- Email: [email protected]
Environmental regulation for optimal biomass yield and photoproduction of hydrogen by Rhodobacter sphaeroides O.U. 001
Int. J. Hrdrogen Energy, Vol. 16, No. 9, pp. 597-601, 1991. Printed in Great Britain.
0360 3199'91 $3.00 + 0.00 Pergamon Press pie. ~' 1991 International Association for Hydrogen Energy.
ENVIRONMENTAL REGULATION FOR OPTIMAL BIOMASS Y I E L D A N D P H O T O P R O D U C T I O N OF H Y D R O G E N BY R H O D O B A C T E R S P H A E R O I D E S O.U. 001" K. SASIKALA,CH. V. RAMANA and P. RAGHUVEERRAO Microbial Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad 500 007, India
(Receivedfor publication 8 May 1991) Abstract--The effect of pH, temperature, light intensity and concentration of carbon source/e donor and nitrogen source on the regulation of biomass yield and photoproduction of hydrogen (the fuel of the future) by a purple non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides O.U. 001, was studied. The importance of culture densily and age on the hydrogen photoproduction is also discussed.
INTRODUCTION Biological hydrogen generation by photosynthetic bacteria, being investigated all over the world as a potential source of renewable and pollution-free fuel involving less capital inputs and simple facilities, was proved promising for large scale generation [1]. Several environmental factors influence the hydrogen production and their optimization is essential to obtain maximal yields. Work in this regard is restricted to one or two parameters on a number of organisms [2, 3], or one or a few parameters in a single organism [4, 5]. However, for a practical system of hydrogen production it is essential to study all of the factors influencing the biomass yield and hydrogen production by an organism with promising potential. Here, we report the results of optimization of pH, temperature, light intensity and concentrations of carbon source/e donor and nitrogen source for both biomass yield and photoproduction of hydrogen by Rhodobacter sphaeroides O.U. 001, an organism with good potential for hydrogen photoproduction [6].
temperature for growth were 7,0 and 30 _+ 2 C respectively.
Biomass yield Biomass yield was determined in terms of dry weight as described earlier [7].
Hydrogen production assay Washed cell suspensions of early stationary phase cultures growing photoheterotrophically were used for hydrogen production assay. Cell suspensions (6 ml) were assayed for hydrogen photoproduction under an atmosphere of argon gas chromatographically [6]. For temperature and light intensity effects, manometric methods [8] were used. Cell suspension 12.2 ml) was taken in the main chamber of the Warburg flask and, in the centre well on a filter paper wick 0.2 ml of 20% (w/v) KOH solution was taken to absorb carbon dioxide. Experimental details are given in the foot notes of respective figures. RESULTS AND DISCUSSION
MATERIALS A N D METHODS
Organism and growth conditions
Hydrogen production was studied as a two step process, i.e. (1) the growth phase to obtain hydrogen generating biomass and (2) hydrogen photoproduction by resting cells from the biomass obtained. In the first step, the culture was provided with a nitrogen source for growth while the hydrogen assay medium was devoid of a nitrogen source. The regulation of the above two processes by some of the important environmental factors is discussed.
A local isolate of purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides O.U. 001 (ATCC 49419; DSM 5864) was grown photoheterotrophically [7] in fully-filled screw cap test tubes (30 ml capacity) with malate (30mM) and sodium glutamate (10mM) as carbon and nitrogen sources respectively. Light was provided by a bank of white fluorescent tubes at an intensity of 4000 lux as measured by a lux meter, pH and
pH
*Dedicated to the loving memory of the late Drs (Mrs) B. Renuka Rao and M. Vinayakumar.
Growth was observed at a wide range of pH (pH 6--9) tested; optimal biomass was obtained at pH 6.8 7.0 (Fig. 1) as also observed for many strains of 597
K. SASIKALA et al.
598
E
3.6E
b
~3
cn 2.Z
"o £n
£
E2
._~ m 1'2
:>,
bt 1 r~ E .o_
E .Q
--9 5 •-•
7'58
[9, 10]. Hydrogen production was optimum at pH 7.0. At p H 6.0, low levels of hydrogen production occurred for 72 h, later a rapid and steady increase was observed (Fig. 2).
