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Effect of pesticides on hydrogen metabolism of Rhodobacter sphaeroides and Rhodopseudomonas palustris
FEMS Microbiology
Ecology
I9 ( 1996)
I -4
Effect of pesticides on hydrogen metabolism of Rhodobacter sphaeroides and Rhodopseudomonas palustris A.V. Chalam, Ch. Sasikala Microbial
Biotechnology
Lnhorato~.
*,
Department
Ch.V. Ramana, P. Raghuveer Rao of Botany
Received 26 April 1995: revised 4 August
Osmnnin
Utlic,ersi@. HJderahad 500 007. Indin
1995; accepted 7 August
1995
Abstract The present study reports the effect of 2.4-D, quinalphos, monocrotophos. captan and carbendazim on the hydrogen metabolism (nitrogenase, photoproduction of hydrogen and hydrogenase activities) of two purple non-sulfur bacteria isolated from paddy soils. In general, the pesticides were found to be inhibitory to both nitrogenase and hydrogen photoproduction activities of both the organisms, and their effect on hydrogenase-mediated reactions varied with the pesticides used and the organisms. Keyrcords: Pesticide effect: Nitrogenase: Rhodopseudomonas palustris
Hydrogenase;
Photoproduction
1. Introduction
Purple non-sulfur
can affect the nitrogen
Anoxygenic phototrophic important role in biological
bacteria (APB) play an H, metabolism in anoxic
environments and contribute significantly to the H, cycle [l]. These bacteria can produce H2 which is greatly nitrogenase-mediated and consume H, by the enzyme uptake hydrogenase [2]. Certain hydrogenases are reversible (H ? + 2H++ 2e-) and under certain assay conditions they can also mediate H2 production. Anoxygenic phototrophic bacteria significantly contribute to soil fertility; particularly in flooded rice fields [3] where perfect anaerobic conditions prevail, these bacteria exist in comparatively high numbers [4]. Constant application of pesticides to rice fields
* Corresponding
of hydrogen:
author.
0168-6496/96/$15.00 0 1996 Federation SD1 0168-6496(95)00065-E
of European
Microbiological
bacteria;
Rhodobacter
sphaemides:
and hydrogen metabolism of soil bacteria. Extensive studies on the effect of some of the commonly used pesticides on the nitrogen fixing chemotrophic bacteria and cyanobacteria have been carried out. Though a few studies on the effect of xenobiotic aromatic compounds [51, antibiotics [6,7] and s-triazine herbicides (atrazine) [8-l I] on growth of APB have been reported to date, to the best of our knowledge there are no reports on the effect of pesticides on the hydrogen metabolism on this group of microorganisms. In the present communication, the effect of a herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), the fungicides captan and carbendazim. and the insecticides quinalphos and monocrotophos on the hydrogen metabolism (nitrogenase, hydrogen photoproduction and hydrogenase activities) of two purple non-sulfur bacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris isolated from paddy fields, is discussed. Societies. All rights reserved
2. Materials
and methods
2.1. Organisms
the present study are the minimum diazotrophic growth inhibitory concentration as has been established in an earlier study [IS]).
and growth conditions
Rhodohacter sphaeroides 1B and Rhodopseudomotzas palustris 3A were isolated as dominating species of purple non-sulfur APB existing in two different paddy soils of Andhra Pradesh. Axenic cultures of these organisms were grown photoheterotrophically in Biebl and Pfennig’s [12] medium with succinate (0.3% w/v) and ammonium chloride (0.04% w/v) as carbon and nitrogen source respectively at 30 & 2°C and 2400 lux. 2.2. Analytical
methods
Growth was monitored by measuring absorbance at 660 nm. Nitrogenase (acetylene reduction), hydrogen photoproduction [ 131 and hydrogenase [ 141 activities of resting cells were studied at regular intervals using gas chromatography. Resting cells of logarithmic phase cultures grown on ammonium chloride and succinate were used for various assays. For assay. 5 ml of resting cell suspension were taken in 15 ml test tubes, sealed with subba seals and flushed with ultrapure argon. Biomass per vessel for Rb. sphaeroides and Rps. palustris were 10 and 6 mg respectively. All these activities were recorded with succinate (0.3% w/v) as electron donor with added pesticide (the concentrations of the pesticides used in
Table I Effect of pesticides pnlusrris 3A
on nittagenase
Pesticides (ppm)
2.4.-D
(acetylene
Nitrogenase
(400)
Quinalphos (100) Monocrotophos (100) Captan (400) Carbendazim (400) ( -) pesticide control
reduction)
activity
’
3. Results and discussion 3.1. l$ect of pesticides reduction actiuity)
on nitrogetrase
(aceelene
Inhibition of nitrogenase activity was observed with all the pesticides for both the organisms (Table 1). Captan totally inhibited nitrogenase activity of both the organisms. Compared to nitrogenase activity of Rps. palmsiris. that of Rb. sphaeroides showed resistance to carbendazim, quinalphos and monocrotophos, while nitrogenase activity of Rps. palustris was less inhibited by 2,4-D. 3.2. Eflect otl hydrogetz photoproductiotl Hydrogen photoproduction was assayed in the absence of combined nitrogen in the medium, under which conditions both nitrogenaseand hydrogenase-mediated hydrogen photoproduction can be observed. As observed for nitrogenase activity, captan totally inhibited hydrogen photoproduction along with nitrogenase activity in both bacteria. Other pesticides did not inhibit both activities. 2,4-D completely inhibited hydrogen photoproduction of Rb.
