Environmental factors on hydrogen production

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Effect Of Some Environmental Factors On Hydrogen Production


Biological hydrogen production can be carried out by photo- and dark-fermentation processes. Hydrogen evolution by dark-fermentation has been treated with little attention, while hydrogen evolution by photosynthetic microorganisms has been extensively studied. Photosynthetic bacteria are favorable candidates for biological hydrogen production due to their high conversion efficiency and versatility in the substrates they can utilize [1].

Photosynthetic bacteria can produce hydrogen at the expense of solar energy and small-chain organic acids as electron donors. The conversion efficiency of light energy to hydrogen, with the supply of an appropriate carbon source, is the key factor for hydrogen production by biological systems. Photosynthetic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process [2].

Two enzymes namely, nitrogenase and hydrogenase play an important role in biohydrogen production. Photofermentation by Purple Non-Sulfur bacteria (PNS) is a major field of research through which the overall yield for biological hydrogen production can be improved significantly by optimization of growth conditions and immobilization of active cellss [3].

Purple non-sulfur bacteria evolve molecular h3 catalyzed by nitrogenase under nitrogen-deficient conditions using light energy and reduced compounds (organic acids).

C6h32O6 + 12h3O+Light energy→12h3 + 6CO2

These photoheterotrophic bacteria have been investigated for their potential to convert light energy into h3 using waste organic compounds as substrate [4]. Among species of photosynthetic bacterium, Rhodobacter sphaeroides (formerly known as Rhodopseudomonas sphaeroides) has been studied most widely for hydrogen production [5]. In this paper we are reporting the effect of some environmental parameters that will affect the growth and hydrogen production by this PNS bacterium R. sphaeroides NCIMB 8253 especially aerobic and anaerobic condition, effect of shaking, effect of media and age of inoculum. The purpose of this paper is to review various parameters of biohydrogen production using PNS bacteria along with several current developments. Suitable process parameters such as carbon and nitrogen ratio, illumination intensity, bioreactor configuration and inoculum age may lead to higher yields of hydrogen generation using PNS bacterium [6].

Materials And Method


R. sphaeroides NCIMB 8253 (freeze-preserved bacteria) was obtained from NCIMB Limited, Scotland. In the preparation of the inoculum, the freeze-preserved bacteria were transferred into medium 27 to grow and activate this bacterium. Then the bacterium was cultured into modified Biebl & Pfennig growth medium for hydrogen production experiment. The amount of inoculum used to inoculate the hydrogen production medium was 10% by volume.

Culture Condition

The medium used for the growth was modified medium of Biebl and Pfennig. The modified medium of Biebl and Pfennig that contains Maliec acid (7.5 mM) as the organic carbon source and sodium glutamate (10 mM) as the nitrogen source (Koku et al. 2003). Solid agar medium was prepared by adding 20g agar Bacteriological No. 1 into 1 liter modified medium of Biebl & Pfennig. The pH of the medium was adjusted to 6.82 with 1 M sodium hydroxide solution. The liquid culture medium used for the hydrogen production was essentially the same as the growth medium except that the concentrations of malate and glutamate were 15 and 2 mM, respectively (Koku et al. 2003). Both medium were sterilized at 121oC for 15 minutes in an autoclave before being used.

The bacterium was grown in liquid medium anaerobically under a light intensity of 3.0 -3.8 klux at 32◦C with tungsten lamp (100 W) as the light source. Argon gas was used to create anaerobic conditions. The bacterium was also cultured on the solid modified medium of Biebl & Pfennig and incubated in anaerobic jar with light intensity of 3.0 -3.8 klux at 32◦C with tungsten lamp (100 W) as the light source.

Hydrogen Fermentation

Hydrogen gas production experiments were done in batch culture systems (100 mL medium fermentation) with 10 % v/v inoculum. The temperature was maintained at 32 ºC, under the illumination of a tungsten lamp (100 W) with light intensity of 3.8 klux. For all hydrogen production experiments, the reactor was flushed with pure argon in order to create an anaerobic atmosphere. After flushing with argon, 10% v/v inoculum of the pre-activated bacteria (in minimal medium of Biebl and Pfennig) was transferred into the hydrogen production medium. During the experiments, the evolved gas was collected and measured volumetrically in a measuring cylinder.


Cells growth analysis. Growth of the culture was monitored by using the optical density (OD) at 660 nm using a Thermo Spectronic spectrophotometer (Model: Genesys 10 UV). Fresh medium was used as blank solution. Cells dry weight was estimated by centrifuging 10 ml of cells suspension at 13 000 rpm for 10 min (KUBOTA 5220) and then the pellet was washed twice with deionized water and dried in an oven at 105 oC until constant weight was attained. A relationship of cells dry weight and OD was obtained by plotting a graph of OD versus cell dry weight. It was found that an optical density of 1.0 at 660 nm corresponded to a cell density of 0.341 g dry weight per liter of culture.

GC analysis. For GC analysis, 1 mL gas samples taken from collected gas by h3 fermentation. The h3 gas produced was analyzed by gas chromatograph (GC) equipped with a helium ionization detector (SRI 8610C GC, USA). Helium was used as the carrier gas. Oven and detector temperatures were 50oC and 150oC, respectively. Percentage of h3 in gas by GC analysis was used to determine the total of h3 collected by each h3 fermentation experiments.

Determination of pH. The pH of the culture medium was measured with Eutech Instruments pH meter (Model: pH 510; pH/ mV/ oC; Cyberscan).

Determination of light intensity. Luxmeter was used to measure the intensity of light used.