Temperature Biomass yield was optimum at a temperature of 30-35°C (Fig. 3). G r o w t h was not observed at temperatures below 20°C or above 45°C. Photoproduction of hydrogen was optimum at a temperature range of 30-40°C (Fig. 4). This wide temperature tolerance of the strain makes it particularly suitable for outdoor experiments in tropical regions.
/ / ~ ~ /
2.0;
Fig. 3. The influence of temperature on the biomass yield of R. sphaeroides O.U. 001. Biomass yield of R. sphaeroides O.U. 001 at various temperatures compared. Other experimental conditions as in Fig. I. The pH of the medium was 7.0.
Light intensity Light was an absolute requirement for growth of the organism under anaerobic conditions [7]. Light intensity influenced growth rate (data not shown) rather than the final biomass that could be obtained. When assayed after 72 h of growth (data not shown), the biomass yield at various light intensities was more or less the same. However, when assayed after 48 h of growth (Fig. 5), there was variation in the biomass obtained. Growth under continuous light was better than that under conditions where light and dark cycles exist (natural sunlight; data not shown). Hydrogen photoproduction was saturated at a light intensity of 5000 lux (Fig. 6). For both growth and photoproduction of hydrogen, light intensity higher than optimum, however, did not cause inhibition unlike the algal hydrogen evolution [11] where high light intensities result in partial inhibition or total cessation of hydrogen evolution.
8-07'0 9.0 2.8
1.61-2~
6-0
g
5.0
0
~6 15 20 25 30 35 40 45 50
Temp (°C)
sphaeroides
2.4-
0
859
Fig. 1. The influence of pH of the growth medium on the biomass yield of R. sphaeroides O.U. 001. Biomass yield of R. sphaeroides O.U. 001 at various pH was compared. Malate (30 mM) and sodium glutamate (10 mM) were the carbon and nitrogen sources respectively. Data pertain to that after 48 h of growth. The temperature and light intensity were 30 _+2°C and 4000 lux respectively.
R.
tl
Q3
6 6"56.87 PH
24
48
72
9'6
1:20 144
Time(hrs) Fig. 2. The influence of initial pH of the assay medium on photoproduction of hydrogen by resting cells of R. sphaeroides O.U. 001. Resting cell suspension (5 ml; 0.56 mg dry wt m l - ' ) of log phase cultures grown anaerobically in light [with malate (30 mM) and sodium glutamate (10 mM) as C and N sources respectively] were assayed for hydrogen photoproduction from malate (30mM). The temperature and light intensity of the assay were 30 + 2°C and 4000 lux respectively. The pH was varied as indicated.
2.4 2-0 ~1.6 \1.2. 0.8.
~ I
~ 0.4.
0
0
o 10 15 2 0 2 5 3 0 3 5 4 0 4 5 5 0 Temp (°C)
Fig. 4. The influence of temperature on photoproduction of hydrogen by R. sphaeroides O.U. 001. Resting cell suspensions (2.2 ml; 0.9 mg dry wt ml 1) were assayed for photoproduction of hydrogen at various temperatures. The hydrogen assay was done manometrically. Results pertain to that after 5 h of anaerobic incubation. The pH and light intensity of the assay were 7.0 and 4000 lux respectively.
H 2 PRODUCTION BY RHODOBACTER SPHAEROIDES
599
o
.10
,/
~£_2.4 /~
D//"
~3
2.0
c~ 1.6
/"
"° 1"2 0.8
~ 0'4 m
0
/
/
/
/"
/
753
Y
(D
//
/
E o ~3
soo
26o0 36oo 6oo
6o0
,/
/
0
-
Fig. 5. The effect of light intensity on the biomass yield of R.
sphaeroides O.U. 001. Experimental conditions as in Fig. 1, except that the light intensity was changed as indicated. The pH of the medium was 7.0.
