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Isolation of non-sulphur photosynthetic bacterial strains efficient in hydrogen production at elevated temperatures
Int. J. HydrogenEnergy,Vol. 16, No. 6, pp. 403~05, 1991.
0360-3199/91 $3.00+ 0.00 PergamonPress plc. InternationalAssociationfor HydrogenEnergy.
Printed in Great Britain.
ISOLATION OF N O N - S U L P H U R PHOTOSYNTHETIC BACTERIAL STRAINS EFFICIENT IN H Y D R O G E N P R O D U C T I O N AT ELEVATED TEMPERATURES S. P. SINGH* and S. C. SRIVASTAVA Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India
(Receivedfor publication 13 December 1990) Abstract--Four strains of non-sulphur photosynthetic bacteria were isolated from root zone associations of aquatic plants like Azolla, Salvinia and Eichhornia, as well as the deep-water rice. Based on the gross cell morphology and pigmentation, the isolates resembled Rhodopseudomonas sp. and have been designated as BHU strains 1 to 4, respectively. When subjected to elevated temperature (from 33-45°C), substantial growth/hydrogen production could be observed only in strains 1 and 4. Strains 2 and 3 on the other hand, showed diminished growth and negligible hydrogen photoproduction. The BHU strains 1 and 4 have been selected as the most active (thermostable) hydrogen producing strains of local origin as far as the Indian tropical climate is concerned.
hornia as these formed a major component of floating
INTRODUCTION The major concern about the efficient hydrogen producing photosynthetic bacteria has been their tolerance against high light irradiance as well as high temperature, Efforts in this direction have yielded positive results only in a few cases. Watanabe et al. [1] made a comparison of hydrogen production by different strains of Rhodopseudomonas originating from Japan and Thailand, and the majority of strains in the latter case exhibited thermostable hydrogen production within a maximum limit of 40°C. The temperature upper limit, extended to 45°C, restricted hydrogen production to only R. sphaeroides B5 isolated from Thailand and SL16 from Korea and KM ! 13 from Japan [2]. Similarly, four strains of photosynthetic bacteria isolated from the Bangkok area were reported to grow well at 40°C but a coculture of strain TR-22R-B and the strict aerobic bacterium, 22TW-S of the same origin, resulted in growth of both the organisms at 45°C, thus suggesting that the growth of photosynthetic bacteria could be encouraged by the associating aerobe at elevated temperature provided some oxygen migrated from the air into the culture media [3]. Thermostable photohydrogen production is reported here in selected strains of the non-sulphur photosynthetic bacterium, Rhodopseudomonas sp., isolated from the root associations of aquatic ferns and deep water rice. EXPERIMENTAL
plant community in local ponds. Deep water rice plant roots were collected from Surha Tal (Lake), Ballia District (about 250 km east). Non-sulphur photosynthetic bacteria were isolated by the conventional enrichment technique under light-anaerobic conditions as reported earlier by Singh et al. [4]. The bacterial strains isolated from the root zone associations of Azolla, Salvinia, Eichhornia and deep water rice plants have been tentatively designated as Rhodopseudomonas sp. BHU strain-I to 4, respectively (BHU for Banaras Hindu University, India).
Growth medium The basal medium as prescribed by Pfennig [5], was used with slight modifications: yeast extract, 0.5 g 1-~; DL-malate, 1.87 g l-l; KH2PO4 ' 1.0 g l ~; MgSO4.7H20, 0.4 g 1-~; NaC1, 0.4 g 1-1; sodium succinate, 2.0 g 1-~ and CaCI 2.2H 20, 0.05 g 1-1. Ammonium chloride (0.1 g/l) was added initially to anaerobic cultures for initiation of growth. All chemicals were of laboratory reagent grade obtained from BDH (U.K. or India). The medium was sterilized at 101b (15 min) and left overnight before bacterial inoculation. The pH of the growth medium was adjusted to 6.8. The concentrated bacterial cell suspensions were transferred anaerobically at a cell density of OD660= 0.3 in 60 ml fresh growth medium in rubberstoppered glass bottles having a gas phase of 95% argon and 5% CO2. Cultures were grown under tungsten illumination (2 kLux) with periodic shaking.
lnocula
Assays of hydrogen production
The bacterial strains were isolated from the root zones of different aquatic plants like Azolla, Salvinia and Eich-
A 0.5 ml samples of gas phase from the light-incubated bacterial cultures was analysed for H 2 in a gas chromatograph (Tracor 540, USA) on TCD mode fitted with Carbosieve-II column and connected to Hewlett-Packard
*Author to whom correspondence should be addressed.
