pyrazinoic acid, pyrazinamide, nicotinic acid, and nicoti- namide). However, the test tubes with the heterocyclic com- pounds were continuously exposed to light ...
Photometabolism of Heterocyclic Aromatic Compounds by Rhodopseudomonas palustris OU 11 C. SASIKALA,* C. V. RAMANA, AND P. RAGHUVEER RAO Microbial Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad 500 007, India Received 19 January 1994/Accepted 9 April 1994
Rhodopseudomonas palustris OU 11 (ATCC 51186; DSM 7375) isolated from a pond of chemical industry effluent could anaerobically photometabolize heterocyclic aromatic compounds belonging to the pyridine and pyrazine groups only after a period of adaptation on pyrazinoic acid of 5 to 6 weeks. Growth on heterocyclic compounds was light dependent. The effects of various concentrations of heterocyclic compounds on growth suggest that higher concentrations of these compounds inhibit growth and are toxic.
Anoxygenic phototrophic bacteria (APB) are nutritionally versatile organisms capable of growth on substances ranging from simple aliphatic organic acids to complex polysaccharides. A number of aromatic compounds are also known to serve as the sole carbon source for growth (2), which is of special interest from the environmental pollution point of view with special respect to industrially produced organic compounds. In nature, nonsulfur purple bacteria seem to be ideal candidates for aromatic compound catabolism because, unlike heterotrophic organisms, phototrophic bacteria do not require energy from catabolism of aromatic compounds (12). Not many purple nonsulfur bacteria are known to degrade aromatic compounds. Rhodopseudomonas palustris (9), Rhodospirillum fulvum (14), and Rhodocyclus purpureus (13) are known to degrade benzoate, which is a universal property for differentiating these species from others of the same genera. Other species known to degrade aromatic compounds are Rhodomicrobium vannielii (20), Rhodocyclus gelatinosus (6), and Rhodobacter capsulatus (in the presence of acetic acid) (4). Aromatic compounds reported to be used by various APB for growth belong to the homocyclic group of compounds (6-12, 15, 16, 20). The only report of growth on heterocyclic aromatic compounds among APB was by Rhodobacter capsulatus degrading purine anaerobically with urea as an intermediate (1, 5); however, purines could serve only as the nitrogen source rather than as the sole carbon source. Rhodopseudomonas palustris failed to grow on the heterocyclic aromatic compound nicotinate (9), while although it could not use thiophene-2-carboxylate as a growth-supporting substrate, washed cell suspensions under anaerobic conditions were able to transform it to (+)-3-hydroxytetrahydrothiophene-2-carboxylate and tetrahydrothiophene-2-carboxylate (18). To the best of our knowledge there are no reports available on the use of heterocyclic aromatic compounds as the sole carbon source by APB in general and by Rhodopseudomonas palustris in particular. In the present communication we report for the first time the growth of Rhodopseudomonas palustris on heterocyclic compounds as the sole organic carbon source. Organism, media, cultural conditions, and analytical methods. Rhodopseudomonas palustris OU 11 (ATCC 51186 and DSM 7375) isolated from a soil sample from a pond of an industrial effluent at Hyderabad, India, was used in the present investigation. It was grown photoheterotrophically in Biebl and *
Pfennig's (3) mineral medium supplemented with an organic carbon source and ammonium chloride (7 mM) as the nitrogen source. The light intensity, temperature, and initial pH for growth were 2,400 lx, 30 ± 2°C, and 7.0, respectively. Growth was carried out in fully filled screw-cap test tubes (15 by 125 mm) and was measured in terms ofA660, and biomass yield was calculated from a graph of absorbance versus dry weight (0.1 absorbance = 0.08 mg [dry weight]/ml). The concentration of heterocyclic aromatic compounds in the medium was estimated spectrophotometrically from their UV absorption after separation of the cells by centrifugation (at 12,000 rpm for 10 min with a Remi centrifuge) and suitable dilutions. Adaptation of Rhodopseudomonas palustris