CURRENT ~r

Vol. 25 (1992), pp. 63-67

Current Microbiology 9 Springer-Verlag New York Inc. 1992

Anoxygenic Degradation of Aromatic Substances by

Rhodopseudomonas palustris P. Khanna, B. Rajkumar, and N. Jothikumar National Environmental Engineering Research Institute, Jawaharlal Nehru Marg, Nagpur, India

Abstract. Three strains of the phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris were isolated from different environments and were evaluated for their aromatic

degradative potential under phototrophic conditions. All three strains (PFR I, PNR4, and MRL 1) utilized benzoate, 4-hydroxybenzoate, 4-aminobenzoate, 4-aminophenol, cinnamate, ferulate, phloroglucinol, and 4-dimethylaminobenzaldehyde in the absence of exogenous CO2.4-Aminobenzoate and 4-aminophenol served as a carbon and nitrogen source for all the three strains. Utilization of 4-aminophenol was enhanced in the presence of 4-hydroxybenzoate. Salicylate was utilized by PFR1 and MRL1 strains, and phenol was utilized by the MRL1 strain only in the presence of exogenous CO2.

Chemical and petroleum industries discharge a variety of toxic organic wastes, including many aromatic substances, which deplete oxygen in the receiving stream and result in highly anoxygenic conditions. Hence, many studies have been devoted to understanding [he fate of aromatic substances under anoxygenic conditions [1, 3, 5, 15]. Phototrophic purple nonsulfur bacteria (PNSB) have been shown to efficiently degrade toxic aromatic substances, utilizing them as sole carbon sources under anoxygenic conditions, deriving energy from light for growth [4-7, 10, 13, 16, 20]. Rhodopseudomonas palustris has been acknowledged as the most versatile of all PNSB with respect to aromatic degradation [6, 10, 20]. This paper describes the evaluation of three strains of R. palustris isolated from different environments for anoxygenic aromatic degradation.

MgC12 9 6H20, 0.02%; CaC12 9 2H20, 0.02%; Na2S203 9 5H2O, 0.004%; trace elements solution SLA [8], 1 ml/L. Yeast extract (100 mg/L) was added to the medium to suffice for vitamin requirements. Aromatic substances at 2 mM final concentration, unless otherwise mentioned, served as the sole source of carbon. Sodium bicarbonate was not added to the medium. Ammonium chloride was omitted when aminoaromatic substances were included in the BSM. The pH of the medium prior to inoculation was 7.2 -+ 0.05.

Materials :and Methods

Growth measurement. Growth was measured in terms of increase in both dry weight [12] and protein [11]. Total cell protein content was estimated after extraction by boiling in 1 N NaOH for 20 rain.

Bacterial strains. Three isolates of PNSB designated as PFR1, PNR4, and MRL 1 were isolated from the samples collected from paddy field soil near Madras, an industrial wastewater disposal stream in Nagpur, and a petrochemical industry wastewater treatment plant near Madras, respectively, through an enrichment technique that used 4-hydroxybenzoate as the sole carbon source. Isolates were identified as different strains ofR. palustris following Truper and Pfenning's procedure [17] and Bergey's Manual of Systematic Bacteriology [9]. Media. Composition (in wt/vol) of basal salts medium (BSM) was as follows: K2HPO4, 0.085%; KH2PO4, 0.015%; NH4C1, 0.1%;

Growth conditions. All cultures were grown photoheterotrophically under anaerobic light conditions in 30 ml screw-cap tubes and transparent glass bottles. Cells grown in BSM supplemented with sodium acetate (0.2% wt/vol) were harvested by centrifugation at 10,000 g (for 20 min) at 4~ washed, and suspended in phosphate buffer to a final density of 30-40/xg cells dry weight/ ml, and served as the inoculum. Inocula (5% vol/vol) were added to culture vessels containing BSM with 2 m M aromatic substance. Culture vessels were incubated at room temperature (30~ -+ 5~ under continuous incandescent illumination of 1500 lx intensity.

Quantitation and catabolism of aromatic substances. Concentrations of all aromatic substances were estimated from their UV absorbance in growth medium by suitably diluting the culture filtrate in 50 mM phosphate buffer following the establishment of standard curves relating concentration to UV absorbance [20]. At different time intervals during growth, the utilization of aromatic substances was monitored by following the UV absorption spectrum of culture filtrates between 200 and 400 nm in a Hitachi 15020 model spectrophotometer.

Address reprint requests to: Prof. P. Khanna, Director, National Environmental Engineering Research Institute, Jawaharlal Nehru Marg, Nagpur 440 020, India.