403
404
S.P. SINGH and S. C. SRIVASTAVA
3392 A integrator. The values have been expresed as/~1 H 2 m g - l dry wt. Dry wt measurements were based on the conventional oven-drying of bacterial cells,
Temperature upshift experiments For comparison of hydrogen production activity, all the four bacterial strains were grown at 33°C and 45°C under the same degree of illumination (2 kLux). Parallel sets were also maintained for the simultaneous measurements of bacterial growth (expressed as/~g protein ml -l culture). Protein was estimated by the method of Lowry et al. [6], modified by Herbert et al. [7]. RESULTS A N D DISCUSSION The very association of Rhodospirillaceae with roots of aquatic plants, as observed here and reported earlier by Singh et aL [4], is in line with the reports of Mitsui et al. [8] that photosynthetic bacteria thrive well with debris of macroalgae, seagrasses and mangrove leaves. As regards light-to-hydrogen energy conversion efficiency [9, 10], it could reach a maximum of 4.9% or 7.9% respectively, Much emphasis has been laid on the feasibility of hydrogen production by such bacteria outdoor subjected to the prevailing daylength (irradiance) as well as temperature. Using a solar simulator, Miyake and Kawamura [10] observed high light energy conversion to hydrogen in R. sphaeroides 8703 at low light intensity corresponding to 50 Wm 2 that decreased to 6.3% if the irradiance was raised by a factor of ten. This eventually puts the efficiency of solar energy conversion as a critical factor in determining hydrogen production by photosynthetic bacteria, and emphasizes the need for the time to isolate/ train such microbes to adapt to elevated irradiance or temperature common to the tropics. The present experiments, have therefore, taken into account the possibility of thermostable hydrogen production by a few strains of Rhodospirillaceae at 33 and 45°C; the temperature range most common to a major part of Indian climate, We have recently reported on the isolation and photohydrogen production in a non-sulphur photosynthetic bacterium resembling Rhodopseudomonas designated as BHU strain 1 [4]. This strain was found growing in association with the roots of water fern Azolla. The Table'l. Effect of temperature on the activity of hydrogen photoproduction by four strains of non-sulphur photosynthetic bacteria Hydrogen photoproduction (pl H 2 h-i mg-i dry wt) Strain no. BHU strain 1 BHU strain 2 BHU strain 3 BHU strain 4
Source Azollaroot zone Salviniaroot zone Eichhorniaroot zone Deep water rice plant root zone
33°C 12 (292)* 10 (283)* 9 (264)* 12 (231)*
45°C 7 (231)* 4 (180)* 2.5 (168)* 15 (200)*
* Values indicate growth yield in terms of #g protein ml-t bacterialculture,
other three strains examined for hydrogen production at elevated temperatures also originated from similar associations of another water fern, Salvinia, water hyacinth and deep water rice plant, respectively. Data presented in Table I display a comparison of relative thermotolerance of BHU strains 1-4 in terms of growth yield and hydrogen production activity as light-anaerobic cultures. As all the strains were routinely grown at 33°C, BHU strains 1 and 4 invariably maintained their superiority in terms of hydrogen production over strains 2 and 3. However, the higher rate of hydrogen production in strain 4 was not accompanied by increased growth yield compared to strain 1. Subsequent to temperature shift-up (45°C) also, strain 4 proved to be on top of others with a rate of 15pl H2h l mg-I dry wt followed by BHU strain 1 (7/~ l H 2 h - l mg I dry wt). The latter, however, showed better growth yield (231/~g protein m l - l culture over strain 4 (200 #g protein ml 1 culture). This again emphasizes that hydrogen production activity may not be strictly