Factors influencing the production of hydrogen by the purple non-sulphur phototrophic bacterium Rhodopseudomonas acidophila KU001.

Rhodopseudomonas acidophila KU001 was isolated from leather industry effluents and the effect of different cultural conditions on hydrogen production was studied. Anaerobic light induced more hydrogen production than anaerobic dark conditions. Growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Resting and growing cells preferred a pH of 6.0 ± 0.24 for hydrogen production. Succinate was the most preferred carbon source for the production of hydrogen while citrate was a poor source of carbon. Acetate and malate were also good carbon sources for hydrogen production under anaerobic light. Among the nitrogen sources, R. acidophila preferred ammonium chloride followed by urea for production of hydrogen. L-tyrosine was the least preferred nitrogen source by both growing and resting cells.

Hydrogen has the highest utilization efficiency, compatible with the biosphere, energy conserving, resource conserving, least capital intensive and most inflation fighting fuel (Ahmet Lokurlu ). However, widespread application of hydrogen as a fuel is limited due to lack of an advanced production, storage, and transportation and utilization technology. At present, hydrogen utilization as a fuel represents only about 5% of the total production and is often obtained as a by‐product from petroleum and chemical industries. Of various methods of hydrogen production such as electrolysis of water, photoelectrolysis, photocatalysis and biophotolysis, phototrophic production of hydrogen proved to be far superior. Photoevolution of hydrogen by phototrophic bacteria is reported to be influenced by a number of factors including cultural conditions. Koku and colleagues (2002) have reported the use of organic acids and carbohydrates for hydrogen production. Lakshmi and Polasa (1993) emphasized the importance of nitrogen source on the growth medium for photoproduction of hydrogen. Glutamic acid was effective as nitrogen source with no inhibition on nitrogenase activity, but may be expensive for industrial use. Response surface methodology (RSM) was employed to evaluate the effect of glutamate concentration on hydrogen production by Rhodobacter capsulatus (Shi and Yu, 2005). Not much information is available on hydrogen production by Rhodopseudomonas acidophila (Eike and Pfennig, 1978). In view of above facts, influence of different carbon and nitrogen sources in production of hydrogen by two anoxygenic phototrophic bacteria was studied and the results are discussed.

The phototrophic bacteria were isolated from the effluent samples by enrichment techniques by inoculating into the medium and incubated anaerobically in the light (2000 lux). Bacteria thus isolated were identified with the help of cultural characteristics (colour, size and shape), carbon and nitrogen requirement, vitamin requirements, absorption spectra analysis, bacteriochlorophylls and carotenoids. Identification keys provided in Bergey's Manual of Systematic bacteriology (1989) were adopted. The basic techniques used in the hydrogen production were those established by Vincenzini and colleagues (1982). Five millilitres of bacterial culture were harvested by centrifugation at 10 000 g for 10 min, washed thrice with 0.3% saline and the cells were suspended in the basal medium devoid of electron donor (acetate, citrate, lactate, malate, succinate, glucose) and nitrogen source (Ammonium chloride, Urea, glycine, thiourea, tyrosine, nitrogen gas). Depending on the experimental conditions different electron donors and nitrogen sources were added at required concentrations. The electron donors were added at a concentration of 1.0% and nitrogen sources were added at 0.5% concentration. Preparation of growing cells were done by taking logarithmic cultures of the organism and was inoculated (1% v/v) into basal medium containing different carbon sources along with nitrogen sources. The resting cells were prepared by following procedure. Cells were grown with succinate and nitrate until mid‐log phase and were harvested by centrifugation (16 000 g for 20 min). The pellet was washed twice and resuspended in basal salts medium. This suspension was then distributed into screw‐cap test tubes (10 × 100 mm) to fill them fully (anaerobic) or into 20 ml medium in a 100 ml conical flask (aerobic) and incubated under light (2400 lux) or dark conditions at 32°C.

To test the hydrogen production activity, the washed cell suspension (2.0 × 106 no of cells) was inoculated into 8 ml of the BPM [Beibl and Pfennig's media (BPM) containing mg l−1 medium of MgSO4.7H2O:200; NaCl:400; NH4Cl:400; CaCl2.2H2O:50; succinate:1000; Yeast extract:200; Ferric citrate solution (0.01 g ml−1) 1.0 ml. The trace element solution 1 ml and cyanocobalamine (Vitamin B12, 0.01 g l−1, 1 ml). The trace element solution contained mg l−1 of ZnCl2:70; MnCl2,4H2O:100; H3BO3:60; CoCl2.6H2O:200; NiCl2.6H2 O:20; NaMO4.2H2O:40 and HCl (25% v/v), 1 ml) in 15 ml capacity rimless test tubes sealed with subaseals and anaerobic conditions were created by evacuating and flushing with nitrogen (100%). Hydrogen produced was measured by injecting 0.5 ml of the gas phase from the reaction vessels with an airtight syringe into a gas chromatograph (Mak Analytica make) fitted with a molecular sieve 5A column (2 m × 1/8” ODSS) to a thermal conductivity detector (TCD). Gas analysis was done with oven temperature at 60°C with argon as carrier gas (flowrate 30 ml min−1), 120 mA detector current. Integrator and recorder were used at highest sensitivity. Before withdrawing each sample, 0.5 ml of nitrogen was injected in the vessel to maintain positive pressure. The amount of hydrogen liberated by the photosynthetic bacterium was calculated from the peak height of the recorder with reference to calibration curve prepared using ultra pure hydrogen.