.... 1~0
2~0 .... 3~0
Fig. 7. The influence of concentration of malate on the biomass yield of R. sphaeroides O.U. 001. Experimental conditions as in Fig. 1, except that the concentration of the malate was changed as indicated.
/o---o
3-0-
/
2.4 1.8\
~
7'5
Malate Conc. (mM)
Light intensity (Lux)
1.2
/
/
/
/
/
-'50
o/
>
£,
, -
1000
--,
2500 6o0 m'oo Light intensity (Lux)
taboo
Fig. 6. The effect of light intensity on the photoproduction of hydrogen by R. sphaeroides O.U. 001. Experimental conditions as in Fig. 4, except that light intensity was varied as indicated. The temperature of the assay was 30 _+ ZC.
Concentration of carbon source~e- donor T h e c o n c e n t r a t i o n of c a r b o n source/e d o n o r (malate) influenced biomass yield (Fig. 7), with a n increasing c o n c e n t r a t i o n of m a l a t e ( 5 - 3 0 m M ) resulting in increased biomass yield. M a l a t e at a c o n c e n t r a t i o n of 30 m M was f o u n d to be o p t i m u m for the p h o t o p r o d u c tion of h y d r o g e n (Fig. 8). A b o v e this c o n c e n t r a t i o n , a slight reduction in h y d r o g e n p h o t o p r o d u c t i o n was observed. However, with lactate, h y d r o g e n p r o d u c t i o n increased with increasing c o n c e n t r a t i o n s (Fig. 9), emphasizing the need to optimize the c o n c e n t r a t i o n of the particular substrate to be employed in commercial production.
0
24
48
72
96
120
144
Time (hr)
Fig. 8. The influence of the concentration of malate on photoproduction of hydrogen by R. sphaeroides O.U. 001. Experimental conditions as in Fig. 2, except that the concentration of malate was changed as indicated. The pH of the assay was 7.0.
mM
~a 2
o10
~
Concentration of nitrogen source A n u m b e r of nitrogen sources could be utilized for growth of this o r g a n i s m [7], o p t i m i z a t i o n of the concentration of sodium g l u t a m a t e which p r o v e d best was carried out, which showed a n o p t i m u m at 1 2 - 1 4 m M (Fig. 10). P h o t o p r o d u c t i o n o f h y d r o g e n was high in the absence of any c o m b i n e d nitrogen source in the m e d i u m (data not shown).
24
48
72 96 Time (hr)
120
144
Fig. 9. The influence of concentration of lactate on photoproduction of hydrogen by R. sphaeroides O.U. 001. Experimental conditions as in Fig. 8, except that lactate was used as the electron donor at the various concentrations indicated.
600
K. SASIKALA eta/.
~4 E
2.0
¸
1.6
~> /
1'2
/,
$2
9 0.8 /
E
/
0,4
//
5
0
8
I0 12 14 I;7 2'0
Sod. Glutamate Conc (raM) Fig. 10. The effect of sodium glutamate (nitrogen source) concentration on the biomass yield of R. sphaeroides O.U. 001. Experimental details as in Fig. 1, except the concentration of sodium glutamate was changed as indicated, The pH of the medium was 7.0. 1.4
Z 8
1'2 16 20 24 28 32 36 Culture age(hr) Fig. 12. The effect of culture age on the photoproduction of hydrogen by R. sphaeroides O.U. 001. Cell suspensions prepared from cultures of various ages were used for the hydrogen production assay. The biomass of all the suspensions was made equal by adjusting to a common O.D. (biomass of 0.5 mg dry wt ml t). Other experimental details as in Fig. 2. The results pertain to that after 24 h of incubation.
1.2 ising for employment outdoors, under the local climatic conditions.
1'0 i0.8 _~
(?.6-
9
0.4-
~
£ 0.2 0
0
0.2 0.3 0.4 0,6 0.7 1.0 1.2 1,6 1'8 2.0 Cell Conc. ( mg dry wt/ml) Fig. 1I. The effect of cell concentration on the photoproduction of hydrogen by R. sphaeroides O.U. 001. Resting cell suspensions (3 ml) were assayed for photoproduction of hydrogen from malate. Experimental conditions as in Fig. 2. Results pertain to that after 24 h of incubation.
