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FEMS Microbiology Letters 61 (1989) 195-198 Published by Elsevier 195 FEMSLE 03693 Selenate reduction by a Pseudornonas species: a new mode of anaerobic respiration J o a n M. Macy, T h o m a s A. Michel a n d D o n a l d G. Kirsch Department of Animal Science, University of California, Davis, CA, U.S.A. Received 9 April 1989 Revision received 9 May 1989 Accepted 10 May 1989 Key words: Pseudomonas sp.; Selenate reduction; Anaerobic respiration 1. SUMMARY The high levels of selenium (selenate, selenite) in agricultural drainage water in the San Joaquin Valley of California, which have led to environmental problems, might be lowered if the selenate/selenite could be reduced to elemental insoluble selenium [1]. Two organisms have been newly isolated which do this in anaerobic coculture. One, a strictly anaerobic, Gram-positive rod, reduces selenite to elemental selenium. The other, a Pseudomonas species, was shown to respire selenate to selenite. Cells grown anaerobically in Minimal Medium on acetate plus selenate oxidized 14C-acetate to 14C02 with concomitant reduction of selenate to selenite and small amounts of elemental selenium. 2. INTRODUCTION Many organisms are known to reduce selenium oxides [for review of literature see 2-4]. No published reports exist, however, suggesting that any Correspondence to: Joan M. Macy, Department of Animal Science, University of California, Davis, CA 95616, U.S.A. selenate- or selenite-reducing organisms are able to use these compounds for anaerobic respiration [2-4]. Since the species of selenium in drainage water are selenate (SeO42-) and selenite (SeO~-), it was important to determine whether organisms exist that can use either of these two compounds to respire when grown under anaerobic conditions. Recently, Oremland and Hollibaugh have shown that selenate might be respired by anaerobic bacteria in sediments [5]. This is the first report of selenate respiration by a pure bacterial culture. 3. MATERIALS AND METHODS Media were prepared according to the method of Hungate [6]. Minimal Medium contained (g/liter; pH 7.2): NaC1, 1.2; KC1, 0.3; NH4C1, 0.3; K H z P O 4 , 0.2; N a z S O 4 , 0.3; MgC12.H20, 0.4; CaC12 • 2H20 , 0.15; NaHCO 3, 0.6; Trace elements 'SL8' [11]; Vitamins (thiamin- HC1, pyridoxine. HC1, niacinamide, pantothenic acid, B12 , biotin, p-aminobenzoic acid, riboflavin, folic acid). Where required medium also contained: ascorbate (0.03%, reducing agent), sodium selenate, 20 mM; sodium acetate, 5 or 20 mM; methanol 20 mM; yeast extract, 0.4%; agar, 2%; medium containing yeast extract is referred to as YE medium. 0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies 196 The Gram-negative rods isolated were identified as Pseudomonas aeruginosa and a Pseudomonas sp. (designated strain AX), using the api 20 E system and rapid N F T system of api Analytab Products, Quebec, Canada. To demonstrate the oxidation of acetate to CO 2 in the presence of selenate, cells were grown 18 h in anaerobic (ga phase: 100% N2) Minimal Medium containing 20 mM sodium acetate, 20 m M sodium selenate and 0.03% ascorbate (reducing agent). Cells were harvested (16000 x g, 20 min), washed once in potassium phosphate (KP) buffer (50 mM, p H 7.4), and suspended in O2-free buffer (KP, 50 mM, pH 7.4, 0.03% ascorbate, gas phase: 100% N2). Aliquots (0.9 ml) of suspension were added (under a stream of N 2 gas) to anaerobic Warburg flasks. Flasks were closed with stoppers. The center well contained 200 /xl of 10% K O H and a 2 × 4 cm filter-paper wick for CO 2 trapping. Flasks were incubated at 28°C. Reactions were started by the addition of 0.1 ml of a solution containing 70 mM selenate and 31.5 mM 14CU-acetate (specific activity: 50 # C i / m m o l ) . Reactions were stopped by placing flasks at 90 o C for 5 min. To achieve maximal trapping of the CO 2 in KOH, flasks were incubated, with shaking (24 h at 60 o C). After cooling to room temperature, radioactive counts in the filter paper wick and K O H were determined by liquid scintillation counting. The counts in 200 ~1 of the reaction mixture were determined in the same way. A further aliquot of the reaction mixture was centrifuged (13000 × g, 40 min), and the amount of radioactivity in 200/~1 of the supernatant was determined. 