Relationship between Hydrogen Consumption, Dehalogenation, and ...

Vol. 57, No. 7

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1991, P. 1929-1934

0099-2240/91/071929-06$02.00/0 Copyright C) 1991, American Society for Microbiology

Relationship between Hydrogen Consumption, Dehalogenation, and the Reduction of Sulfur Oxyanions by Desulfomonile tiedjei KIM A. DEWEERD, FRANK CONCANNON,

AND

JOSEPH M. SUFLITA*

Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019 Received 15 January 1991/Accepted 13 April 1991

Resting-cell suspensions of Desulfomonile tiedjei consumed H2 with 3-chloro-, 3-bromo-, and 3-iodobenzoate electron acceptors with rates of 0.50, 0.44, and 0.04 ,umol h-1 mg-I, respectively. However, benzoate and 3-fluorobenzoate were not metabolized by this bacterium. In addition, H2 uptake was at least fourfold faster when sulfate, sulfite, or thiosulfate was available as the electron acceptor instead of a haloaromatic substrate. When sulfite and 3-chlorobenzoate were both available for this purpose, the rate of H2 uptake by D. tiedjei was intermediate between that obtained with either electron acceptor alone. Hydrogen concentrations were reduced to comparably low levels when either 3-chlorobenzoate, sulfate, or sulfite was available as an electron acceptor, but significantly less H2 depletion was evident with benzoate or nitrate. Rates of 3-chlorobenzoate dechlorination increased from an endogenous rate of 14.5 to 17.1, 74.0, 81.1, and 82.3 nmol h-1 mg-' with acetate, pyruvate, H2, and formate, respectively, as the electron donors. Sulfite and thiosulfate inhibited dehalogenation, but sulfate and NaCl had no effect. Dehalogenation and H2 metabolism were also inhibited by acetylene, molybdate, selenate, and metronidazole. Sulfite reduction and dehalogenation were inhibited by the same respiratory inhibitors. These results suggest that the reduction of sulfite and dehalogenation may share part of the same electron transport chain. The kinetics of H2 consumption and the direct inhibition of dehalogenation by sulfite and thiosulfate in D. tiedjei cells clearly indicate that the reduction of sulfur oxyanions is favored over aryl dehalogenation for the removal of reducing equivalents under anaerobic conditions. Such findings confirm that dehalogenation represents a novel type of anaerobic respiration and may help explain why the process is slower in sulfate-rich environments.

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Aryl reductive dehalogenation is the initial and often the rate-limiting reaction for the biodegradation of halogenated aromatic compounds in anoxic habitats (15). Anaerobic dehalogenation reactions have been studied in various habitats and are generally found in environmental samples where methanogenesis occurs, but they are found less frequently when sulfate- or nitrate-reducing conditions predominate (9, 10, 15-17, 22). In fact, the presence of sulfur oxyanions has been shown to inhibit dehalogenation in environmental samples or by a dehalogenating anaerobe, Desulfomonile tiedjei (5, 9, 10, 16, 17, 22). Earlier work has also shown that aryl reductive dehalogenation reactions are enhanced in anoxic aquifer slurries by the addition of organic acids and alcohols (10, 16). It was hypothesized that this stimulation was due to a transient increase in H2 concentrations in the slurries and that H2 was an electron donor for dehalogenation (16). Similarly, H2 was the proposed electron donor for dehalogenation by a reconstructed consortium containing D. tiedjei because of an apparent change in electron flow from methanogenesis to dechlorination when 3-chlorobenzoate was present (7). However, H2 inhibited dehalogenation by D. tiedjei when the cell was cultured with pyruvate and 3-chlorobenzoate in a complex medium amended with rumen fluid (17). Recent reports now show that slow growth of D. tiedjei can occur in a mineral salts medium amended with acetate and 3-chlorobenzoate with either H2 or formate as the electron donor (6, 18). These results suggested that the oxidation of H2, formate, or acetate coupled to dehalogenation could supply sufficient energy for growth of D. tiedjei (6, 18). However, results obtained with growing cultures and complex consortia fail to *