Cell concentration for hydrogen photoproduction With an increasing concentration of cells (0.2-1.6 mg dry wt m1-1), hydrogen production increased. Optimal cell concentration for hydrogen production was 1.6-1.8 mg dry wt ml L A higher cell mass resulted in lowering of hydrogen photoevolution (Fig. 11), indicating that higher densities of cells were not favourable for hydrogen evolution. Age of the culture for hydrogen photoproduction Cultures growing photoheterotrophically under anaerobic conditions were harvested at various intervals of time, and the optical density of the cell suspension was adjusted to 0.15 at 6 6 0 n m (equal to 0.56mg dry wt ml ~) and subjected to hydrogen production assay, which revealed that mid log phase cultures (20 h old) were better producers of hydrogen (Fig. 12). The data generated in this investigation will form a basis for the designing and fabrication of a reactor for large scale generation of biomass and photohydrogen production by this organism, which is particularly prom-
Acknowledgements--The authors wish to thank the Department of Non-conventional Energy Sources, New Delhi, for financial support. K.S. thanks the U.G.C., New Delhi, and Ch.V.R. thanks the C.S.I.R. New Delhi, for the award of research fellowships. REFERENCES 1. J. S. Kim, K. Ito and H. Takahashi, Production of molecular hydrogen in outdoor batch cultures of Rhodopseudomonas sphaeroides. Agric. Biol. Chem. 46, 937-941 (1982). 2. K. Watanabe, J. S. Kim, K. Ito, L Buranakarl, T. Kampee and H. Takahashi, Therm-stable nature of hydrogen production by non-sulfur purple photosynthetic bacteria isolated in Thailand. Agric. Biol. Chem. 45, 217-222 (1981). 3. P. Stevens, C. Vertonghen, P. Devos and J. De Ley, The effect of temperature and light intensity on hydrogen gas production by different Rhodopseudomonas capsulata strains. Biotechnol. Lett. 6, 277 282 (1984). 4. P. Hillmer and H. Gest, Hydrogen metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: hydrogen production by growing cultures. J. Bacteriol. 129, 724-731 (1977). 5. P. Hillmer and H. Gest, Hydrogen metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: production and utilization of hydrogen by resting cells. J. Bacteriol. 129, 732 739 (1977). 6. K. Sasikala, Ch. V. Ramana, P. Raghuveer Rao and M. Subrahmanyam, Effect of gas phase on the photoproduction of hydrogen and substrate conversion efficiency in the photosynthetic bacterium Rhodobacter sphaeroides O.U. 001. Int. J. Hydrogen Energy 15, 795-797 (1990). 7. K. Sasikala and Ch. V. Ramana, Production of hydrogen by photosynthetic purple non-sulfur bacteria: isolation, characterization, identification and growth of Rhodobacter sphaeroides O.U. 001. Proc. Ind. Nam. Sci. Acad. B56, 235-240 (1990). 8. W. W. Umbreit, R. H. Burris and J. F. Stauffer, Manometric techniques, 4th edn. Burgess, Minneapolis (1964).
It 2 PRODUCTION BY RHODOBACTER SPHAEROIDES 9. J. t:. lmhoff, Genus Rhodobacter. In J. T. Stanley, N. Pfennig and J. G. Holts (eds) Bergey's Manual o/ Systematic Bacteriology, vol. 3, pp. 1663-1672. Williams and Wilkins, Baltimore (1989). 10. K. Sasaki and S. Nagai, The optimum pH and temperature for the aerobic growth of Rhodopseudomonas gelatinosa and
6()1
vitamin BI2 and ubiquinone formation on a starch medium J. Ferment. Technol. 57, 383 386 (1979). II. B. Mahro and L H. Grimme, Hydrogen photoproduction by green algae: the significance of anaerobic preincubation periods and of high light intensities for hsdrogen production of Chorella fU.sca, Arch. Mi(rohiol. 132, 82 ~6 (19821
0360 3199'91 $3.00 + 0.00 Pergamon Press pie. ~' 1991 International Association for Hydrogen Energy.
