APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1990, 0099-2240/90/123871-03$02.00/0 Copyright © 1990, American Society for Microbiology

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Vol. 56, No. 12

Anaerobic Phototrophic Metabolism of 3-Chlorobenzoate by Rhodopseudomonas palustris WS17 VARSHA S. KAMAL

AND

R. CAMPBELL WYNDHAM*

Institute of Biology, Carleton University, Ottawa, Ontario, Canada KJS 5B6 Received 13 April 1990/Accepted 30 September 1990

A mixed phototrophic culture was found to reductively metabolize 3-chlorobenzoate in the presence of benzoate following adaptation for a period of 7 weeks. The dominant bacterial isolate from this mixed culture, identified as Rhodopseudomonas palustris WS17, metabolized 3-chlorobenzoate completely in the presence of benzoate and light and in the absence of oxygen. ['4C]3-chlorobenzoate was converted to 14CO2 and cell biomass. The results of tests conducted on several other isolates of R. palustris indicated that adaptation to 3-chlorobenzoate metabolism is a common phenomenon in this species.

the type strain of R. palustris ATCC 17001 were cultivated in a complex medium at 25°C in screw-cap culture bottles or tubes under anaerobic conditions in the presence of light. Complex medium P contained the following (in milligrams per liter): K2HPO4, 1,710; NaH2PO4, 340; NH4Cl, 500; MgSO4, 400; CaCl2, 50; NaCl, 500; yeast extract (Difco), 500; 10 ml of a 100x concentrated trace element stock solution. Benzoate (1 mM) and 3-chlorobenzoate (0.5 mM) were added as carbon sources, and their concentrations in cell-free media were determined by high pressure liquid chromatography (HPLC; Waters 501/UV-481 detector at 254 nm, with a C18 column and methanol-water-acetic acid [50:49.5:0.5] solvent). Cultures were illuminated with a 250-W infrared-red lamp at a distance of 1 m. Growth was determined by measuring the optical density at 660 nm and total protein (3). Enrichment of a mixed, phototrophic, dehalogenating culture. Benzoate degradation in the mixed, phototrophic culture began immediately following each transfer, reaching a maximum rate of 0.4 mM per day (Fig. 1). 3-Chlorobenzoate metabolism occurred only after the culture was acclimatized for 7 weeks through two transfers. The maximum 3-chlorobenzoate degradation rate achieved in the mixed, dehalogenating culture was 0.23 mM per day. The culture showed enrichment of the purple non-sulfur group of bacteria, in particular, cell types that resembled R. palustris in morphology and in the production of swarmer cells. Characterization of the dehalogenating culture. Anaerobic plate counts on medium P or on ATCC yeast extract medium 112 incubated in GasPaks (BBL Microbiology Systems) in the presence of light yielded red-pigmented isolates WS8, a nonmotile coccus; WS9, a Rhodospirillum species; and WS17, a Rhodopseudomonas species. When these isolates were grown as pure cultures in liquid medium P, only WS17 exhibited significant degradation of the aromatic substrates. Isolate WS17 was a gram-negative bacterium with a budding life cycle characteristic of R. palustris. Transmission electron micrographs of WS17 (Fig. 2) showed the lamellar structure of photosynthetic membranes lying parallel to the cytoplasmic membrane. These characteristics and growth with benzoate identified WS17 as R. palustris. Requirements for 3-chlorobenzoate metabolism by R. palustris WS17. 3-Chlorobenzoate metabolism by WS17 growing under aerobic-light, aerobic-dark, and anaerobic-light conditions in medium P is shown in Fig. 3. The halogenated