and photoproduction
% Inhibition
of hydrogen Hydrogen
activities
of Rh. sphoeroides
photoproduction
IB and Rps.
a ^ 9 Inhibition
Rb
R/x
Rh
Rps
Rb
RPS
Rb
RPS
2290 3206 2478 0 4378 4752
1224 190 245 0 734 208 I
52 33 48 100 8 0
41 76 88 100 65 0
0 89 73 0 186 170
66 0 33 0 72 99
100 48 56 I00 LX+) 0
33 100 67 100 27 0
’ . nmol ethylene formed/vessel.
’ ’ , ~1 hydrogen photoproduced/vessel. (+ ), = R enhancement Rb = Rhodobacter
Results expressed 30 + 2°C.
in photoproduction of hydrogen. and Rps = Rhodopselrdomo,2ns pnlustris. are average of experiments done in triplicate and pertain to that after 24 h of light (2400 lux) anaerobic .spheroides
incubation
at
A. V. Chnlunt et al. / FEMS Microbiology
sphaeroides while nitrogenase activity was still observed. Similarly, quinalphos has completely inhibited hydrogen photoproduction of Rps. palustris, while nitrogenase activity was still observed. All the other biocides partially inhibited both hydrogen photoproduction and nitrogenase activity in both bacteria to different extents, as did 2,4-n for Rps. palustris and quinalphos for Rb. sphaeroides. The only exception was for Rb. sphaeroides where carbendazim stimulated photoproduction of hydrogen though slightly inhibited nitrogenase activity. In general the results suggest that pesticides are inhibitory to both nitrogenase and hydrogen photoproduction activities. 3.3. Effect reactions
of pesticides
on hydrogenuse-mediated
Hydrogenase activity of resting cells of purple bacteria was assayed in the presence of ammonium chloride, a condition under which nitrogenase-mediated reactions are totally inhibited [2]. In the absence of added pesticides, hydrogenase of Rps. palustris was in the evolutionary mode while for Rb. sphaeroides it was in the uptake mode (Table 2). The effect of pesticides varied with the pesticide used and the organism. 2,4-D enhanced hydrogenase activities (either production or uptake activities) of both the organisms. Monocrotophos and quinalphos stimulated hydrogenase (hydrogen production) activity by Rps. palustris, while an inhibitory effect (on hydrogen uptake
Ecology 19 (19961 l-4
3
activity) was observed for Rb. sphaeroides. In contrast, carbendazim and captan stimulated uptake hydrogenase activity by Rps. palustris and hydrogen production activity by Rb. sphaeroides. Most of the pesticides used in the present study were aromatic compounds (2,4-D, quinalphos, captan and carbendazim). Though Rps. palustris is known to photobiodegrade and metabolize a variety of aromatic compounds [ 16- 181, under the experimental conditions tested none of these compounds were photobiodegraded (data not shown). However, it is reported that carbendazim was photoassimilated by another strain of Rps. palustris as sole carbon and nitrogen source [ 191. Pesticides, most of which are xenobiotic, often accumulate under subsurface soils where anaerobic conditions prevail and have a diverse influence on the microbial activity and diversity within these anoxic ecosystems. Apart from photoproduction of hydrogen being a potential source of fuel in the future [2], nitrogen fixation and hydrogen metabolism play an important role in improving soil fertility [l]. Our results suggest a probable influence of some of the commonly used pesticides on hydrogen metabolism and nitrogen fixation of purple non-sulfur bacteria occurring in anoxic ecosystems of paddy fields. However, under natural conditions, several biotic and abiotic factors are also to be taken into consideration.