64

CURRENT MICROBIOLOGY Vol. 25 (1992)

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Fig. 1. (a) Growth of R. palustris PFRI (O), PNR4 (El), and MRL1 (A) strains on 4aminophenol, utilizing it as both sole carbon and nitrogen source. Residual concentrations of 4-aminophenol during the growth of PFRI (Q), PNR4 ( I ) , and MRL1 (A) strains. (b) The UV absorption spectra of BSM amended with 4aminophenol (as the sole carbon and the nitrogen source) prior to inoculation ( ) and after 30 days of growth of R. palustris PFR1 ( - - - ) under anaerobic light conditions, after appropriate dilution. (c) Growth of R. palustris PFR1 (O), PNR4 ([2]), and MRL1 (&) strains on 4aminophenol in the presence of 4-hydroxybenzoate. Residual concentrations of 4-aminophenol during the growth of PFR1 (O), PNR4 ( I ) , and MRL1 (&) strains and of 4-hydroxybenzoate during the growth of PFR1 ( 0 - - - - ( 3 ) , PNR4 (E3------C]), and MRL1 (&. . . . &) strains.

P. Khanna et al.: Anoxygenic Aromatic Degradation by R. palustris

Results and Discussion T h r e e s t r a i n s o f R. palustris w e r e i s o l a t e d t h r o u g h a n e n r i c h m e n t c u l t u r e t e c h n i q u e a n d w e r e f o u n d to grow on aromatic substances that have not been reported previously. The results of various aromatic substances tested for anoxygenic aromatic degradation by the strains PFR1, PNR4, and MRL1 are p r e s e n t e d in T a b l e 1. T h i s is t h e first r e p o r t to s h o w the utilization of phloroglucinol, salicylate, phenol, 4-aminophenol, 4-aminobenzoate, and 4-dimethyla m i n o b e n z a l d e h y d e b y R. palustris. U t i l i z a t i o n o f a m i n o a r o m a t i c s u b s t a n c e s 4 - a m i n o p h e n o l (Fig. l a a n d b) a n d 4 - a m i n o b e n z o a t e (Fig. 2a a n d 2b) as s o l e c a r b o n as w e l l as n i t r o g e n s o u r c e s is a n i n t e r e s t i n g m e t a b o l i c : c a p a b i l i t y o f all t h r e e s t r a i n s o f R. palustris s t u d i e d . 4 - D i m e t h y l a m i n o b e n z a l d e h y d e was u t i l i z e d b y all t h r e e s t r a i n s as a c a r b o n s o u r c e o n l y , a n d n o t as a n i t r o g e n s o u r c e . A d d i t i o n o f 0.4 m M 4hydroxybenzoate to a 4-aminophenol-supplemented m e d i u m i n c r e a s e d t h e r a t e o f u t i l i z a t i o n o f 4a m i n o p h e n o l (Fig. l c ) . A 4 - a m i n o p h e n o l c o n c e n t r a t i o n a b o v e 1 m M w a s i n h i b i t o r y to t h e c u l t u r e s .

65

Table 1. Utilization of aromatic substances for photoheterotrophic growth of strains ofR. palustris Growth substrate (raM)

PFR1

PNR4

MRL1

4-Aminobenzoate (2) 4-Aminophenol (1) Aniline (1) Benzoate (2) Cinnamate (2) 4-Cresol (1) 4-Dimethylaminobenzaldehyde (2) 2-4,Dimethylphenol (1) Ferulate (2) 2-Hydroxybenzoate (2) [salicylate] 3-Hydroxybenzoate (2) 4-Hydroxybenzoate (2) Phenol (1) Phloroglucinol (2)

++ ++ +++++ +++++ ++

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Fig. 2. (a) Growth ofR. palustris PFR1 (O), PNR4 (23), and MRL1 (A) strains on 4-aminobenzoate utilizing it as both sole carbon and nitrogen source. Residual concentrations of 4-aminobenzoate during the growth of PFR1 (O), PNR4 (ll), and MRL1 (A) strains. (b) The UV absorption spectra of BSM amended with 4-aminobenzoate (as the sole carbon and nitrogen source) prior to inoculation ( ) and after 30 days of growth of R. palustris PFR1 ( - - - ) under anaerobic light conditions, after appropriate dilution.

CURRENT MICROBIOLOGYVO1. 25 (1992)

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P. Khanna et al.: Anoxygenic Aromatic Degradation by R. palustris