Rhodopseudomonas acidophila could produce hydrogen over a pH range of 4.5–7.5. Growth of the organism started from pH 3.5 but hydrogen production could not be recorded till pH 4.5. Rhodopseudomonas acidophila opted for pH 6.0 ± 0.24 for maximum production of hydrogen by growing cells and resting cells. Incubation period of 120 h was optimum for production of hydrogen (Fig. 1). There were variations in the initial pH and final pH but the variations were minute and not greater than 0.52(±). Table 1 shows the cultural characteristics of the organism based on which the organism was identified. The bacterium under investigation showed preference towards carbon source present in the medium for production of hydrogen under anaerobic light (Table 2). Rhodopseudomonas acidophila opted succinate followed by acetate and malate as carbon source for hydrogen production under anaerobic light. Citrate was a poor source of a carbon for R. acidophila. It could grow only on acetate, citrate, lactate, malate, succinate and glucose. Rhodopseudomonas acidophila produced good amount of hydrogen in most of the carbon sources tried in anaerobic dark (Table 3). Succinate induced higher amount of hydrogen followed by glucose. Malate and lactate were almost of same nutritive value for hydrogen production. Citrate and acetate were also good sources of carbon. Growing cells produced more amount of hydrogen than resting cells except in glucose, which induced more amount of hydrogen in resting cells.

Figure 1

Effect of pH on production of hydrogen by growing and resting cells of R. acidophila using succinate and nitrate.

Table 1

Cultural characteristics of the phototrophic bacterium R. acidophila.

Name of the organism	Shape	Size (µm)	Amax	Carotenoid
R. acidophila	Rod	0.8–1.2	380,464,492,528,592,804,860	Lycopene
Carbon/Nitrogen source	Utilization		Carbon/Nitrogen source	Utilization
Acetate	+		Lactose	−
Citrate	+		Surcrose	−
Lactate	+		Mannitol	−
Malate	+		Ammonium chloride	+
Succinate	+		Urea	+
Glucose	+		Glycine	+
Glycerol	−		Thiourea	+
Tartarate	−		L‐tyrosine	+
Benzoate	−		Nitrogen gas	+
Fructose	−		L‐arginine	−
Nitrate	+

Table 2

Effect of carbon source on hydrogen production (ml/volume of the reaction vessel) by R. acidophila under anaerobic light conditions with nitrate as nitrogen source.

Carbon source	Incubation period in h
		48	72	96	120	144	162	192
Acetate	R		–	0.42 ± 0.18	1.24 ± 0.14	1.68 ± 0.24	1.82 ± 0.28	2.2 ± 0.08	1.44 ± 0.14
	G	0.32 ± 0.06	0.48 ± 0.12	0.58 ± 0.10	1.48 ± 0.20	2.86 ± 0.32	3.28 ± 0.22	4.1 ± 0.16	3.06 ± 0.14
Citrate	R	–	0.4 ± 0.06	0.2 ± 0.04	0.86 ± 0.08	1.04 ± 0.22	1.42 ± 0.18	0.96 ± 0.14	0.58 ± 0.08
	G	–	0.16 ± 0.04	0.28 ± 0.08	1.02 ± 0.32	1.88 ± 0.42	2.98 ± 0.12	1.6 ± 0.06	1.24 ± 0.28
Lactate	R	–	–	0.18 ± 0.02	0.68 ± 0.18	0.8 ± 0.07	0.86 ± 0.32	0.52 ± 0.16	0.36 ± 0.08
	G	0.18 ± 0.08	0.36 ± 0.08	0.52 ± 0.16	2.18 ± 0.24	3.11 ± 0.32	3.52 ± 0.16	2.68 ± 0.25	1.28 ± 0.32
Malate	R	–	0.12 ± 0.04	0.22 ± 0.04	0.72 ± 0.14	0.96 ± 0.16	1.08 ± 0.16	1.20 ± 0.24	0.52 ± 0.18
	G	0.24 ± 0.04	0.36 ± 0.08	0.58 ± 0.18	1.86 ± 0.38	2.34 ± 0.52	3.21 ± 0.28	3.84 ± 0.38	2.06 ± 0.42
Succinate	R	–	0.18 ± 0.04	0.38 ± 0.08	1.84 ± 0.18	2.08 ± 0.36	2.58 ± 0.18	1.78 ± 0.32	1.36 ± 0.22
	G	0.36 ± 0.14	0.54 ± 0.28	0.82 ± 0.16	2.48 ± 0.18	3.32 ± 0.44	4.3 ± 0.24	3.56 ± 0.58	2.84 ± 0.68
Glucose	R	–	0.22 ± 0.06	0.36 ± 0.12	0.78 ± 0.08	1.06 ± 0.34	0.96 ± 0.28	0.68 ± 0.14	0.46 ± 0.20
	G	0.18 ± 0.06	0.34 ± 0.06	0.52 ± 0.12	1.72 ± 0.14	2.38 ± 0.22	1.4 ± 0.28	0.96 ± 0.34	0.66 ± 0.32