Acknowledgements Table 2 Effect of pesticides on the hydrogenase-mediated sphaeroides 1B and Rps. pulustris 3A
activities of Rb.
Pesticides
Hydrogenase activity ( ~1 hydrogen produced/consumed per vessel) Rb. .sphneroides
Rp.r. palustris
2.4.-D (400) Quinalphos (100) Monocrotophos (100) Captan (400) Carbendazim (400) (- ) pesticide control
C-)41 c-)6 C-18 (+)26 (t) 16.5
(+)31 (+I 11.5 (+) 18 C-)5 (-) 12
(-)
(+I
15
10
(+I. = hydrogen production by hydrogenase. (- ). = uptake of hydrogen by hydrogenase. Other experimental details as in Table 1 except that the medium contained ammonium chloride (3 mM).
P.R.R. and Ch.S. thank the UGC, New Delhi, for the award of Professor Emeritus and Research Scientistship respectively. Ch.V.R. thanks the CSIR, New Delhi, for the award of Research Associateship. A.V.C. thanks the ICAR and CSIR, New Delhi for the award of research fellowship. ICAR and CSIR are acknowledged for providing financial support.
References [ll Schlegel. H.G. and Schneider, K. (1985). Microbial metabolism of hydrogen. In. Comprehensive Biotechnology (Moo-Young, M.. Ed.). pp. 435-457. Pergamon Press. Oxford.
[2] Sasikala, K.. Ramana. Ch.V., Raghuveer Rao, P. and Kovats, K. (1993) Anoxygenic phototrophic bacteria : Phyriology and advances in hydrogen production technology. Adv. Appl. Microbial. 38. 7-l l-295. [3] Habte. M. and Alexander, M. (1980) Nitrogen fixation by photosynthetic bacteria in lowland rice culture. Appl. Environ. Microbial. 39, 342-347. [4] Kobayashi. M., Takahashi, E. and Kawaguchi, K. (1967) Distribution of nitrogen-fixing microorganisms in paddy
and
diuron
resistant atrains of Rhodopsrudorrlorrtrs Weed Sci. 32. 664-669. Biebl. H. and Pfennig. N. (1981) Isolation of members of the Family Rhodospirillaceae. In: The Prokaryotes (Starr. M.P.. Stolp. H.. Truper. H.G.. Balows, A. and Schlegel. H.G.. Eda.). Vol. I. pp. 267-173. Springer Verlag. New York. Saaihala, K.. Ramana, Ch.V., Raghuveer Rao. P. and Subrahmanyam. M. (1990) Photoproduction of hydrogen, nitrogenase and hydrogenaae activities of free and immobilized whole cells of Rhodnbncter sphoeroidrs O.U. 001. FEMS Microbial. Lett. 73, 13-28. Tel-Or. E.. Luijk, L.W. and Packer, L. (1977) An inducible hydrogenase in cyanobacteria enhances nitrogen fixation. FEBS Lett. 78. 49-52. Chalam. A.V.. Sasikala, Ch.. Ramana, Ch.V.. Jaya Sri. K. and Raghuveer Rao. P. (1995) Effect of pesticides on the diarotrophic growth and nitrogenase activity of purple nonsulfur bacteria. Bull. Environ. Contam. Toxicol.. in press. Harwood. C.S. and Gibson, J. (1988) Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhorlopselrd~rl1orlcr.v pcdustris. Appl. Environ. Microbial. 54, 712-717. Sasikala, Ch.. Ramana. Ch.V. and Raghuveer Rao. P. (1994) Photometabolism of heterocyclic aromatic compounds by Rtrorict~.srudt,/rrorrris palustris OU I I Appl. Environ. Microbiol. 60. 2187-2190. Sasikala. Ch.. Ramana, Ch.V. and Raghuveer Rao. P. (1994) Nitrogen fixation by Rphod~pseudo,?lurlcl.v pcdmtr-is OU I I with aromatic compounds as carbon source/electron donors. FEMS Microbial. Lett. 131. 75-78. Rajkumur, B. and Lalithakumari, D. (1992) Carbendazim as the sole carbon and nitrogen source for a photorrophic bacterium. Biomed. Lett. 47. 337-346. .\pharroide.s.