to photometabolize heterocyclic aromatic compounds. While studying the aromatic (both homo- and heterocyclic) degradation potential of the local isolate of Rhodopseudomonas palustris OU 11, it was observed that the bacterium could utilize a variety of homocyclic aromatic compounds for growth (after 6 days of anaerobic incubation) but could not simultaneously utilize any of the heterocyclic aromatic compounds tested (pyrazine, pyrazinoic acid, pyrazinamide, nicotinic acid, and nicotinamide). However, the test tubes with the heterocyclic compounds were continuously exposed to light and the absorbance was observed every day. After about 5 to 6 weeks, growth appeared only in tubes with pyrazinoic acid, and even 15 weeks of incubation did not result in growth in tubes with other
TABLE 1. Utilization of heterocyclic aromatic compounds for photoheterotrophic growth of Rhodopseudomonas palustris OU l la (0.5 mM)
A660
Growthb
Generation
Pyridine Nicotinic acid Nicotinamide Pyrazine Pyrazinoic acid Pyrazinamide Aminopyrazine None (inoculum control)
0.06 0.25 0.14 0.17 0.23 0.26 0.21 0.06
+ + + + + +
NGC 50 87 17 29 45 48 NA
NAd
a Log-phase cultures of Rhodopseudomonas palustris grown on pyrazinoic acid were used as the initial inocula. All media contained bicarbonate (0.1% [wt/vol]), and the values of final absorbance are expressed. h +, utilized; -, not utilized. ' NG, no growth. d NA, not applicable.
Corresponding author. 2187
2188
APPL. ENVIRON. MICROBIOL.
NOTES
0*5
100
cQL4
.80
2
(n
~~~~~~~~c U)
E
(0
0Q3
x60 °
0~~~~~~~~
/ 40° 0-2 o~~~~~~~4 0. < O-lT ~~~20 L)
0-0
0
e(d
2
4
6
8
0 10
Time (days) FIG. 2. Photoheterotrophic growth of Rhodopseudomonas palustris OU 11 on pyrazinoic acid. The medium contained 1.0 mM (100%) pyrazinoic acid and 0.1% (wt/vol) bicarbonate. Symbols: 0, growth (A660); X, pyrazinoic acid concentration.
L.. 0
Uf) n
AMINO PYRAZINE .,,,O '_
--I
Wavelength (nm)2W
H~N
N2
Ie
_
.
N
350
FIG. 1. Spectrum of various heterocyclic aromatic compounds after fresh culture medium containing 0.5 suitable dilutions. Symbols: mM heterocyclic aromatic compound; -, culture supernatant following growth of Rhodopseudomonaspalustris for 6 days, by which time final biomass yields were obtained. ,
--
compounds. These experiments were repeated several times, and every time growth was observed on pyrazinoic acid alone after 5 to 6 weeks of adaptation. The preadapted organism grew well on subculturing with pyrazinoic acid as the carbon source, and when this culture was used as the initial inoculum the organism could photometabolize a variety of heterocyclic aromatic compounds (Table 1). Photometabolism of heterocyclic aromatic compounds. In the present investigation, heterotrophic utilization of two groups (pyridine and pyrazine) of compounds belonging to six-member nitroheterocyclic compounds as the sole organic carbon source was studied under light anaerobic and dark aerobic conditions. Growth was not observed under dark aerobic conditions (data not shown), suggesting that catabolism of heterocyclic compounds is light dependent. Pyridine could not be photometabolized; however, its substituted derivatives nicotinic acid and nicotinamide were metabolized. Growth occurred under light anaerobic conditions on pyrazine and its derivatives (Table 1). To the best of our knowledge, there is only one report on the biodegradation of a member of the pyrazine group of compounds (pyrazinamide) by a Pseudomonas sp. (17) when the compound was utilized as the sole source of nitrogen. The presence of carbonate or bicarbonate in the medium was not found to be essential for growth to occur on pyrazinoic