Results And Discussion

Effect Of Shaking

The experiments to study the effect of shaking was done two times and the results show that hydrogen production in static culture is better than shake culture which was almost no hydrogen or gas was produced in shake culture. Table 1 shows the h3 production in different culture conditions. In static culture, h3 produced in aerobic condition was 39 mL, and 58.5 mL under anaerobic condition.

Figure 1 shows the trend of gas produced by different culture conditions. From the graph, it is shown that the volume of gas produced is higher by using static culture compared to shake culture. This results was opposite with what Kim et al. 1982 found in his experiment. He reported that the cells had the tendency to flocculate. The flocculation would retard hydrogen production because of the decrease in the efficiency of light absorption. This usually happen in large scale of hydrogen production. We assume that stirring will increase the tendency of to be used in second stage of metabolism to produce other products, and that's why static culture was better than liquid culture.

Effect Of Different Culture Condition (Aerobic Versus Anaerobic)

The effect of the aerobic and anaerobic condition to the cultures was studied to compare h3 production and the growth of the cellss. Figure 2 shows the trends of different or the same culture condition used for growth and h3 production. The graph of the total gas produced versus time shows that the culture with aerobic condition for inoculum and anaerobic condition for h3 fermentation shows the highest of the total gas produced compared to other condition, with 63.60 mL of gas. But from the percentage of h3 production and total of gas produced from Table 2 shows that both culture conditions for inoculum and h3 production in anaerobic condition was the highest compared to other culture condition.

*AN-AN = Anaerobic growth and Anaerobic h3 fermentation, *AN-AE = Anaerobic growth and Aerobic h3 fermentation, *AE-AN = Aerobic growth and Anaerobic h3 fermentation, *AE-AE = Aerobic growth and Aerobic h3 fermentation.

However when the inoculum was prepared in aerobic condition and h3 fermentation in anaerobic condition the yield of h3 decreased to 112.92 mLg-1 (Yh3/S) and Yh3/S, 54.71 mLg-1 was obtain when the inoculum was prepared in anaerobic and h3 fermentation was prepared in aerobic condition. The lowest yield of h3 was obtained when both inoculum and h3 fermentation was prepared in aerobic condition which Yh3/S, 46.22 mLg-1. It was found that the lag phase has been shortened when same medium was used for inoculum and h3 fermentation but no effect of h3 volume if different media were used for inoculum and h3 fermentation.

Effect Of Medium And Inoculum Age

h3 production was studied using two different media, growth and h3 production medium. The effect of inoculum age was also investigated using 24 hours and 48 hours inoculum. Figure 3 (a) and 3 (b) show the plot of total gas produced versus time. The results showed that culture with production medium that was inoculated with 24 hours inoculum grown in growth medium produced the highest of % of h3 (20.76%), the highest yields of h3 produced per substrate consumed, Yh3/S, (138.05 mLg-1) and the highest rate of h3 produced (9.6 x 10-4 L/L/j) compared to other cultures. It was found that the lag phase has been shortened when same medium was used for inoculum and h3 fermentation but no effect of h3 volume if different media were used for inoculum and h3 fermentation.

(a) 24 hours inoculum

(b) 48 hours inoculum.

Figure 3. Total gas produced by cultures with different medium and inoculum age

Table 3 shows the comparison of the % of h3, total of h3 produced, yields of h3 produced per substrate consumed, Yh3/S, and the rate of h3 produced by different cultures.


This study showed that growth and h3 production of the PNS bacteria, R.sphaeroides NCIMB 8253 was affected by some parameters. h3 production by R.sphaeroides NCIMB 8253 should be carried out in static culture under anaerobic condition. The use of 24 hours inoculum and the right media for growth and h3 production gave better results. The work is still in the preliminary stage and will study many other factors such as pH, carbon and nitrogen source, light intensities and source, effect of NH4+ and age of inoculum as this bacteria has the bright potential in biological hydrogen production. Once the optimum parameters achieved, this research will be proceed in larger scale using 7L photo bioreactor.


The sincere gratitude goes to Assoc. Prof. Dr. Mohd. Sahaid Kalil and Dr Nurina Anuar for their support and guidance and also for Ministry of Science and Technology (MOSTI), for financial support under Science Fund grant 02-01-02-SF0176.


[1] Koku H, Eroglu I, Gunduz U, Yucel M, Turker L, ”Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides”. Int J Hydrogen Energy, 2002. (27), pp. 1315-1329.

[2] Barbosa, M. J., Rocha, J. M. S., Tramper, J., & Wijffels, R. H, “Acetate as a carbon source for hydrogen production by photosynthetic bacteria”. Journal of Biotechnology, 2001. (85), pp. 25-33.

[3] Basak, N. &Das, D. “The prospect of purple non-sulfur (PNS) photosynthetic bacteria for hydrogen production: the present state of the art”. World J Microbiol Biotechnol, 2007. (23), pp.31-42.

[4] Levin, D.B., Pittb, L. & Loveb, M, “Biohydrogen production: prospects and limitations to practical application”. International Journal of Hydrogen Energy, 2004. (29), pp. 173- 185

[5] Fang, H.H.P. Zhu, H. & Zhang, T. “Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides”. International Journal of Hydrogen Energy, 2006 (31) pp. 2223 - 2230

[6] Basak, N. &Das, D. “The prospect of purple non-sulfur (PNS) photosynthetic bacteria for hydrogen production: the present state of the art”. World J Microbiol Biotechnol, 2007. (23), pp.31-42.