[IZ]
[ 131
[I41
[I51
[ 161
[l7]
[ 181
[I91
Ecology
I9 ( 1996)
I -4
Effect of pesticides on hydrogen metabolism of Rhodobacter sphaeroides and Rhodopseudomonas palustris A.V. Chalam, Ch. Sasikala Microbial
Biotechnology
Lnhorato~.
*,
Department
Ch.V. Ramana, P. Raghuveer Rao of Botany
Received 26 April 1995: revised 4 August
Osmnnin
Utlic,ersi@. HJderahad 500 007. Indin
1995; accepted 7 August
1995
Abstract The present study reports the effect of 2.4-D, quinalphos, monocrotophos. captan and carbendazim on the hydrogen metabolism (nitrogenase, photoproduction of hydrogen and hydrogenase activities) of two purple non-sulfur bacteria isolated from paddy soils. In general, the pesticides were found to be inhibitory to both nitrogenase and hydrogen photoproduction activities of both the organisms, and their effect on hydrogenase-mediated reactions varied with the pesticides used and the organisms. Keyrcords: Pesticide effect: Nitrogenase: Rhodopseudomonas palustris
Hydrogenase;
Photoproduction
1. Introduction
Purple non-sulfur
can affect the nitrogen
Anoxygenic phototrophic important role in biological
bacteria (APB) play an H, metabolism in anoxic
environments and contribute significantly to the H, cycle [l]. These bacteria can produce H2 which is greatly nitrogenase-mediated and consume H, by the enzyme uptake hydrogenase [2]. Certain hydrogenases are reversible (H ? + 2H++ 2e-) and under certain assay conditions they can also mediate H2 production. Anoxygenic phototrophic bacteria significantly contribute to soil fertility; particularly in flooded rice fields [3] where perfect anaerobic conditions prevail, these bacteria exist in comparatively high numbers [4]. Constant application of pesticides to rice fields
* Corresponding
of hydrogen:
author.
0168-6496/96/$15.00 0 1996 Federation SD1 0168-6496(95)00065-E
of European
Microbiological
bacteria;
Rhodobacter
sphaemides:
and hydrogen metabolism of soil bacteria. Extensive studies on the effect of some of the commonly used pesticides on the nitrogen fixing chemotrophic bacteria and cyanobacteria have been carried out. Though a few studies on the effect of xenobiotic aromatic compounds [51, antibiotics [6,7] and s-triazine herbicides (atrazine) [8-l I] on growth of APB have been reported to date, to the best of our knowledge there are no reports on the effect of pesticides on the hydrogen metabolism on this group of microorganisms. In the present communication, the effect of a herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), the fungicides captan and carbendazim. and the insecticides quinalphos and monocrotophos on the hydrogen metabolism (nitrogenase, hydrogen photoproduction and hydrogenase activities) of two purple non-sulfur bacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris isolated from paddy fields, is discussed. Societies. All rights reserved
2. Materials
and methods
2.1. Organisms
the present study are the minimum diazotrophic growth inhibitory concentration as has been established in an earlier study [IS]).
and growth conditions
Rhodohacter sphaeroides 1B and Rhodopseudomotzas palustris 3A were isolated as dominating species of purple non-sulfur APB existing in two different paddy soils of Andhra Pradesh. Axenic cultures of these organisms were grown photoheterotrophically in Biebl and Pfennig’s [12] medium with succinate (0.3% w/v) and ammonium chloride (0.04% w/v) as carbon and nitrogen source respectively at 30 & 2°C and 2400 lux. 2.2. Analytical
methods
Growth was monitored by measuring absorbance at 660 nm. Nitrogenase (acetylene reduction), hydrogen photoproduction [ 131 and hydrogenase [ 141 activities of resting cells were studied at regular intervals using gas chromatography. Resting cells of logarithmic phase cultures grown on ammonium chloride and succinate were used for various assays. For assay. 5 ml of resting cell suspension were taken in 15 ml test tubes, sealed with subba seals and flushed with ultrapure argon. Biomass per vessel for Rb. sphaeroides and Rps. palustris were 10 and 6 mg respectively. All these activities were recorded with succinate (0.3% w/v) as electron donor with added pesticide (the concentrations of the pesticides used in
Table I Effect of pesticides pnlusrris 3A
on nittagenase
Pesticides (ppm)
2.4.-D
(acetylene
Nitrogenase
(400)
Quinalphos (100) Monocrotophos (100) Captan (400) Carbendazim (400) ( -) pesticide control
reduction)
activity
’
3. Results and discussion 3.1. l$ect of pesticides reduction actiuity)
on nitrogetrase
(aceelene
Inhibition of nitrogenase activity was observed with all the pesticides for both the organisms (Table 1). Captan totally inhibited nitrogenase activity of both the organisms. Compared to nitrogenase activity of Rps. palmsiris. that of Rb. sphaeroides showed resistance to carbendazim, quinalphos and monocrotophos, while nitrogenase activity of Rps. palustris was less inhibited by 2,4-D. 3.2. Eflect otl hydrogetz photoproductiotl Hydrogen photoproduction was assayed in the absence of combined nitrogen in the medium, under which conditions both nitrogenaseand hydrogenase-mediated hydrogen photoproduction can be observed. As observed for nitrogenase activity, captan totally inhibited hydrogen photoproduction along with nitrogenase activity in both bacteria. Other pesticides did not inhibit both activities. 2,4-D completely inhibited hydrogen photoproduction of Rb.