acid; however, the presence of bicarbonate increased the biomass yield (data not shown), and for growth on heterocyclic aromatic compounds, bicarbonate (0.1%) was routinely used in the medium. Uptake of heterocyclic aromatic compounds. Direct evidence of the utilization of heterocyclic aromatic compounds comes from the study of the disappearance of these compounds from the medium, as can be seen in the UV absorption spectra of media before and after growth (Fig. 1). Figure 2 shows the change in substrate concentration and growth of Rhodopseudomonas palustns on pyrazinoic acid. At the end of the growth phase, it was observed that only 50 to 60% of the substrate was consumed and was converted into the biomass. Similarly, the catabolism of all the other heterocyclic aromatic compounds was not complete, with only 50 to 60% being utilized (Fig. 1) and converted to biomass (Table 2). In this study, we rule out nutrient limitation, since the same concentrations of the nutrients have supported growth on compounds such as benzoate malate and succinate to much higher biomass yields. Wright and Madigan (20) have observed such growth of Rhodomicrobium vannielii on the methoxylated benzoate derivatives vanillate and syringate. They found that this effect was a result of catabolism of these compounds leading to significant inhibition of bacteriochlorophyll synthesis. Similarly, catabolism of the heterocyclic compounds also must inhibit the
TABLE 2. Biomass yield of Rhodopseudomonas palustris heterocyclic aromatic compoundsa Carbon sourceelectron donor
Concn of
substrate
(0.5 mM)
consumed
Nicotinic acid Nicotinamide Pyrazine Pyrazinoic acid Pyrazinamide Aminopyrazine
0.26 0.19 0.26 0.25 0.28 0.28
(mM)
on
Molar growth yield
Biomass yield
(g [dry wt]/mmol
(mg [dry wt]/ml)
of substrate
consumed) 0.538 0.526 0.500 0.584 0.564 0.535
0.14 0.10 0.13 0.146 0.158 0.15
a All media contained bicarbonate, and the results days of growth under light anaerobic conditions.
were
determined after 6
NOTES
VOL. 60, 1994
N
rCONzH2
NH2
Nicotinic acid
Nicotinamide
Pyrazine
Pyrazinoic acid
Pyrazinamide
Aminopyrazine
2189
Nc
Arrinopyrazine
0'1
Pyrazinamid
\
",VN COOH CO yN
?_~ Nll
Further photo-anaerobic biodegradation
Pyrazinoic acid
03 02
0001"
Pyrazinee FIG. 3. Hypothetical scheme for photodegradation of various pyrazine derivatives by Rhodopseudomonas palustns. This scheme shows carbon gain by pyrazine and aminopyrazine (carbon number of four), explaining the observed molar growth yields similar to those of pyrazinoic acid and pyrazinamide, which have a carbon number of five.
synthesis of some molecules essential for the bacteria to grow, though we have not looked into the matter yet. The molar biomass yields obtained (Table 2) are more than the theoretical yields for the heterocyclic aromatic compounds consumed. This may be due to the bicarbonate used in the medium, which eventually must have shared in building up the biomass, since such an involvement of CO2 in the catabolism of heterocyclic aromatic compounds (as observed earlier for hypoxanthine degradation) is known (19). The biomass calculated in terms of molar yield remained almost the same for all the compounds tested, though they contain various numbers of carbons. Though we have no experimental results to account for this discrepancy, it probably reflects the extent of involvement of bicarbonate in the catabolism of these compounds. Pyrazinoic acid and pyrazinamide (carbon number, five) must involve more CO2 than do nicotinic acid and nicotinamide (carbon number, six) in their metabolism. The same molar growth yields of pyrazinoic acid and pyrazinamide (both with a carbon number of five) and pyrazine and aminopyrazine (both with a carbon number of four) can be plausibly explained by the assumption that these compounds are degraded after being converted to pyrazinoic acid (Fig. 3). Aminopyrazine and pyrazine gain one carbon from bicarbonate, and hence their molar growth yields equal those of the other two, which have a carbon atom more. Effects of concentrations of heterocyclic aromatic compounds. The tolerance of Rhodopseudomonas palustris OU 11 of heterocyclic aromatic compounds varied from compound to compound (Fig. 4). Unlike homocyclic aromatic compounds like cinnamate, which could support growth of Rhodopseudomonas palustris up to a concentration of 12 mM (12), heterocyclic aromatic compounds were growth inhibitory at concentrations as low as 4 mM (data not shown). Increasing the concentrations from 0.05 to 1 mM increased growth on pyrazine, pyrazinoic acid, and aminopyrazine, while growth inhibition was observed for the amide forms (nicotinamide and pyrazinamide) at a concentration of 1 mM, suggesting that amide forms are toxic at much lower concentrations.
