, we investigated the effects of variables of coexistent chemical and biological reactions (concentrations of PCE, hydrogen peroxide, and fer- rous iron and the initial cell number) on mineralization of PCE. Chemicals. PCE and DCAA-sodium salt were purchased from Aldrich Chemical Co. (Milwaukee, Wis.); 14 C[1-2]-PCE was obtained from Sigma Chemical Co. (St. Louis, Mo.); hy- drogen peroxide (30%) was purchased from Fisher Scientific (Fair Lawn, N.J.); and Ecolite scintillation cocktail was pur- * Corresponding author. Mailing address: Center for Hazardous Waste Remediation and Research, University of Idaho, Moscow, ID 83844-0904. Phone: (208) 885-7461. Fax: (208) 885-7908. E-mail: tfhess @uidaho.edu. † Publication number 99301 of the Idaho Agricultural Experiment Station. 2784 on July 24, 2020 by guest http://aem.asm.org/ Downloaded from