14C-U-acetate label in poly-fl-hydroxybutyrate (PHB) was determined in a separate experiment. Cells were incubated with 14C-U-acetate for 2 h as described and harvested by centrifugation (20000 x g, 30 min). PHB was extracted according to Smibert [8], dissolved in chloroform and radioactive counts were determined by liquid scintillation counting. To demonstrate the reduction of selenate to selenite, during the oxidation of acetate to CO2, the suspension prepared for the labeling experiment described above was incubated with 3.15 mM acetate and 7.0 mM selenate. Ascorbate was not included in the anaerobic incubation buffer because it interfered with the selenite analyses (see below). The suspension was incubated at 28°C. Portions of the suspension were removed at various times, and the reaction stopped by placing the material at 90 o C, for 5 min. Samples were centrifuged (20000 × g, 30 min). The supernatant was removed and frozen. The supernatant was analyzed for concentrations of selenate, selenite and acetate. Selenate ion and selenite ion were analyzed by single-column ion chromatography (HPLC), using a 4 mM p-hydroxybenzoic acid/sodium borate mobile phase [9]. A preconcentration column (Ani o n / R guard cartridge, Wescan) was used in place of a sample loop. A mobile phase of pH 8.0 was used for the analysis of selenate, while a mobile phase of pH 7.2 was used for the analysis of selenite. Elemental selenium concentration was calculated as the difference between selenate used and selenite formed. Acetate was determined using the acetic acid UV test kit of Boehringer Mannheim. 4. RESULTS A N D DISCUSSION The first step toward demonstration of selenate respiration was the isolation, under anaerobic conditions, of selenate-reducing bacteria. Material from biological reactors for selenium removal [10], was inoculated into anaerobic methanol-acetate enrichment cultures (Minimal medium plus sodium selenate, 20 mM; sodium acetate, 5 mM; methanol, 20 mM). Within a week a red color, indicating the presence of elemental selenium, was visible in the enrichment cultures. The culture was transferred (every two days: 10% inoculum) at least thirty times before organisms from the stable anaerobic enrichment were isolated. Organisms from the enrichment were isolated in YE Medium (plus 2% agar) [8], and three pure cultures were obtained: (1) Pseudomonas sp AX 92) Pseudomonas aeruginosa and (3) a Gram-positive strictly anaerobic rod-shaped bacterium designated strain 'E'. In YE Medium (plus 20 mM sodium selenate), a co-culture of the Pseudomonas sp. plus the strict anaerobe rapidly selenate to elemental selenium. Co-cultures of the P. aeruginosa and the strict anaerobic did not reduce selenate to elemental 197 selenium, thus this p s e u d o m o n a d was not further studied. The anaerobe (strain ' E ' ) grew in anaerobic Y E M e d i u m in the absence of selenate or selenite. Selenate when added was not reduced, selenite when added was reduced to elemental selenium. Characteristics of the anaerobe will be described elsewhere. The Pseudomonas sp. A X only grew in the anaerobic Y E M e d i u m when selenate was present, only small a m o u n t s of elemental selenium were formed. G r o w t h with selenite did not occur. Anaerobic growth with nitrate (20 m M ) also occurred. These results suggest that the Pseudornonas sp. A X respires selenate, forming selenite and that in coculture, selenite formed by the Pseudomonas sp. A X is reduced by the anaerobe to elemental selenium. T o prove rigorously that the Pseudomonas sp. A X can respire using selenate as the terminal electron acceptor, it was first necessary to determine whether the organism can grow anaerobically on acetate plus selenate, in Minimal Medium. Theoretically, this should occur (equation [1]) while complete oxidation of acetate to C O 2 in the absence of an electron acceptor is not possible (equation [2]) CH3COO- +H + +4SeO2- ~ 2 C O 2 +4SeO 2- +2H20 ( A G o, = _ 556 kJ/mol acetate) equation [1] CH3COO- + H + + 2H 2° ---,2CO2 + 4H 2 ( AG °' = - + 112 kJ/mol acetate) equation [2] (Free energies of formation for selenate and selenite f r o m [11]; for the remaining c o m p o u n d s f r o m [12].) G r o w t h of the Pseudomonas sp. A X in anaerobic Minimal M e d i u m with acetate (20 m M ) and selenate (20 m