correlated with growth rate/yield, and the efficiency is strain specific. Such a specificity is quite evident when one compares hydrogen production by all the four strains at 45°C. It may also be mentioned that BHU strains 1-4 inhabited almost the same type of habitat with heavy load of organics but differed a great deal in terms of their thermostable growth/hydrogen production. Strains originating from either Salvinia or Eichhornia, on the other hand, proved thermosensitive as reflected by diminished growth and extremely low rates of hydrogen production. Similar comparisons made by others showed that majority of Rhodopseudomonas strains from Thailand showed almost equal activity rates of hydrogen production at 30 ° or 40°C compared to those originating from either Japan or Thailand itself [l]. Kim et al. [2] in a subsequent study, also observed higher rates of hydrogen production at 40°C in R. sphaeroides B5 from Thailand over strains SL1, SL3, SL16 from Korea and K M l l 3 and TN3 from Japan. However, thermotolerant hydrogen production could be noticed in cultures of strain B5, SL 16 and K M I I 3 at 45°C. The stimulated high temperature growth of a photosynthetic bacterium (strain TR-22R-B) by the co-existing aerobe (strain 22TW-S) in culture, has been attributed to the input of some oxygen from air [3]. In conclusion, therefore, we can suggest the suitability of BHU strains 1 and 4 for photohydrogen generation outdoor in regions with temperature ranging between 33 ° and 45°C. Work is in progress to look for light to hydrogen conversion efficiency in such strains also at high irradiance.
Acknowledgements--This work was supported by a research grant from the Department of Non-Conventional Energy Sources, Government of India, New Delhi. Authors are grateful to Dr K. D. Pandey for technical details and helpful discussion. REFERENCES 1. K. Watanabe, J. S. Kim, K. Ito, L. Buranakarl, T. Kampee and H. Takahashi, Thermostable nature of hydrogen
ISOLATION OF NON-SULPHUR PHOTOSYNTHETIC BACTERIA
2.
3.
4.
5.
production by non-sulfur purple photosynthetic bacteria isolated in Thailand. Agric. Biol. Chem. 45, 217-222 (1981). J. S. Kim, H. Yamauchi, K. Ito and H. Takahashi, Selection of a photosynthetic bacterium suitable for hydrogen production in outdoor cultures among strains isolated in the Seoul, Taegu, Sendai and Bangkok areas. Agric. Biol. Chem. 46, 1469-1474 (1982). L. Buranakarl, H. Yoneyama, T. Iwata, J. S. Kim, S.I. Okuda, T. Kampee, K. Izaki and H. Takahashi, Growth of non-sulfur purple photosynthetic bacteria under high temperature. Nihon Biseibutsu Seitai Gakkaiho (Bull. Japanese Soc. Microbial Ecol.) 2, 13-19 (1987). S. P. Singh, S. C. Srivastava and K. D. Pandey, Photoproduction of hydrogen by a non-sulphur bacterium isolated from root zones of water fern Azolla pinnata. Int. J. Hydrogen Energy 15, 403-406 (1990). N. Pfennig, Photosynthetic bacteria. A. Rev. Microbiol. 21, 285-324 (1967).
405
6. O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951). 7. D. Herbert, P. J. Phipps and R. E. Strange, Chemical analysis of microbial cells. In J. R. Norris and D. W. Ribbons (eds), Methods in Microbiology, Vol. VB, pp. 209344. Academic Press, London (1971). 8. A. Mitsui, C. Hill and D. Rosner, The association of photosynthetic microorganisms with debris of macroalgae, seagrasses and mangrove leaves. Abst. Amer. Soc. Microbiol., pp. 24 (1974). 9. P. F. Weaver, Photoconversion of organic substrates into hydrogen using photosynthetic bacteria. Proc. Energy from Biomass and Wastes V Conf., pp. 489-497. Institute of Gas Technology, Chicago (1981). 10. J. Miyake and S. Kawamura, Efficiency of light energy conversion to hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides. Int. J. Hydrogen Energy 12, 147-149 (1987).