Chlorinated aromatics are pollutants of major concern because of their toxicity and recalcitrance. Microorganisms have, rarely, evolved effective means of metabolizing these compounds; however, both aerobic and anaerobic microbial communities have been found to degrade haloaromatics in the environment. In contrast to the aerobic pathways of metabolism, the mechanisms of anaerobic metabolism of chloroaromatics are not well understood. The reasons for this include the difficulty of isolating and cultivating pure cultures of anaerobes able to dehalogenate and grow on chlorinated aromatics, and the apparent lability of reductive aromatic catabolic enzymes. There are now many anaerobic dehalogenation studies in the literature dealing with consortia of methanogens and the sulfate- and nitrate-reducing bacteria (9, 12, 13, 15, 17, 18), but there are relatively few descriptions of pure cultures able to metabolize halogenated substrates. The latter include denitrifying bacteria (2, 11, 16, 19) and a sulfate-reducing bacterium, DCB-1 (14). The earliest studies of anaerobic biodegradation of aromatics were conducted with the photosynthetic bacterium Rhodopseudomonas palustris. Dutton and Evans suggested a reductive ring cleavage pathway through cyclohexane carboxylate and pimelic acid (4, 5). Several researchers have supported this general mechanism, with the additional observation that intermediates in the pathway were conjugated to coenzyme A (1, 6, 7, 11, 19). The range of substrates metabolized by R. palustris was recently extended to many phenolic, dihydroxylated, and methoxylated aromatic acids as well as aromatic aldehydes, making R. palustris one of the most versatile of the anaerobic bacteria with respect to anaerobic, aromatic degradation (8). In this report we extend the range of known substrates for R. palustris to include 3-chloro- and 3-bromo benzoate, adding another phenotype to the known range of bacterial metabolic types capable of reductive dehalogenation of aromatic compounds. Media, culture conditions, and analytical methods. A mixed anaerobic culture of bacteria dominated by phototrophic purple sulfur and non-sulfur bacteria was kindly provided by Wilfred Hahn and Virginia Corbett (Activite Biotechnology Development Corporation, Ottawa, Ontario, Canada). The culture was enriched from sediments with no prior exposure to halogenated aromatics. The mixed culture, isolates, and *

Corresponding author. 3871

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Days FIG. 1. Benzoate (0) and 3-chlorobenzoate (40) degradation by a mixed, phototrophic culture. The times of transfer of 10% of the culture to fresh medium are indicated by vertical arrows.

substrate was metabolized by WS17 with benzoate as a cosubstrate only under anaerobic-light conditions. There was no 3-chlorobenzoate degradation under aerobic conditions. Similar experiments conducted with medium P lacking benzoate resulted in growth of the culture on the yeast extract component, but no 3-chlorobenzoate degradation occurred (data not presented). This result indicates the

FIG. 2. Transmission electron micrograph of R. palustris WS17, fixed with 4% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in ethanol and then propylene oxide, and embedded in Epon. Thin sections were observed in a Philips 400 microscope. A longitudinal section showing the photosynthetic membrane (PM) lying parallel to the cytoplasmic membrane and what are most likely carboxysomes (CS).

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Days FIG. 3. Influence of cosubstrate and culture conditions on the degradation of 3-chlorobenzoate by R. palustris WS17. Growth was carried out in medium P in the presence of benzoate under anaerobic-light (0), aerobic-light (A), and aerobic-dark (A) conditions.