ENVIRONMENTAL REGULATION FOR OPTIMAL BIOMASS Y I E L D A N D P H O T O P R O D U C T I O N OF H Y D R O G E N BY R H O D O B A C T E R S P H A E R O I D E S O.U. 001" K. SASIKALA,CH. V. RAMANA and P. RAGHUVEERRAO Microbial Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad 500 007, India
(Receivedfor publication 8 May 1991) Abstract--The effect of pH, temperature, light intensity and concentration of carbon source/e donor and nitrogen source on the regulation of biomass yield and photoproduction of hydrogen (the fuel of the future) by a purple non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides O.U. 001, was studied. The importance of culture densily and age on the hydrogen photoproduction is also discussed.
INTRODUCTION Biological hydrogen generation by photosynthetic bacteria, being investigated all over the world as a potential source of renewable and pollution-free fuel involving less capital inputs and simple facilities, was proved promising for large scale generation [1]. Several environmental factors influence the hydrogen production and their optimization is essential to obtain maximal yields. Work in this regard is restricted to one or two parameters on a number of organisms [2, 3], or one or a few parameters in a single organism [4, 5]. However, for a practical system of hydrogen production it is essential to study all of the factors influencing the biomass yield and hydrogen production by an organism with promising potential. Here, we report the results of optimization of pH, temperature, light intensity and concentrations of carbon source/e donor and nitrogen source for both biomass yield and photoproduction of hydrogen by Rhodobacter sphaeroides O.U. 001, an organism with good potential for hydrogen photoproduction [6].
temperature for growth were 7,0 and 30 _+ 2 C respectively.
Biomass yield Biomass yield was determined in terms of dry weight as described earlier [7].
Hydrogen production assay Washed cell suspensions of early stationary phase cultures growing photoheterotrophically were used for hydrogen production assay. Cell suspensions (6 ml) were assayed for hydrogen photoproduction under an atmosphere of argon gas chromatographically [6]. For temperature and light intensity effects, manometric methods [8] were used. Cell suspension 12.2 ml) was taken in the main chamber of the Warburg flask and, in the centre well on a filter paper wick 0.2 ml of 20% (w/v) KOH solution was taken to absorb carbon dioxide. Experimental details are given in the foot notes of respective figures. RESULTS AND DISCUSSION
MATERIALS A N D METHODS
Organism and growth conditions
Hydrogen production was studied as a two step process, i.e. (1) the growth phase to obtain hydrogen generating biomass and (2) hydrogen photoproduction by resting cells from the biomass obtained. In the first step, the culture was provided with a nitrogen source for growth while the hydrogen assay medium was devoid of a nitrogen source. The regulation of the above two processes by some of the important environmental factors is discussed.
A local isolate of purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides O.U. 001 (ATCC 49419; DSM 5864) was grown photoheterotrophically [7] in fully-filled screw cap test tubes (30 ml capacity) with malate (30mM) and sodium glutamate (10mM) as carbon and nitrogen sources respectively. Light was provided by a bank of white fluorescent tubes at an intensity of 4000 lux as measured by a lux meter, pH and
pH
*Dedicated to the loving memory of the late Drs (Mrs) B. Renuka Rao and M. Vinayakumar.
Growth was observed at a wide range of pH (pH 6--9) tested; optimal biomass was obtained at pH 6.8 7.0 (Fig. 1) as also observed for many strains of 597
K. SASIKALA et al.
598
E
3.6E
b
~3
cn 2.Z
"o £n
£
E2
._~ m 1'2
:>,
bt 1 r~ E .o_
E .Q
--9 5 •-•
7'58
[9, 10]. Hydrogen production was optimum at pH 7.0. At p H 6.0, low levels of hydrogen production occurred for 72 h, later a rapid and steady increase was observed (Fig. 2).