R, resting cells; G, growing cells; –, no hydrogen production.

Table 3

Effect of carbon source on hydrogen production (ml/volume of the reaction vessel) by R. acidophila under anaerobic dark conditions with nitrate as nitrogen source.

Carbon	Incubation period in h
		24	48	72	96	120	144	162	192
Acetate	R	–	–	–	–	–	0.06 ± 0.02	–	–
	G	–	–	0.05 ± 0.01	0.08 ± 0.02	1.12 ± 0.12	2.22 ± 0.62	1.32 ± 0.34	–
Citrate	R	–	–	–	–	0.24 ± 0.06	0.33 ± 0.12	–	–
	G	–		0.06 ± 0.02	1.12 ± 0.14	1.5 ± 0.32	1.8 ± 0.18	0.83 ± 0.12	–
Lactate	R	–	–	–	1.43 ± 0.36	1.54 ± 0.24	1.08 ± 0.14	0.72 ± 0.18	0.6 ± 0.14
	G	0.48 ± 0.14	0.82 ± 0.06	1.22 ± 0.18	1.36 ± 0.34	1.72 ± 0.24	1.58 ± 0.16	0.84 ± 0.22	0.48 ± 0.12
Malate	R	0.22 ± 0.04	0.36 ± 0.06	0.55 ± 0.12	0.86 ± 0.18	0.92 ± 0.12	0.58 ± 0.14	0.22 ± 0.08	–
	G	0.18 ± 0.08	0.33 ± 0.12	0.56 ± 0.14	1.12 ± 0.32	1.86 ± 0.28	1.65 ± 0.14	0.93 ± 0.22	0.48 ± 0.14
Succinate	R	0.24 ± 0.04	0.48 ± 0.12	0.66 ± 0.22	1.38 ± 0.36	1.88 ± 0.28	1.2 ± 0.06	0.66 ± 0.18	0.54 ± 0.08
	G	0.36 ± 0.08	0.54 ± 0.16	0.92 ± 0.24	1.28 ± 0.14	2.18 ± 0.42	1.56 ± 0.32	1.08 ± 0.16	0.86 ± 0.14
Glucose	R	–		1.11 ± 0.32	2.07 ± 0.18	2.09 ± 0.14	2.16 ± 0.36	0.74 ± 0.18	0.42 ± 0.12
	G	–	0.07 ± 0.02	1.56 ± 0.34	1.92 ± 0.48	1.48 ± 0.16	1.08 ± 0.14	0.96 ± 0.28	0.54 ± 0.14

R, resting cells; G, growing cells; –, no hydrogen production.

Rhodopseudomonas acidophila preferred ammonium chloride followed by urea for production of hydrogen in anaerobic light (Table 4). Growing cells produced more amount of hydrogen than resting cells. Resting cells of R. acidophila preferred L‐tyrosine as nitrogen source, while growing cells failed to respond positively for tyrosine. Rhodopseudomonas acidophila produced least amount of hydrogen when nitrogen gas served as nitrogen source. Thiourea and tyrosine failed to induce hydrogen production in R. acidophila under anaerobic dark (Table 5). The growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Ammonium chloride could support good amount of hydrogen production and 120 h of incubation period was optimum. Nitrogen gas also supported good amount of hydrogen production, and required only 96 h of incubation period. Of all the nitrogen sources for hydrogen production, L‐tyrosine was the least preferred by both the resting cells and growing cells. In general, resting cells showed a lag period in hydrogen production than growing cells whether in anaerobic light or anaerobic dark conditions. This trend was seen in all the electron donor and nitrogen sources tried in the study. Nitrogen gas was utilized for the production of hydrogen; the mechanism behind this is not clear and is contrary to many other observations on this group of phototrophic bacteria. Succinate induced more amounts of hydrogen production in light and dark conditions than and other electron donors. Carbon sources are known to influence hydrogen production through nitrogenase enzyme by causing variation in electron donation capabilities of the cofactor compounds to nitrogenase, which could be the probable mechanism behind higher amounts of hydrogen production in succinate containing medium. This is a preliminary information but significant because very less data have been reported from this particular species of Rhodopseudomonas. There are no reports of this organism's hydrogen producing abilities so far. To date only one report has been published, which was in 1978 (Eike and Pfennig, 1978). The organism could produce hydrogen at a pH of 4.5 that could be exploited in effluents, which are acidic in nature. Further, work under different and modified conditions are required to fully establish the organism's hydrogen producing abilities.