There hats been no report on the utilization of 4aminophenol and 4-aminobenzoate as sole carbon as well as nitrogen sources. However, there are reports on 2-aminobenzoate (anthranilic acid) degradation by denitrifying Pseudomonas sp. into N H 2 and CO2 with simultaneous reduction of NO3 to NO{- to N2 gas [2, 21]. NH4C1 inhibited the utilization of aminoaromatic substances by all three strains. Probably this might be the reason for lack of growth on 4aminobenzoate in an earlier study [6]. Phloroglucinol supported good growth of all three strains of R. palustris, as shown in Fig. 3. It was reported earlier [19] that phloroglucinol is catabolized via dihydrophloroglucinol to acetate in R. gelatinosa, a mechanism similar to that found in tureen bacteria [14, 18]. Salicylate supported the growth of PFR1 and MRL1 strains (Fig. 4), while phenol supported the growth of the MRL1 strain only. Phenol above I mM concentration completely inhibited the growth of the MRL1 strain. All three strains did not require exogenous COg for growth on tested aromatic substances (except for phenol utilization by the MRLI strain), unlike the results mentioned in other reports [6, 13, 20]. The results of the present study confirm the potential of R. palustris to degrade several aromatic substances under the laboratory conditions as pure cultures, and indicate the possible role of PNSB in detoxifying and mineralizing toxic aromatic and other organic substances in the natural environment, where diverse physiochemical conditions and organisms are present. The utilization of aminoaromatic substances as sole carbon as well as nitrogen sources by all three strains of R. palustris reveals an important metabolic capability of the bacterium that should be further investigated in detail. The anoxygenic aromatic degradation potential ofR. palustris could be effectively used for the development of an effluent treatment system for detoxifying industrial wastewater by use of sunlight as the energy source. ACKNOWLEDGMENTS

The authors thank Prof. D. Lalithakumari, Centre for Advanced Studies in Botany, University of Madras, and Dr. S. Kamakshiammal, Scientist, NEERI, Madras Zonal Laboratory, for their help and advice with experimental work. B. Rajkumar thanks the Department of Biotechnology, Government of India, for providing a research fellowship.

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2. Braun K, Gibson DT (1984) Anaerobic degradation of 2aminobenzoate (anthranilic acid) by denitrifying bacteria. Appl Environ Microbiol 48:102-107 3. Dagley S (1971) Catabolism of aromatic compounds by microorganisms. Adv Microb Physiol 6:1-46 4. Dutton PL, Evans WC (1969) The metabolism of aromatic compounds by Rhodopseudomonas palustris. Biochem J 113:525-536 5. Evans WC, Fuchs G (1988) Anaerobic degradation of aromatic compounds. Annu Rev Microbiol 42:289-317 6. HarwoodCS, Gibson J (1988) Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris. Appl Environ Microbiol 54:712-717 7. Hegeman GD (1967) The metabolism ofp-hydroxybenzoate by Rhodopseudomonas palustris and its regulation. Arch Microbiol 59:143-148 8. ImhoffJF (1988) Anoxygenic phototrophic bacteria. In: Austin B (ed) Methods in aquatic bacteriology. New York: John Wiley & Sons, pp 207-240 9. Imhoff JF, Truper HG (1989) Purple nonsulfur bacteria. In: Staley JT, Bryant MP, Pfennig N, Holt JG (eds) Bergey's manual of systematic bacteriology, Vol. 3. Baltimore: The Williams and Wilkins Co., pp 1658-1682 10. Kamal VS, Wyndham RC (1990) Anaerobic phototrophic metabolism of 3-chlorobenzoate by Rhodopseudornonas palustris WS 17. Appl Environ Microbiol 56:3871-3873 11. Lowry OH, R0sebrough NJ, Farr AL, Randall RS (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 12. Madigan MT, Gest H (1979) Growth of the photosynthetic bacterium Rhodopseudomonas capsulata chemoautotrophically in darkness with H2 as the energy source. J Bacteriol 137:524-530 13. Madigan MT, Gest H (1988) Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source. FEMS Microbiol Ecol 53:53-58 14. Patel TR, Jure KG, Jones GA (1981) Catabolism ofphloroglucinol by the rumen anaerobe Coprococcus. Appl Environ Microbiol 42:1010-1017 15. Sleat R, Robinson JP (1984) The bacteriology of anaerobic degradation of aromatic compounds. J Appl Bacteriol 57:381-394 16. Tanaka H, Maeda H, Suzuki H, Kamibayashi A, Tonomura K (1982) The metabolism of thiophene-2-carboxylate by a photosynthetic bacterium. Agric Biol Chem 46:1429-1438 17. Truper HG, Pfenning N (1981) Characterization and identification of the anoxygenic phototrophic bacteria. In: Starr MF, Stolp H, Truper HG, Balows A, Schlegel HG (eds) The prokaryotes--a hand book on habitats, isolation and identification of bacteria, Vol 1. New York: Springer-Verlag, pp 299-312 18. Tsai DG, Jones GA (1975) Isolation and identification of rumen bacteria capable of anaerobic phloroglucinol degradation. Can J Microbiol 21:794-801 19. Whittle PJ, Lunt DO, Evans WC (1976) Anaerobic photometabolism of aromatic compounds by Rhodopseudomonas sp. Biochem Soc Trans 4:490-491 20. Wright GE, Madigan MT (1991) Photocatabolism of aromatic compounds by the phototrophic purple bacterium Rhodomicrobium vannielii. Appl Environ Microbiol 57:2069-2073 21. Ziegler K, Braun K, Bockler A, Fuchs G (1987) Studies on the anaerobic degradation of benzoic acid and 2-aminobenzoic acid by a denitrifying Pseudomonas strain. Arch Microbiol 149:62-69