Abstract: The aim of this study was to investigate the influence of some environmental factors on bacterial metabolism. Fermentative hydrogen production by C. acetobutylicum, using glucose as the substrate. The effect of initial pH (4-8), inoculum size (1-20% (v/v)) and glucose concentration (1-30 g L-1) on hydrogen production were studied. The optimum cultivation temperature for hydrogen production was at 30 °C. The results show that substrate concentration and inoculum size resulted in hydrogen yield (YP/S) of 391 mL g-1 glucose utilized with maximum hydrogen productivity of 77.5 mL/L/h. Higher substrate concentration or inoculum size adversely affects hydrogen production, which decreases hydrogen yield by 15% to 334 mL g-1 glucose utilized when 30% (v/v) inoculum size was used. The use of 30 g L-1 substrate concentration resulted in a 75% decrease to 97 mL g-1 glucose supplied. Concluded that proper Xo/So enhanced the hydrogen production.
INTRODUCTION
The trend of global environmental goals of lower emissions and carbon dioxide reduction and depletion of known crude oil reserves have intensified the development of non-polluting or clean energy carriers from renewable resources. Among recognized alternatives to fossil fuels, hydrogen is regarded as a clean energy carrier because water is the only product of its combustion. Moreover, its energy value of 231 BTU mol-1 (244 kJ mol-1) is higher than that of any hydrocarbon. Although it can be generated from both renewable and non-renewable resources, more than 96% of the H2 produced worldwide depends on fossilized energy in one form or another (Wunschiers and Lindblad, 2002). By the year 2002, producers were generating about 45 million tonnes of H2 from fossil fuel (Gorman, 2002).
In contrast, microbial production of H2, also referred to as biohydrogen production, is a much less capital and energy-intensive process. In particular, H2 production by anaerobic fermentation is technically much simpler and can generate H2 from a large a number of carbohydrate materials obtained in refuse and waste products (Nandi, and Sengupta, 1998). It is superior in terms of the rate of production to photosynthetic H2 production and it can also make use of the existing infrastructure for biogas and fermentative acetone-butanol production without elaborate adjustments (Das and Veziroglu, 2001).
Clostridia have the capacity to utilize a wide variety of sugars, thus broadening the spectrum of potential carbon sources for H2 production. Optimizing fermentation conditions that favour clostridial growth should therefore, essentially enhance H2 production. However, what controls the mechanisms of clostridial H2 production has not been determined in detail, since the culture conditions that affect H2 production are only beginning to be ascertained (Logan et al., 2002). To date, studies have shown that iron availability, H2 partial pressure, substrate concentration, temperature (Zhang et al., 2003) and the carbon-to-nitrogen ratio (Lin and Lay, 2004) all in some way affect the H2-producing potential of a microorganism. Nonetheless, most clostridia produce both organic solvents (alcohols and ketones) and acids. Since alcohol production involves the consumption of H2 in the form reducing equivalents such as NADH, it is inevitable that fermentation conditions that favour the metabolism of sugar to alcohols reduce H2 production.
Clostridium acetobutylicum NCIMB13357 is a known alcohol-producing microorganism. Only one study was conducted using this microorganism for fermentative acetone, butanol and ethanol (ABE) production (Kalil et al., 2003). Its hydrogen-producing potential, however, has not been reported. Hence this study was conducted to investigate the influence of culture parameters on H2 production by the microorganism. Hence, this study was conducted to investigate the influence of some environmental factors have been reported to affect on bacterial metabolism like initial culture pH, substrate concentration and inoculum size on hydrogen production by this microorganism.
MATERIALS AND METHODS
Microorganism and culture conditions: C. aceto- butylicum NCIMB 13357 was purchased from a British culture collection, NCIMB Ltd. Scotland, UK. This study was conducted in Chemical Lab, Department of Chemical and Bioprocess Engineering: Universiti Kebangsaan Malaysia at the period between 2006 to 2008. The bacterium was cultivated in anaerobic condition in Reinforced Clostridial Medium (RCM) for 24 h at 30 °C. Liquid medium of RCM was used for inoculum preparation. The growth of culture in RCM was monitored by measuring an optical density at 600 nm using a spectrophotometer. Only inoculum with Optical Density (OD) values greater than 0.4-0.6 after 18 h cultivation was used as inoculum. An inoculum of 10% (v/v) was used throughout this study.
Cultivation medium: New medium was formulated in the lab to be used for hydrogen production by bacterium species we used in this study have the following composition in (g L-1): glucose (5), yeast extract (5), L-Cystine. HCl (1.0) and bacteriological agar (0.5).
The initial anaerobic condition in the reactor after inoculation inside the anaerobic glove box was established by replacing the gaseous phase with nitrogen at start of cultivation. Then incubated at 30 °C in temperature controlled water bath without shaking. The evolved gas was monitored and collected in a gas collection cylinder and the volume of evolved gas was measured at room temperature by the water displacement method (Morimoto et al., 2004) in a graduated cylinder (inverted), that had been filled with water of pH 3 or less in order to prevent dissolution of the gas components.