0360-3199/91 $3.00+ 0.00 PergamonPress plc. InternationalAssociationfor HydrogenEnergy.
Printed in Great Britain.
ISOLATION OF N O N - S U L P H U R PHOTOSYNTHETIC BACTERIAL STRAINS EFFICIENT IN H Y D R O G E N P R O D U C T I O N AT ELEVATED TEMPERATURES S. P. SINGH* and S. C. SRIVASTAVA Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India
(Receivedfor publication 13 December 1990) Abstract--Four strains of non-sulphur photosynthetic bacteria were isolated from root zone associations of aquatic plants like Azolla, Salvinia and Eichhornia, as well as the deep-water rice. Based on the gross cell morphology and pigmentation, the isolates resembled Rhodopseudomonas sp. and have been designated as BHU strains 1 to 4, respectively. When subjected to elevated temperature (from 33-45°C), substantial growth/hydrogen production could be observed only in strains 1 and 4. Strains 2 and 3 on the other hand, showed diminished growth and negligible hydrogen photoproduction. The BHU strains 1 and 4 have been selected as the most active (thermostable) hydrogen producing strains of local origin as far as the Indian tropical climate is concerned.
hornia as these formed a major component of floating
INTRODUCTION The major concern about the efficient hydrogen producing photosynthetic bacteria has been their tolerance against high light irradiance as well as high temperature, Efforts in this direction have yielded positive results only in a few cases. Watanabe et al. [1] made a comparison of hydrogen production by different strains of Rhodopseudomonas originating from Japan and Thailand, and the majority of strains in the latter case exhibited thermostable hydrogen production within a maximum limit of 40°C. The temperature upper limit, extended to 45°C, restricted hydrogen production to only R. sphaeroides B5 isolated from Thailand and SL16 from Korea and KM ! 13 from Japan [2]. Similarly, four strains of photosynthetic bacteria isolated from the Bangkok area were reported to grow well at 40°C but a coculture of strain TR-22R-B and the strict aerobic bacterium, 22TW-S of the same origin, resulted in growth of both the organisms at 45°C, thus suggesting that the growth of photosynthetic bacteria could be encouraged by the associating aerobe at elevated temperature provided some oxygen migrated from the air into the culture media [3]. Thermostable photohydrogen production is reported here in selected strains of the non-sulphur photosynthetic bacterium, Rhodopseudomonas sp., isolated from the root associations of aquatic ferns and deep water rice. EXPERIMENTAL
plant community in local ponds. Deep water rice plant roots were collected from Surha Tal (Lake), Ballia District (about 250 km east). Non-sulphur photosynthetic bacteria were isolated by the conventional enrichment technique under light-anaerobic conditions as reported earlier by Singh et al. [4]. The bacterial strains isolated from the root zone associations of Azolla, Salvinia, Eichhornia and deep water rice plants have been tentatively designated as Rhodopseudomonas sp. BHU strain-I to 4, respectively (BHU for Banaras Hindu University, India).
Growth medium The basal medium as prescribed by Pfennig [5], was used with slight modifications: yeast extract, 0.5 g 1-~; DL-malate, 1.87 g l-l; KH2PO4 ' 1.0 g l ~; MgSO4.7H20, 0.4 g 1-~; NaC1, 0.4 g 1-1; sodium succinate, 2.0 g 1-~ and CaCI 2.2H 20, 0.05 g 1-1. Ammonium chloride (0.1 g/l) was added initially to anaerobic cultures for initiation of growth. All chemicals were of laboratory reagent grade obtained from BDH (U.K. or India). The medium was sterilized at 101b (15 min) and left overnight before bacterial inoculation. The pH of the growth medium was adjusted to 6.8. The concentrated bacterial cell suspensions were transferred anaerobically at a cell density of OD660= 0.3 in 60 ml fresh growth medium in rubberstoppered glass bottles having a gas phase of 95% argon and 5% CO2. Cultures were grown under tungsten illumination (2 kLux) with periodic shaking.
lnocula
Assays of hydrogen production
The bacterial strains were isolated from the root zones of different aquatic plants like Azolla, Salvinia and Eich-
A 0.5 ml samples of gas phase from the light-incubated bacterial cultures was analysed for H 2 in a gas chromatograph (Tracor 540, USA) on TCD mode fitted with Carbosieve-II column and connected to Hewlett-Packard
*Author to whom correspondence should be addressed.