necessity for benzoate as a cosubstrate for complete 3-chlorobenzoate degradation. Uptake and mineralization of ['4C]3-chlorobenzoate. Metabolism of 3-chlorobenzoate by WS17 was confirmed by supplementing medium P with 0.5 mM [ring-U-14C]3-chlorobenzoate (Sigma Radiochemicals; final activity, 2 MBq/ mmol). Analysis was done on replicate 13.5-ml serumstoppered tubes by removing 1 ml of the culture, filtering it through polycarbonate filters, washing, and measuring the activity in an LKB Beta-Rak scintillation counter. The filtrate from the sample was used in HPLC analysis for counting the remaining substrate. 1'4CO2 from the remaining culture volume was trapped in 1 ml of ethanolamine by double-vial respirometry. All counts were corrected on a per-culture-volume (13.5 ml) basis. The rate of metabolism of 3-chlorobenzoate increased to a maximum of 0.045 mM per day between 3 and 7 days of incubation, with corresponding increases in incorporation of label into biomass and 14CO2 (Fig. 4). The label found in the CO2 fraction reached a maximum after 6 days and then remained constant. This may be explained by the fact that 14CO2 produced from [14C]3-chlorobenzoate was simultaneously fixed by the growing cells. A similar reduction in CO2 yield from 0.7 to 0.5 mol/mol of succinate in Rhodospirillum rubrum was reported by Ormerod (10). At the end of the growth phase, approximately 75% of the ring carbon atoms of 3-chlorobenzoate were incorporated into cellular biomass, while some 20% were mineralized to 14CO2. Incorporation of this large proportion of organic carbon into biomass may be due to the assimilation of 14C-labeled intermediates into biomass, the simultaneous fixation of 14CO2, or irreversible binding of reactive intermediates of chlorobenzoate metabolism with cellular constituents. We have not yet observed labeled metabolites of 3-chlorobenzoate in HPLC profiles. The results for the metabolism and assimilation of 3-chlorobenzoate indicate that the chlorinated substrate can serve as a significant source of carbon for biosynthesis by R. palustris WS17 (Fig. 3 and 4). That this may represent a selective advantage for WS17 or other R. palustris strains is evident in the enrichment for dehalogenating populations

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This work was carried out with the support of the Natural Sciences and Engineering Research Council of Canada.

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FIG. 4. [ring-U-14C]3-chlorobenzoate metabolism by R. palustris WS17. Activity is expressed as counts per minute per incubation tube (13.5-ml culture volume) for [14C]3-chlorobenzoate (0), 14C in cell biomass (A), and 14CO2 released into the medium (A).

that we observed in the mixed culture (Fig. 1). A selective advantage to chlorobenzoate metabolism may exist in the form of additional reductant for phototrophic growth or additional carbon or both. We have observed the growth of R. palustris WS17 on plates in a diffusion gradient of 3-chlorobenzoate by using the auxanography technique (8); however, the chlorinated compound alone was a poor growth substrate relative to benzoate. Metabolism of chlorobenzoate may require benzoate to act as an inducer of the reductive ring fission pathway in R. palustris. Halogenated aromatic metabolism in R. palustris. The distribution of the 3-chlorobenzoate metabolic phenotype in R. palustris was examined in several isolates. The ATCC 17001 strain of R. palustris, inoculated in medium P and grown anaerobically in the light, metabolized 1 mM benzoate but did not photometabolize 3-chlorobenzoate (data not shown). No attempt was made to adapt this culture through exposure to 3-chlorobenzoate. However, several independent isolates of R. palustris, enriched on medium P with 0.5 mM benzoate and 0.5 mM 3-chlorobenzoate from greenhouse pond water, were able to photometabolize the chlorinated substrate at rates similar to that of isolate WS17 (data not shown). The mixed phototrophic culture enriched with 3-chlorobenzoate (Fig. 1) and R. palustris WS17 were also capable of complete degradation of 3-bromobenzoate. These results indicate that metabolism of halogenated aromatics by R. palustris may not be a rare phenomenon. Wastewaters held in lagoons which are open to sunlight and which are receiving high loadings of organic matter, such as pulp mill effluents, food manufacturing waste streams, and detergent wastes, often support copious mats of phototrophic bacteria. In these environments complex mixtures of aromatic substrates, in some cases including halogenated aromatics, may be important substrates for phototrophic growth. R. palustris may contribute to the removal of chlorinated aromatics under these conditions. We gratefully acknowledge the contribution of Wilfred Hahn, who instrumental in focusing our attention on the phototrophic bacteria and their metabolic diversity. was