Analytical methods: The gas composition was determined by gas chromatography (Shimadzu Co., Kyoto, GC-8A) under the following conditions: column: Porapack-Q, carrier gas: Nitrogen, flow rate: 33 mL min-1; column temperature: 50 °C, injection temperature: 100 °C, detector temperature: 50 °C, detector: Thermal Conductivity Detector (TCD). The soluble glucose concentration was measured at the end of each batch experiment for the calculation of the amount of glucose consumed by DNS method modified by Miller (1959) using spectrophotometer (UV 1601IPC, Shimadzu corporation- Japan) optical density (OD550 nm). Individual batch experiments were observed until the hydrogen production from each bottle stopped. Final medium pH was measured by pH meter (Mettler Teldo) and final biomass was measured by Spectrophotometer (UV 1601IPC, Shimadzu Corporation-Japan) at optical density (OD600 nm). All of these data were the average (mean) of three trials.
RESULTS
Effect of initial pH: pH is one of the important controlling factors of anaerobic fermentation process. The results shown in Fig. 1a demonstrate the effect of initial pH on bacterial conversion of glucose to hydrogen gas. At acidic initial pH, low hydrogen was produced, whereas the quantity of produced hydrogen increased as the initial pH near to neutral pH. As shown in Fig. 1a, increasing the pH at intervals of 0.5 unit`s affected H2 production. The full utilization of 1 g of glucose at initial pH of 4.0 was 357 mL g-1 glucose utilized but only 50 mL g-1 glucose supplied was obtained, suggested that at acidic pH, the bacterial productivity of hydrogen by this species was inhibited.
As described earlier that optimal pH gave the highest hydrogen yield and that also shown in Fig. 1b which indicated that percentage of glucose consumption was increased with culture pH and the maximum of 87% of glucose was consumed within 22 h of batch process at pH of 7.0. Increased or decreased initial pH reduces the percentage of glucose consumed and finally affect adversely on total evolved hydrogen. Furthermore, the results shown in Fig. 1d revealed that neutral pH enhanced the biomass concentration and the maximum of 1.34 g L-1 was obtained at pH of 7.0. Above results showed that between pH 4.5 and 7.0, H2 production was enhanced by every 1 unit increase in pH. While the highest yield of 391 mL g-1 glucose utilized (340 mL g-1 glucose supplied), was obtained at pH of 7.0. Further increased in culture pH reduce the hydrogen yield to 360 mL g-1 glucose utilized. Increasing the biomass shown to have positive effect on the bacterial productivity of hydrogen as shown in Fig. 1c. The results shown in Fig. 1c indicated that the bacterial productivity of hydrogen was increased from 11.5 mL/L/h at pH of 4.0 to 77.5 mL/L/h at pH of 7.0 then decreased to 50 mL/L/h at pH of 8.0 indicating that H2 production by C. acetobutylicum NCIMB13357 was favored by a neutral initial pH.
The results shown in Table 1 shown that the hydrogen
yield from glucose found in this study is higher than the 125 mL g-1
glucose supplied for the pure culture of Enterobacter aerogenes
(Tanisho et al., 1989) and of 275 mL g-1 glucose supplied,
using E. cloacae by Kumur and Das (2000) and also than 175-288
mL g-1 glucose supplied, using C. butyricum reported
by Kataoka et al. (1997). Whereas by using mixed culture, the results
of this study was higher than that of 213 mL g-1 glucose supplied
reported by Lin and Chang (1999). Results of this study and those reported
for pure culture seem to suggest that the maximum hydrogen yield by glucose
fermentation is varied, depend on the bacterial species used.
| Table 1: | Comparison of hydrogen yield using glucose as substrate |
| Hydrogen yield: (mL g-1 glucose supplied) | |
Compared with reported hydrogen yield per carbohydrate supplied, but
with varied pH and carbon source using mixed culture, Table
2 shows that the results of this study was lower than the yield reported
by Fang et al. (2006) of 346 mL g-1 carbohydrate supplied
but higher than other reported values, suggested that medium pH play an
important role in pure culture but the lower yield in mixed culture it
might because that not all hydrogen producing organisms would be active
in selected pH.
| Table 2: | Comparison of Hydrogen yield using different substrate |
| Hydrogen yield: (mL g-1 carbohydrate supplied), b: Continuous experiments | |
The results shown in Table 4 were used to calculate
different yields like YP/X (mL/g/L): (H2 mL g-1
Biomass L-1), YX/S: (Biomass production per
g glucose supplied) and YH2/s (conversion of H2
(mL) to H2 (g) per g glucose utilized). These results showed
that optimum pH was necessary for bacterial growth which reversed to enhance
both bacterial growth as well as hydrogen production.
| Table 3: | Results of initial pH on glucose consumption and H2P (mL/L/h) by C. acetobutylicum NCIMB13357 |
| [Glucose]: 5 g L-1, inoculum size 10% (v/v) I pH 7.0, Temp. 30 °C | |
| Table 4: | Results of initial pH on H2 production by C. acetobutylicum NCIMB13357 |
| Y1P/S : (H2 mL g-1 glucose supplied) (mL g-1), Y2P/S (mL g-1) (Utilized): (H2 mL g-1 glucose utilized), [Biomass] (g L-1): Biomass production g L-1 culture, YP/X (mL/g/L): (H2 mL g-1 Biomass L-1), YX/S: (Biomass production per g glucose supplied), YH2 /s: (conversion of H2 (mL) to H2 (g) per g glucose utilized) [Glucose], 5 g L-1, inoculum size 10% (v/v), I pH 7.0, Temp. 30 °C | |
Finally the result in Table 3 and 4 was in agreement also with results which showed that H2 production by Clostridia is completely inhibited below a pH range of 4 to 5 (Bahl et al., 1986; Dabrock et al., 1992; Wu and Lin, 2004).