403
404
S.P. SINGH and S. C. SRIVASTAVA
3392 A integrator. The values have been expresed as/~1 H 2 m g - l dry wt. Dry wt measurements were based on the conventional oven-drying of bacterial cells,
Temperature upshift experiments For comparison of hydrogen production activity, all the four bacterial strains were grown at 33°C and 45°C under the same degree of illumination (2 kLux). Parallel sets were also maintained for the simultaneous measurements of bacterial growth (expressed as/~g protein ml -l culture). Protein was estimated by the method of Lowry et al. [6], modified by Herbert et al. [7]. RESULTS A N D DISCUSSION The very association of Rhodospirillaceae with roots of aquatic plants, as observed here and reported earlier by Singh et aL [4], is in line with the reports of Mitsui et al. [8] that photosynthetic bacteria thrive well with debris of macroalgae, seagrasses and mangrove leaves. As regards light-to-hydrogen energy conversion efficiency [9, 10], it could reach a maximum of 4.9% or 7.9% respectively, Much emphasis has been laid on the feasibility of hydrogen production by such bacteria outdoor subjected to the prevailing daylength (irradiance) as well as temperature. Using a solar simulator, Miyake and Kawamura [10] observed high light energy conversion to hydrogen in R. sphaeroides 8703 at low light intensity corresponding to 50 Wm 2 that decreased to 6.3% if the irradiance was raised by a factor of ten. This eventually puts the efficiency of solar energy conversion as a critical factor in determining hydrogen production by photosynthetic bacteria, and emphasizes the need for the time to isolate/ train such microbes to adapt to elevated irradiance or temperature common to the tropics. The present experiments, have therefore, taken into account the possibility of thermostable hydrogen production by a few strains of Rhodospirillaceae at 33 and 45°C; the temperature range most common to a major part of Indian climate, We have recently reported on the isolation and photohydrogen production in a non-sulphur photosynthetic bacterium resembling Rhodopseudomonas designated as BHU strain 1 [4]. This strain was found growing in association with the roots of water fern Azolla. The Table'l. Effect of temperature on the activity of hydrogen photoproduction by four strains of non-sulphur photosynthetic bacteria Hydrogen photoproduction (pl H 2 h-i mg-i dry wt) Strain no. BHU strain 1 BHU strain 2 BHU strain 3 BHU strain 4
Source Azollaroot zone Salviniaroot zone Eichhorniaroot zone Deep water rice plant root zone
33°C 12 (292)* 10 (283)* 9 (264)* 12 (231)*
45°C 7 (231)* 4 (180)* 2.5 (168)* 15 (200)*
* Values indicate growth yield in terms of #g protein ml-t bacterialculture,
other three strains examined for hydrogen production at elevated temperatures also originated from similar associations of another water fern, Salvinia, water hyacinth and deep water rice plant, respectively. Data presented in Table I display a comparison of relative thermotolerance of BHU strains 1-4 in terms of growth yield and hydrogen production activity as light-anaerobic cultures. As all the strains were routinely grown at 33°C, BHU strains 1 and 4 invariably maintained their superiority in terms of hydrogen production over strains 2 and 3. However, the higher rate of hydrogen production in strain 4 was not accompanied by increased growth yield compared to strain 1. Subsequent to temperature shift-up (45°C) also, strain 4 proved to be on top of others with a rate of 15pl H2h l mg-I dry wt followed by BHU strain 1 (7/~ l H 2 h - l mg I dry wt). The latter, however, showed better growth yield (231/~g protein m l - l culture over strain 4 (200 #g protein ml 1 culture). This again emphasizes that hydrogen production activity may not be strictly