M ) did indeed occur. The organism was subcultured (10% inoculum) at least thirty times. The culture reached an absorbance (600 nm) of roughly 0.4 and the generation time was approximately four hours. After about 24 h, the culture had a slightly red color (i.e., elemental selenium), which was associated with the cells. While growth in Minimal M e d i u m on the nonfermentable c a r b o n source, acetate, implied that respiration with selenate occurred, it was necessary to demonstrate that acetate is oxidized to 320000 240000 160000 80000 0 ' " 0 " ' 15 " " ' 30 " ' 45 " ' 60 ' ' 75 " " ' 90 Time (minutes) Fig. 1. Oxidation of 14C-U-acetate (3.15 mM) to 14CO2 in the presence of selenate (7 mM) by the Pseudomonas sp. AX grown in Minimal Medium plus 20 mM acetate and 20 mM selenate and suspended (0.17 mg protein per ml) in O2-free phosphate buffer (50 mM, pH 7.4). dpm: acetate (A), CO2 (m), cells (e). C O 2 with c o n c o m i t a n t stoichiometric reduction of selenate to selenite (Figs. 1 and 2). As shown in Fig. 1, a large percentage of the acetate was f o u n d associated with the cells; 31.6% of this acetate was recovered in PHB. Acetate not incorporated into ceils was oxidized to C O 2. During the first 50 rain of the experiment, roughly 60% of the added acetate was recovered as CO2; during this time recovery of label was also approximately 90% or higher. After 50 min a m o u n t of label recovered as C O 2 decreased significantly, as did total a m o u n t of label recovered. This is p r o b a b l y due to the difficulty of trapping increasing a m o u n t s of C O 2, resulting in decreased C O 2 trapping efficiency and decreased recovery of label. In Fig. 2 can be seen the reduction of selenate during the oxidation of acetate to C O 2. Small a m o u n t s of elemental selenium were formed during the first 25 min of incubation. Thereafter, selenate was reduced only to selenite, in accordance with the stoichiometry of the reaction as written in equation [1]. 198 ACKNOWLEDGEMENTS 12 We t h a n k J.L. I n g r a h a m , B.W. H o l l o w a y a n d D.R. Lovely for critical review of this m a n u s c r i p t , a n d W. Pfeiffer for helpful discussions. 10 REFERENCES P. ~ 4- 2 0 0 25 50 75 100 125 Time (minutes) Fig. 2. Reduction of selenate (7 raM) to selenite, during the oxidation of acetate (3.15 mM) to CO2 by a suspension of the Pseudornonas sp. AX (see legend Fig. 1). Selenate (o), selenite (A), selenium (n), acetate (/,). T h u s the Pseudomonas sp. A X is able to carry out a n a e r o b i c respiration of selenate, f o r m i n g primarily selenite. This is the first report describing this m o d e of respiration b y a pure bacterial culture. T h e selenate-respiring Pseudomonas sp. A X together with the selenite-reducing a n a e r o b e (strain ' E ' ) m a y prove to be the ideal m e a n s of r e m o v i n g selenium oxides from c o n t a m i n a t e d agricultural drainage water, e n a b l i n g reutilization of the water. [1] Saiki, M.K. and Lowe, T.P. (1987) Arch. Environ. Contam. Toxicol. 16, 657-670. [2] Maiers, D.T., Wichlacz, P.L., Thompson, D.L., Bruhn, D.F. (1988) Appl. Environ. Microbiol. 54, 2591-2593. [3] Burton, G.A., Giddings, T.H., DeBrine, P., Fall, R. (1987) Appl. Environ. Microbiol. 53, 185-188. [4] Doran, J.W. (1982) Adv. Microb. Ecol. 6, 1-32. [5] Oremland, R.S., Hollibaugh, J.T. (1988) Abstract AS51A, Am. Geophys. Union (Dec.). [6] Hungate, R.E. (1970) in Methods in Microbiology (Norris. J.R. and Robbins, D.W., eds.), vol. 3B, pp. 117-132, Academic Press, New York. [7] Pfennig, N., Widdel, F., Triiper, H.G. (1981) in The Prokaryotes (Starr, M.P., Stolp, H., TriPper, H.G., Balows, A., Schlegel, H.G., eds.), vol 1, pp. 926-940, SpringerVerlag, New York. [8] Smibert, R.M., Krieg, N.R. (1981) in Manual of Methods for General Bacteriology, pp, 421-422, American Society for Microbiology. [9] Mehra, H,C. and Frankenberger, W.T. (1988) Chromatographia 25, 585-588. [10] Groves, R., Greenaway, P., Smith, B., Kovac, K., Taylor, E. (1988) Performance Evaluation of Research Pilot Plant for Selenium Removal; California Department of Water Resources (April). [111 Mc Keown, B. and Marinas B. (1986) in Symposium on Selenium in the Environment, pp. 7-15, CSU, Fresno. [12] Thauer, R.K., Jungermann, K., Decker, K. (1977) Bacteriol. Rev. 41, 100-180.