distinguish clearly the effect that these electron donors and alternate electron acceptors have on the growth of the requisite bacteria, the induction of presumed dehalogenating enzymes, or the electron transfer reactions during dehalogenation. To separate growth and induction from metabolism, we used resting-cell suspensions of D. tiedjei to monitor both H2 consumption and dehalogenation activity in the presence of various electron donors and acceptors. MATERIALS AND METHODS Growth of the culture. D. tiedjei was grown with 40 mM pyruvate, 2 mM 3-chlorobenzoate, and 0.05% yeast extract in a defined medium that was described previously (3). Liquid samples were periodically taken to monitor the dehalogenation of 3-chlorobenzoate by high-pressure liquid chromatography (HPLC). For resting-cell experiments, actively dehalogenating cultures of D. tiedjei in the early stationary phase were harvested by centrifugation at 15,000 x g for 20 min at 4°C. Washed-cell preparations. Cells were harvested as indicated above, and the pellet was suspended in anoxic PIPES [piperazine-N,N'-bis-(2-ethanesulfonic acid)] buffer (50 mM, pH 7.0), centrifuged, and suspended in the same buffer at a concentration of 0.2 g (wet weight) per ml of buffer. The washed-cell suspensions were prepared with all materials inside an anaerobic glove box (Coy Laboratories, Ann Arbor, Mich.). Portions of the cell suspension (0.3 ml) were added to 45 ml of the same buffer in 60-ml serum bottles, which were subsequently sealed with black rubber stoppers. The bottles were then removed from the glove box; the headspace in each was evacuated, replaced with 100% N2 gas three times, and set to 70 kPa overpressure unless otherwise indicated. Each serum bottle was amended with

Corresponding author. 1929

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APPL. ENVIRON. MICROBIOL.

DEWEERD ET AL.

anoxic, filter-sterilized, stock solution of either benzoate, 3-fluorobenzoate, 3-chlorobenzoate, 3-bromobenzoate, 3-iodobenzoate, Na2SO3, Na2SO4, Na2S203, or Na2S204 to give an initial concentration of 2 mM. Other compounds and their concentrations used in various experiments included NaCl (4 mM), formate (5 mM), acetate (5 mM), and pyruvate (5 mM) and the metabolic inhibitors acetylene (100%), metronidazole (1 mM), Na2MoO4 (2 mM), and Na2SeO4 (2 mM). For the acetylene inhibition experiment, the concentrated cell suspension was preincubated under an acetylene atmosphere for 30 min to inhibit hydrogenase activity before the cells were dispensed. Serum bottles without added H2 were pressurized with N2 gas at 70 kPa. Each bottle containing the concentrated cell suspension and various additions was incubated for 30 min at 35°C. Samples were taken periodically by syringe, and the concentrations of halogenated substrates, benzoate, and sulfur oxyanions in spent culture fluids were determined by using HPLC procedures as previously described (1, 5, 9). Analytical. The gas pressure in each bottle was monitored by using a pressure transducer (Omega Series PX136; Omega Engineering, Inc., Stamford, Conn.) fixed to an 18-gauge needle, which was inserted through the rubber stopper. Each bottle was initially vented to atmospheric pressure, and the transducer output (millivolts) was recorded. Either H2 or N2 gas (5 ml) was added to the serum bottle by syringe to give an initial positive pressure. Each bottle was then incubated at 35°C with agitation in an orbital shaker (3/8-in. [ca. 0.93-cm] metric radius) at 200 rpm.

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Autoclaved controls maintained a constant positive pressure and did not exhibit dehalogenation activity, confirming that the pressure change in nonsterile bottles was the result of bacterial activity. The headspace gas was electronically monitored every 30 or 60 min by a computer-controlled device designed specifically for that purpose. The pressure change in the bottles was proportional to the amount of headspace gas evolved or consumed. The pressure transducer used for the experiment had an electrical output that was proportional to the gas pressure in the serum bottle (1 mV/kPa). The changes in transducer output were processed through a switching circuit and digital-analog input-output module to the computer. The computer was used to initiate the time schedule for recording the electrical output from each transducer and also as a work station for data collection and analysis. A standard curve of injected H2 versus pressure confirmed that the transducer response was linear up to 100 kPa of overpressure. The rate of H2 consumption was determined by converting the gas pressure measurements into the molar quantity of H2 used per minute and normalizing this rate to the amount of total cellular protein present in bottles. The concentration of H2 in serum bottles attained by incubation of cells with various electron donors and acceptors was determined with a gas chromatograph equipped with either a thermal conductivity detector (>500 ppm of H2) or a mercury vapor detector (