Temperature Biomass yield was optimum at a temperature of 30-35°C (Fig. 3). G r o w t h was not observed at temperatures below 20°C or above 45°C. Photoproduction of hydrogen was optimum at a temperature range of 30-40°C (Fig. 4). This wide temperature tolerance of the strain makes it particularly suitable for outdoor experiments in tropical regions.
/ / ~ ~ /
2.0;
Fig. 3. The influence of temperature on the biomass yield of R. sphaeroides O.U. 001. Biomass yield of R. sphaeroides O.U. 001 at various temperatures compared. Other experimental conditions as in Fig. I. The pH of the medium was 7.0.
Light intensity Light was an absolute requirement for growth of the organism under anaerobic conditions [7]. Light intensity influenced growth rate (data not shown) rather than the final biomass that could be obtained. When assayed after 72 h of growth (data not shown), the biomass yield at various light intensities was more or less the same. However, when assayed after 48 h of growth (Fig. 5), there was variation in the biomass obtained. Growth under continuous light was better than that under conditions where light and dark cycles exist (natural sunlight; data not shown). Hydrogen photoproduction was saturated at a light intensity of 5000 lux (Fig. 6). For both growth and photoproduction of hydrogen, light intensity higher than optimum, however, did not cause inhibition unlike the algal hydrogen evolution [11] where high light intensities result in partial inhibition or total cessation of hydrogen evolution.
8-07'0 9.0 2.8
1.61-2~
6-0
g
5.0
0
~6 15 20 25 30 35 40 45 50
Temp (°C)
sphaeroides
2.4-
0
859
Fig. 1. The influence of pH of the growth medium on the biomass yield of R. sphaeroides O.U. 001. Biomass yield of R. sphaeroides O.U. 001 at various pH was compared. Malate (30 mM) and sodium glutamate (10 mM) were the carbon and nitrogen sources respectively. Data pertain to that after 48 h of growth. The temperature and light intensity were 30 _+2°C and 4000 lux respectively.
R.
tl
Q3
6 6"56.87 PH
24
48
72
9'6
1:20 144
Time(hrs) Fig. 2. The influence of initial pH of the assay medium on photoproduction of hydrogen by resting cells of R. sphaeroides O.U. 001. Resting cell suspension (5 ml; 0.56 mg dry wt m l - ' ) of log phase cultures grown anaerobically in light [with malate (30 mM) and sodium glutamate (10 mM) as C and N sources respectively] were assayed for hydrogen photoproduction from malate (30mM). The temperature and light intensity of the assay were 30 + 2°C and 4000 lux respectively. The pH was varied as indicated.
2.4 2-0 ~1.6 \1.2. 0.8.
~ I
~ 0.4.
0
0
o 10 15 2 0 2 5 3 0 3 5 4 0 4 5 5 0 Temp (°C)
Fig. 4. The influence of temperature on photoproduction of hydrogen by R. sphaeroides O.U. 001. Resting cell suspensions (2.2 ml; 0.9 mg dry wt ml 1) were assayed for photoproduction of hydrogen at various temperatures. The hydrogen assay was done manometrically. Results pertain to that after 5 h of anaerobic incubation. The pH and light intensity of the assay were 7.0 and 4000 lux respectively.
H 2 PRODUCTION BY RHODOBACTER SPHAEROIDES
599
o
.10
,/
~£_2.4 /~
D//"
~3
2.0
c~ 1.6
/"
"° 1"2 0.8
~ 0'4 m
0
/
/
/
/"
/
753
Y
(D
//
/
E o ~3
soo
26o0 36oo 6oo
6o0
,/
/
0
-
Fig. 5. The effect of light intensity on the biomass yield of R.
sphaeroides O.U. 001. Experimental conditions as in Fig. 1, except that the light intensity was changed as indicated. The pH of the medium was 7.0.
.... 1~0
2~0 .... 3~0
Fig. 7. The influence of concentration of malate on the biomass yield of R. sphaeroides O.U. 001. Experimental conditions as in Fig. 1, except that the concentration of the malate was changed as indicated.