Table 4

Effect of nitrogen source on hydrogen production (ml/volume of the reaction vessel) by R. acidophila under anaerobic light conditions with succinate as carbon source.

Nitrogen	Incubation period in h
		24	48	72	96	120	144	162	192
Ammonium	R	–	0.06 ± 0.02	0.22 ± 0.08	0.36 ± 0.14	0.48 ± 0.08	0.3 ± 0.12	0.2 ± 0.06	–
chloride	G	–	0.18 ± 0.06	0.44 ± 0.08	1.6 ± 0.22	1.2 ± 0.16	0.78 ± 0.04	0.34 ± 0.14	0.12 ± 0.06
Urea	R	–	–	0.12 ± 0.04	0.3 ± 0.06	0.42 ± 0.18	0.26 ± 0.06	0.06 ± 0.02	–
	G	–	–	0.24 ± 0.04	0.48 ± 0.06	1.2 ± 0.18	0.62 ± 0.22	0.28 ± 0.08	0.12 ± 0.06
Glycine	R	–	–	–	–	0.06 ± 0.02	0.22 ± 0.08	0.13 ± 0.06	–
	G	–	0.22 ± 0.08	0.39 ± 0.12	0.6 ± 0.14	0.75 ± 0.08	0.81 ± 0.16	0.38 ± 0.04	0.16 ± 0.0
Thiourea	R	–	–	0.12 ± 0.04	0.32 ± 0.06	0.44 ± 0.12	0.22 ± 0.08	0.16 ± 0.04	–
	G	–	–	–	0.22 ± 0.06	0.4 ± 0.08	0.86 ± 0.16	0.68 ± 0.22	0.42 ± 0.08
L‐tyrosine	R	–	0.22 ± 0.04	0.66 ± 0.22	0.98 ± 0.14	0.72 ± 0.08	0.77 ± 0.12	1.56 ± 0.22	0.74 ± 0.14
	G	–	0.12 ± 0.06	0.28 ± 0.08	0.48 ± 0.08	0.24 ± 0.06	–	–	–
Nitrogen	R	–	–	–	0.14 ± 0.04	–	–	–	–
Gas	G	–	0.16 ± 0.04	0.27 ± 0.12	0.76 ± 0.04	0.48 ± 0.08	0.36 ± 0.12	0.22 ± 0.08	–

R, resting cells; G, growing cells; –, no hydrogen production.

Table 5

Effect of nitrogen source on hydrogen production (ml/volume of the reaction vessel) by R. acidophila under anaerobic dark conditions with succinate as carbon source.

Nitrogen		24	48	72	96	120	144	162	192
Ammonium	R	–	–	–	0.12 ± 0.04	0.14 ± 0.04	0.26 ± 0.08	0.2 ± 0.04
chloride	G	–	–	0.12 ± 0.06	0.28 ± 0.12	0.36 ± 0.08	0.58 ± 0.16	0.4 ± 0.08	0.18 ± 0.02
Urea	R	–	–	–	0.12 ± 0.04	0.24 ± 0.08	0.08 ± 0.02	–	–
	G	–	0.14 ± 0.04	0.32 ± 0.08	0.38 ± 0.12	0.42 ± 0.12	0.22 ± 0.06	0.06 ± 0.02	–
Thiourea	R	–	–	–	–	–	–	–	–
	G	–	–	–	–	–	–	–	–
Glycine	R	–	–	–	0.16 ± 0.08	0.22 ± 0.04	–	–	–
	G	–	–	–	0.08 ± 0.02	0.42 ± 0.12	0.28 ± 0.08	0.08 ± 0.02	–
Tyrosine	R	–	–	–	–	–	–	–	–
	G	–	–	–	–	–	–	–	–
Nitrogen gas	R	–	–	0.12 ± 0.04	0.15 ± 0.04	0.18 ± 0.04	0.08 ± 0.02	–	–
	G	–	0.08 ± 0.02	0.22 ± 0.08	0.54 ± 0.24	0.56 ± 0.18	0.32 ± 0.08	0.18 ± 0.06

R, resting cells; G, growing cells; –, no hydrogen production.