and photoproduction
% Inhibition
of hydrogen Hydrogen
activities
of Rh. sphoeroides
photoproduction
IB and Rps.
a ^ 9 Inhibition
Rb
R/x
Rh
Rps
Rb
RPS
Rb
RPS
2290 3206 2478 0 4378 4752
1224 190 245 0 734 208 I
52 33 48 100 8 0
41 76 88 100 65 0
0 89 73 0 186 170
66 0 33 0 72 99
100 48 56 I00 LX+) 0
33 100 67 100 27 0
’ . nmol ethylene formed/vessel.
’ ’ , ~1 hydrogen photoproduced/vessel. (+ ), = R enhancement Rb = Rhodobacter
Results expressed 30 + 2°C.
in photoproduction of hydrogen. and Rps = Rhodopselrdomo,2ns pnlustris. are average of experiments done in triplicate and pertain to that after 24 h of light (2400 lux) anaerobic .spheroides
incubation
at
A. V. Chnlunt et al. / FEMS Microbiology
sphaeroides while nitrogenase activity was still observed. Similarly, quinalphos has completely inhibited hydrogen photoproduction of Rps. palustris, while nitrogenase activity was still observed. All the other biocides partially inhibited both hydrogen photoproduction and nitrogenase activity in both bacteria to different extents, as did 2,4-n for Rps. palustris and quinalphos for Rb. sphaeroides. The only exception was for Rb. sphaeroides where carbendazim stimulated photoproduction of hydrogen though slightly inhibited nitrogenase activity. In general the results suggest that pesticides are inhibitory to both nitrogenase and hydrogen photoproduction activities. 3.3. Effect reactions
of pesticides
on hydrogenuse-mediated
Hydrogenase activity of resting cells of purple bacteria was assayed in the presence of ammonium chloride, a condition under which nitrogenase-mediated reactions are totally inhibited [2]. In the absence of added pesticides, hydrogenase of Rps. palustris was in the evolutionary mode while for Rb. sphaeroides it was in the uptake mode (Table 2). The effect of pesticides varied with the pesticide used and the organism. 2,4-D enhanced hydrogenase activities (either production or uptake activities) of both the organisms. Monocrotophos and quinalphos stimulated hydrogenase (hydrogen production) activity by Rps. palustris, while an inhibitory effect (on hydrogen uptake
Ecology 19 (19961 l-4
3
activity) was observed for Rb. sphaeroides. In contrast, carbendazim and captan stimulated uptake hydrogenase activity by Rps. palustris and hydrogen production activity by Rb. sphaeroides. Most of the pesticides used in the present study were aromatic compounds (2,4-D, quinalphos, captan and carbendazim). Though Rps. palustris is known to photobiodegrade and metabolize a variety of aromatic compounds [ 16- 181, under the experimental conditions tested none of these compounds were photobiodegraded (data not shown). However, it is reported that carbendazim was photoassimilated by another strain of Rps. palustris as sole carbon and nitrogen source [ 191. Pesticides, most of which are xenobiotic, often accumulate under subsurface soils where anaerobic conditions prevail and have a diverse influence on the microbial activity and diversity within these anoxic ecosystems. Apart from photoproduction of hydrogen being a potential source of fuel in the future [2], nitrogen fixation and hydrogen metabolism play an important role in improving soil fertility [l]. Our results suggest a probable influence of some of the commonly used pesticides on hydrogen metabolism and nitrogen fixation of purple non-sulfur bacteria occurring in anoxic ecosystems of paddy fields. However, under natural conditions, several biotic and abiotic factors are also to be taken into consideration.