/o---o
3-0-
/
2.4 1.8\
~
7'5
Malate Conc. (mM)
Light intensity (Lux)
1.2
/
/
/
/
/
-'50
o/
>
£,
, -
1000
--,
2500 6o0 m'oo Light intensity (Lux)
taboo
Fig. 6. The effect of light intensity on the photoproduction of hydrogen by R. sphaeroides O.U. 001. Experimental conditions as in Fig. 4, except that light intensity was varied as indicated. The temperature of the assay was 30 _+ ZC.
Concentration of carbon source~e- donor T h e c o n c e n t r a t i o n of c a r b o n source/e d o n o r (malate) influenced biomass yield (Fig. 7), with a n increasing c o n c e n t r a t i o n of m a l a t e ( 5 - 3 0 m M ) resulting in increased biomass yield. M a l a t e at a c o n c e n t r a t i o n of 30 m M was f o u n d to be o p t i m u m for the p h o t o p r o d u c tion of h y d r o g e n (Fig. 8). A b o v e this c o n c e n t r a t i o n , a slight reduction in h y d r o g e n p h o t o p r o d u c t i o n was observed. However, with lactate, h y d r o g e n p r o d u c t i o n increased with increasing c o n c e n t r a t i o n s (Fig. 9), emphasizing the need to optimize the c o n c e n t r a t i o n of the particular substrate to be employed in commercial production.
0
24
48
72
96
120
144
Time (hr)
Fig. 8. The influence of the concentration of malate on photoproduction of hydrogen by R. sphaeroides O.U. 001. Experimental conditions as in Fig. 2, except that the concentration of malate was changed as indicated. The pH of the assay was 7.0.
mM
~a 2
o10
~
Concentration of nitrogen source A n u m b e r of nitrogen sources could be utilized for growth of this o r g a n i s m [7], o p t i m i z a t i o n of the concentration of sodium g l u t a m a t e which p r o v e d best was carried out, which showed a n o p t i m u m at 1 2 - 1 4 m M (Fig. 10). P h o t o p r o d u c t i o n o f h y d r o g e n was high in the absence of any c o m b i n e d nitrogen source in the m e d i u m (data not shown).
24
48
72 96 Time (hr)
120
144
Fig. 9. The influence of concentration of lactate on photoproduction of hydrogen by R. sphaeroides O.U. 001. Experimental conditions as in Fig. 8, except that lactate was used as the electron donor at the various concentrations indicated.
600
K. SASIKALA eta/.
~4 E
2.0
¸
1.6
~> /
1'2
/,
$2
9 0.8 /
E
/
0,4
//
5
0
8
I0 12 14 I;7 2'0
Sod. Glutamate Conc (raM) Fig. 10. The effect of sodium glutamate (nitrogen source) concentration on the biomass yield of R. sphaeroides O.U. 001. Experimental details as in Fig. 1, except the concentration of sodium glutamate was changed as indicated, The pH of the medium was 7.0. 1.4
Z 8
1'2 16 20 24 28 32 36 Culture age(hr) Fig. 12. The effect of culture age on the photoproduction of hydrogen by R. sphaeroides O.U. 001. Cell suspensions prepared from cultures of various ages were used for the hydrogen production assay. The biomass of all the suspensions was made equal by adjusting to a common O.D. (biomass of 0.5 mg dry wt ml t). Other experimental details as in Fig. 2. The results pertain to that after 24 h of incubation.
1.2 ising for employment outdoors, under the local climatic conditions.
1'0 i0.8 _~
(?.6-
9
0.4-
~
£ 0.2 0
0
0.2 0.3 0.4 0,6 0.7 1.0 1.2 1,6 1'8 2.0 Cell Conc. ( mg dry wt/ml) Fig. 1I. The effect of cell concentration on the photoproduction of hydrogen by R. sphaeroides O.U. 001. Resting cell suspensions (3 ml) were assayed for photoproduction of hydrogen from malate. Experimental conditions as in Fig. 2. Results pertain to that after 24 h of incubation.