03 02
.~ .~ 0-5 1.0 0X05 0Q1
0-05 0-1
1-0 FIG. 4. Photoheterotrophic growth of Rhodopseudomonas palustris
0Q5
OU 11 at various concentrations of heterocyclic aromatic compounds. Results were determined after 6 days of growth under anaerobic light conditions. Values on the x axis indicate the A660, and values on the y axis indicate the millimolar concentration of the compound. For experimental details, see Table 1.
A large number of heterocyclic aromatic compounds occur in nature, and to date very little information on their biodegradation is available (2). Our results have revealed an important metabolic capability of Rhodopseudomonas palustris and have opened the scope for further studies of anaerobic photobiodegradation of heterocyclic aromatic compounds by APB in terms of the metabolic pathways involved as well as their potential utilization in waste treatment. Pyridine, pyrazine, and their derivatives are of particular interest for applied research because they are widely used and produced in pharmaceutical industries and are let out at low concentrations in wastewater. The fact that these heterocyclic aromatic compounds are utilized under anaerobic conditions is very important, since a variety of these compounds disposed of in the subsurface environments contaminate groundwater by leaching and many of them are considered either toxic or carcinogenic even at low concentrations. The growth of Rhodopseudomonas palustris due to degradation of heterocyclic aromatic compounds suggests its possible role (and that of APB in general) in nature as a biodegrader of aromatic compounds, particularly heterocyclic compounds. C.S. thanks the UGC, New Delhi, India, for the award of Scientistship, and C.V.R. thanks the CSIR, New Delhi, India, for the award of Research Associateship. P. R. Rao thanks the U.G.C. for the award of Professor Emeritus. We also thank the ICAR, CSIR, and UGC Government of India for their financial support. We greatly acknowledge P. Appa Rao, University of Hyderabad, and
2190
APPL. ENVIRON. MICROBIOL.
NOT'ES
J. Annapurna, IICT, Hyderabad, India, for helping in spectrophotometric analysis. 10. REFERENCES 1. Aretz, W., H. Kaspari, and J. H. Klemme. 1978. Utilization of purines as nitrogen source by facultative phototrophic bacteria. FEMS Microbiol. Lett. 4:249-253. 2. Berry, D. F., A. J. Francis, and J. M. Bollag. 1987. Microbial metabolism of homocyclic and heterocyclic aromatic compounds under anaerobic conditions. Microbiol. Rev. 51:43-59. 3. Biebl, H., and N. Pfennig. 1981. Isolation of members of the family Rhodosprillaceae, p. 267-273. In M. P. Starr, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes, vol. 1. Springer Verlag, New York. 4. Blasco, R., and F. Castillo. 1992. Light-dependent degradation of nitrophenols by the phototrophic bacterium Rhodobacter capsulatus ElFl. Appl. Environ. Microbiol. 58:690-695. 5. Busse, W., H. Kaspari, and J. H. Klemme. 1984. Urea: an intermediate of aerobic and anaerobic purine degradation in Rhodopseudomonas capsulatus. FEMS Microbiol. Lett. 25:3336. 6. Dutton, P. L., and W. C. Evans. 1978. Metabolism of aromatic compounds by Rhodosprillaceae, p. 719-726. In R. K. Clayton and W. R. Sistrom (ed.), The photosynthetic bacteria. Plenum Press, New York. 7. Elder, J. E. D., and D. J. Kelly. 1992. Anaerobic degradation of trans-cinnamate and w-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a (3-oxidation mechanism. Arch. Microbiol. 157:148-154. 8. Gibson, K. J., and J. Gibson. 1992. Potential early intermediates in anaerobic benzoate degradation by Rhodopseudomonas palustris. Appl. Environ. Microbiol. 58:696-698. 9. Harwood, C. S., and J. Gibson. 1988. Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic
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