LITERATURE CITED 1. Balba, M. T., and W. C. Evans. 1977. The methanogenic fermentation of aromatic substrates. Biochem. Soc. Trans. 5:302-304. 2. Blake, C. L., and G. D. Hegeman. 1987. Plasmid pCBI carries genes for anaerobic benzoate catabolism in Alcaligenes xylosoxidans subsp. denitrificans PN-1. J. Bacteriol. 169:4878-4883. 3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 4. Dutton, P. L., and W. C. Evans. 1968. The photometabolism of benzoic acid by Rhodopseudomonas palustris: a new reductive pathway. Biochem. J. 109:5p. 5. Dutton, P. L., and W. C. Evans. 1969. The metabolism of aromatic compounds by Rhodopseudomonas palustris. Biochem. J. 113:525-535. 6. Guyer, M., and G. Hegeman. 1969. Evidence for a reductive pathway for the anaerobic metabolism of benzoate. J. Bacteriol. 99:906-907. 7. Harwood, C. S., and J. Gibson. 1986. Uptake of benzoate by Rhodopseudomonas palustris grown anaerobically in light. J. Bacteriol. 165:504-509. 8. Harwood, C. S., and J. Gibson. 1988. Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris. Appl. Environ. Microbiol. 54:712-717. 9. Horowitz, A., J. M. Suflita, and J. M. Tiedje. 1983. Reductive dehalogenation of halobenzoates by anaerobic lake sediment microorganisms. Appl. Environ. Microbiol. 45:1459-1465. 10. Ormerod, J. G. 1956. The use of radioactive carbon dioxide in the measurement of carbon dioxide fixation in Rhodospirillum rubrum. Biochem. J. 64:373-380. 11. Schennen, U., K. Braun, and H.-J. Knackmuss. 1985. Anaerobic degradation of 2-fluorobenzoate by benzoate-degrading, denitrifying bacteria. J. Bacteriol. 161:321-325. 12. Sharak Genthner, B. R., W. A. Price II, and P. H. Pritchard. 1989. Anaerobic degradation of chloroaromatic compounds in aquatic sediments under a variety of enrichment conditions. Appl. Environ. Microbiol. 55:1466-1471. 13. Sharak Genthner, B. R., W. A. Price II, and P. H. Pritchard. 1989. Characterization of anaerobic dechlorinating consortia derived from aquatic sediments. Appl. Environ. Microbiol. 55:1472-1476. 14. Shelton, D. R., and J. M. Tiedje. 1984. Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acid. Appl. Environ. Microbiol. 48:840-848. 15. Suflita, J. M., A. Horowitz, D. R. Shelton, and J. M. Tiedje. 1982. Dehalogenation: a novel pathway for the anaerobic biodegradation of haloaromatic compounds. Science 218:11151117. 16. Taylor, B. F., W. L. Hearn, and S. Pincus. 1979. Metabolism of monofluoro- and monochlorobenzoates by a denitrifying bacterium. Arch. Microbiol. 122:301-306. 17. Tiedje, J. M., S. A. Boyd, and B. Z. Fathepure. 1987. Anaerobic degradation of chlorinated aromatic hydrocarbons. Dev. Ind. Microbiol. 27:117-127. 18. Urbanek, M., T. Strycek, C. Wyndham, and M. Goldner. 1989. Use of a bromobenzoate for cross-adaptation of anaerobic bacteria in Lake Ontario sediments for degradation of chlorinated aromatics. Lett. Appl. Microbiol. 9:191-194. 19. Williams, R. J., and W. C. Evans. 1975. The metabolism of benzoate by Moraxella species through anaerobic nitrate respiration. Biochem. J. 148:1-10.

Anaerobic phototrophic metabolism of 3-chlorobenzoate by Rhodopseudomonas palustris WS17.

A mixed phototrophic culture was found to reductively metabolize 3-chlorobenzoate in the presence of benzoate following adaptation for a period of 7 w...
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