Cell concentration for hydrogen photoproduction With an increasing concentration of cells (0.2-1.6 mg dry wt m1-1), hydrogen production increased. Optimal cell concentration for hydrogen production was 1.6-1.8 mg dry wt ml L A higher cell mass resulted in lowering of hydrogen photoevolution (Fig. 11), indicating that higher densities of cells were not favourable for hydrogen evolution. Age of the culture for hydrogen photoproduction Cultures growing photoheterotrophically under anaerobic conditions were harvested at various intervals of time, and the optical density of the cell suspension was adjusted to 0.15 at 6 6 0 n m (equal to 0.56mg dry wt ml ~) and subjected to hydrogen production assay, which revealed that mid log phase cultures (20 h old) were better producers of hydrogen (Fig. 12). The data generated in this investigation will form a basis for the designing and fabrication of a reactor for large scale generation of biomass and photohydrogen production by this organism, which is particularly prom-
Acknowledgements--The authors wish to thank the Department of Non-conventional Energy Sources, New Delhi, for financial support. K.S. thanks the U.G.C., New Delhi, and Ch.V.R. thanks the C.S.I.R. New Delhi, for the award of research fellowships. REFERENCES 1. J. S. Kim, K. Ito and H. Takahashi, Production of molecular hydrogen in outdoor batch cultures of Rhodopseudomonas sphaeroides. Agric. Biol. Chem. 46, 937-941 (1982). 2. K. Watanabe, J. S. Kim, K. Ito, L Buranakarl, T. Kampee and H. Takahashi, Therm-stable nature of hydrogen production by non-sulfur purple photosynthetic bacteria isolated in Thailand. Agric. Biol. Chem. 45, 217-222 (1981). 3. P. Stevens, C. Vertonghen, P. Devos and J. De Ley, The effect of temperature and light intensity on hydrogen gas production by different Rhodopseudomonas capsulata strains. Biotechnol. Lett. 6, 277 282 (1984). 4. P. Hillmer and H. Gest, Hydrogen metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: hydrogen production by growing cultures. J. Bacteriol. 129, 724-731 (1977). 5. P. Hillmer and H. Gest, Hydrogen metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: production and utilization of hydrogen by resting cells. J. Bacteriol. 129, 732 739 (1977). 6. K. Sasikala, Ch. V. Ramana, P. Raghuveer Rao and M. Subrahmanyam, Effect of gas phase on the photoproduction of hydrogen and substrate conversion efficiency in the photosynthetic bacterium Rhodobacter sphaeroides O.U. 001. Int. J. Hydrogen Energy 15, 795-797 (1990). 7. K. Sasikala and Ch. V. Ramana, Production of hydrogen by photosynthetic purple non-sulfur bacteria: isolation, characterization, identification and growth of Rhodobacter sphaeroides O.U. 001. Proc. Ind. Nam. Sci. Acad. B56, 235-240 (1990). 8. W. W. Umbreit, R. H. Burris and J. F. Stauffer, Manometric techniques, 4th edn. Burgess, Minneapolis (1964).
It 2 PRODUCTION BY RHODOBACTER SPHAEROIDES 9. J. t:. lmhoff, Genus Rhodobacter. In J. T. Stanley, N. Pfennig and J. G. Holts (eds) Bergey's Manual o/ Systematic Bacteriology, vol. 3, pp. 1663-1672. Williams and Wilkins, Baltimore (1989). 10. K. Sasaki and S. Nagai, The optimum pH and temperature for the aerobic growth of Rhodopseudomonas gelatinosa and
6()1
vitamin BI2 and ubiquinone formation on a starch medium J. Ferment. Technol. 57, 383 386 (1979). II. B. Mahro and L H. Grimme, Hydrogen photoproduction by green algae: the significance of anaerobic preincubation periods and of high light intensities for hsdrogen production of Chorella fU.sca, Arch. Mi(rohiol. 132, 82 ~6 (19821