Acknowledgements Table 2 Effect of pesticides on the hydrogenase-mediated sphaeroides 1B and Rps. pulustris 3A
activities of Rb.
Pesticides
Hydrogenase activity ( ~1 hydrogen produced/consumed per vessel) Rb. .sphneroides
Rp.r. palustris
2.4.-D (400) Quinalphos (100) Monocrotophos (100) Captan (400) Carbendazim (400) (- ) pesticide control
C-)41 c-)6 C-18 (+)26 (t) 16.5
(+)31 (+I 11.5 (+) 18 C-)5 (-) 12
(-)
(+I
15
10
(+I. = hydrogen production by hydrogenase. (- ). = uptake of hydrogen by hydrogenase. Other experimental details as in Table 1 except that the medium contained ammonium chloride (3 mM).
P.R.R. and Ch.S. thank the UGC, New Delhi, for the award of Professor Emeritus and Research Scientistship respectively. Ch.V.R. thanks the CSIR, New Delhi, for the award of Research Associateship. A.V.C. thanks the ICAR and CSIR, New Delhi for the award of research fellowship. ICAR and CSIR are acknowledged for providing financial support.
References [ll Schlegel. H.G. and Schneider, K. (1985). Microbial metabolism of hydrogen. In. Comprehensive Biotechnology (Moo-Young, M.. Ed.). pp. 435-457. Pergamon Press. Oxford.
[2] Sasikala, K.. Ramana. Ch.V., Raghuveer Rao, P. and Kovats, K. (1993) Anoxygenic phototrophic bacteria : Phyriology and advances in hydrogen production technology. Adv. Appl. Microbial. 38. 7-l l-295. [3] Habte. M. and Alexander, M. (1980) Nitrogen fixation by photosynthetic bacteria in lowland rice culture. Appl. Environ. Microbial. 39, 342-347. [4] Kobayashi. M., Takahashi, E. and Kawaguchi, K. (1967) Distribution of nitrogen-fixing microorganisms in paddy
and
diuron
resistant atrains of Rhodopsrudorrlorrtrs Weed Sci. 32. 664-669. Biebl. H. and Pfennig. N. (1981) Isolation of members of the Family Rhodospirillaceae. In: The Prokaryotes (Starr. M.P.. Stolp. H.. Truper. H.G.. Balows, A. and Schlegel. H.G.. Eda.). Vol. I. pp. 267-173. Springer Verlag. New York. Saaihala, K.. Ramana, Ch.V., Raghuveer Rao. P. and Subrahmanyam. M. (1990) Photoproduction of hydrogen, nitrogenase and hydrogenaae activities of free and immobilized whole cells of Rhodnbncter sphoeroidrs O.U. 001. FEMS Microbial. Lett. 73, 13-28. Tel-Or. E.. Luijk, L.W. and Packer, L. (1977) An inducible hydrogenase in cyanobacteria enhances nitrogen fixation. FEBS Lett. 78. 49-52. Chalam. A.V.. Sasikala, Ch.. Ramana, Ch.V.. Jaya Sri. K. and Raghuveer Rao. P. (1995) Effect of pesticides on the diarotrophic growth and nitrogenase activity of purple nonsulfur bacteria. Bull. Environ. Contam. Toxicol.. in press. Harwood. C.S. and Gibson, J. (1988) Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhorlopselrd~rl1orlcr.v pcdustris. Appl. Environ. Microbial. 54, 712-717. Sasikala, Ch.. Ramana. Ch.V. and Raghuveer Rao. P. (1994) Photometabolism of heterocyclic aromatic compounds by Rtrorict~.srudt,/rrorrris palustris OU I I Appl. Environ. Microbiol. 60. 2187-2190. Sasikala. Ch.. Ramana, Ch.V. and Raghuveer Rao. P. (1994) Nitrogen fixation by Rphod~pseudo,?lurlcl.v pcdmtr-is OU I I with aromatic compounds as carbon source/electron donors. FEMS Microbial. Lett. 131. 75-78. Rajkumur, B. and Lalithakumari, D. (1992) Carbendazim as the sole carbon and nitrogen source for a photorrophic bacterium. Biomed. Lett. 47. 337-346. .\pharroide.s.
[IZ]
[ 131
[I41
[I51
[ 161
[l7]
[ 181
[I91