correlated with growth rate/yield, and the efficiency is strain specific. Such a specificity is quite evident when one compares hydrogen production by all the four strains at 45°C. It may also be mentioned that BHU strains 1-4 inhabited almost the same type of habitat with heavy load of organics but differed a great deal in terms of their thermostable growth/hydrogen production. Strains originating from either Salvinia or Eichhornia, on the other hand, proved thermosensitive as reflected by diminished growth and extremely low rates of hydrogen production. Similar comparisons made by others showed that majority of Rhodopseudomonas strains from Thailand showed almost equal activity rates of hydrogen production at 30 ° or 40°C compared to those originating from either Japan or Thailand itself [l]. Kim et al. [2] in a subsequent study, also observed higher rates of hydrogen production at 40°C in R. sphaeroides B5 from Thailand over strains SL1, SL3, SL16 from Korea and K M l l 3 and TN3 from Japan. However, thermotolerant hydrogen production could be noticed in cultures of strain B5, SL 16 and K M I I 3 at 45°C. The stimulated high temperature growth of a photosynthetic bacterium (strain TR-22R-B) by the co-existing aerobe (strain 22TW-S) in culture, has been attributed to the input of some oxygen from air [3]. In conclusion, therefore, we can suggest the suitability of BHU strains 1 and 4 for photohydrogen generation outdoor in regions with temperature ranging between 33 ° and 45°C. Work is in progress to look for light to hydrogen conversion efficiency in such strains also at high irradiance.
Acknowledgements--This work was supported by a research grant from the Department of Non-Conventional Energy Sources, Government of India, New Delhi. Authors are grateful to Dr K. D. Pandey for technical details and helpful discussion. REFERENCES 1. K. Watanabe, J. S. Kim, K. Ito, L. Buranakarl, T. Kampee and H. Takahashi, Thermostable nature of hydrogen
ISOLATION OF NON-SULPHUR PHOTOSYNTHETIC BACTERIA
2.
3.
4.
5.
production by non-sulfur purple photosynthetic bacteria isolated in Thailand. Agric. Biol. Chem. 45, 217-222 (1981). J. S. Kim, H. Yamauchi, K. Ito and H. Takahashi, Selection of a photosynthetic bacterium suitable for hydrogen production in outdoor cultures among strains isolated in the Seoul, Taegu, Sendai and Bangkok areas. Agric. Biol. Chem. 46, 1469-1474 (1982). L. Buranakarl, H. Yoneyama, T. Iwata, J. S. Kim, S.I. Okuda, T. Kampee, K. Izaki and H. Takahashi, Growth of non-sulfur purple photosynthetic bacteria under high temperature. Nihon Biseibutsu Seitai Gakkaiho (Bull. Japanese Soc. Microbial Ecol.) 2, 13-19 (1987). S. P. Singh, S. C. Srivastava and K. D. Pandey, Photoproduction of hydrogen by a non-sulphur bacterium isolated from root zones of water fern Azolla pinnata. Int. J. Hydrogen Energy 15, 403-406 (1990). N. Pfennig, Photosynthetic bacteria. A. Rev. Microbiol. 21, 285-324 (1967).
405
6. O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951). 7. D. Herbert, P. J. Phipps and R. E. Strange, Chemical analysis of microbial cells. In J. R. Norris and D. W. Ribbons (eds), Methods in Microbiology, Vol. VB, pp. 209344. Academic Press, London (1971). 8. A. Mitsui, C. Hill and D. Rosner, The association of photosynthetic microorganisms with debris of macroalgae, seagrasses and mangrove leaves. Abst. Amer. Soc. Microbiol., pp. 24 (1974). 9. P. F. Weaver, Photoconversion of organic substrates into hydrogen using photosynthetic bacteria. Proc. Energy from Biomass and Wastes V Conf., pp. 489-497. Institute of Gas Technology, Chicago (1981). 10. J. Miyake and S. Kawamura, Efficiency of light energy conversion to hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides. Int. J. Hydrogen Energy 12, 147-149 (1987).