According to Bahl et al. (1982) results, the drop in culture pH
was the critical factor for major shifting from acids production to solvent
production. They reported that at neutral pH and glucose or nitrogen limitation,
the main product was acids, CO2 and H2 by using
C. acetobutylicum species, but when pH drop down because of acid
production and reach to certain concentration the bacteria start forming
the solvent and stop first metabolites production. They reported also
that solvent production was not observed when C. acetobutylicum
was grown in continuous culture with glucose or nitrogen limitation at
pH values of 6.5 or 5.7. They stated that this bacterium is governed by
physiological parameter. The optimal pH for their study for solvent production
was found to be 4.3. From above finding of Bahl et al. (1982),
the result of present study was strongly agreed with Bahl et al.
(1982) regarding the initial pH and its effect on both metabolites and
the final growth. It concluded that the pH for optimal H2 production
was dependent on the hydrogen-producing microorganism(s). The highest
productivity and hydrogen yield were obtained at the optimum pH of 7.0;
all data were shown in Table 3 and 4.
| Table 5: | Results of inoculum size on glucose consumption and H2P (mL/L/h) by C. acetobutylicum NCIMB13357 |
| [Glucose]: 5 g L-1, initial pH 7.0, Temp. 30 °C | |
| Table 6: | Results of inoculum size (%) (v/v), on H2 production by C. acetobutylicum NCIMB13357 |
| Y1P/S : (H2 mL g-1 glucose supplied) (mL g-1), Y2P/S (mL g-1) (Utilized): (H2 mL/g glucose utilized), [Biomass] (g L-1): Biomass production g L-1 culture, YP/X (mL/g/L): (H2 mL g-1 Biomass L-1), YX/S: (Biomass production per g glucose supplied), YH2 /s: (conversion of H2 (mL) to H2 (g) per g glucose utilized) [Glucose], 5 g L-1, initial pH 7.0, Temp. 30 °C | |
Effect of inoculum size: The results shown in Table
5 and 6 shown that hydrogen production was affected
by the inoculum size. Obtained data of tested range shows how the importance
to choose the proper inoculum size to influent substrate. The results
of using 5 g L-1 of glucose in formulated medium and 10% inoculum
size gave the highest hydrogen yield of 391 mL g-1 glucose
utilized. Decreased or increased of inoculum size lead to decrease of
hydrogen yield. The results shown in Fig. 2a indicated
that inoculum size from 1 to 7% (v/v), the hydrogen yield was same with
maximum of 283 mL g-1 glucose utilized using inoculum size
of 7% (v/v) but further increased to 10% (v/v), the yield was enhanced
resulted of 391 mL g-1 glucose utilized then started to decreased
to 331 mL g-1 glucose utilized using 30% (v/v). The results
shown in Table 6 shown that the inoculum size should
be compatible with available substrate for maximum bacterial productivity
due to substrate limitation would affect adversely on bacterial growth
and finally on its metabolites. The reason for that mainly attributed
to both glucose concentration and inoculum size in fermentation medium
and as inoculum size increased the batch process finished early due to
exhausted of available substrate and that was clear from Fig.
2c regarding the bacterial productivity which the maximum of 88 mL/L/h
was obtained by using inoculum size of 30% (v/v). These results of bacterial
productivity of hydrogen indicated that as the inoculum size increased,
the bacterial metabolites would increased which are acids and that would
affected adversely due to dropping in culture pH due to higher acids production
that would made the culture more favorable for solvent production (second
growth phase) and stop the first metabolites production (acids and gases).
The shift from acid production to solvent phase was clear from the final
pH of culture medium and that connected with the hydrogen yield which
was maximized at inoculum size of 10%. The results shown in Fig.
2b indicated that the percentage of consumed glucose at 30% inoculum
size was higher than others and that due to higher biomass concentration
which resulted to increase the final biomass as shown in Fig.
2d. The results shown in Fig. 2d shown that final
biomass concentration was increased as the inoculum size increased but
that increase not enhanced the hydrogen yield suggested that inoculum
size should be used in proper percentage to culture volume to maximize
the bacterial productivity of hydrogen and not to enforce the bacterial
shift from acid phase to solvent phase.
The results shown in Table 6 were used to calculate different yields like YP/X (mL/g/L): (H2 mL g-1 Biomass L-1), YX/S: (Biomass production per g glucose supplied) and YH2/s (conversion of H2 (mL) to H2 (g) per g glucose utilized). These results showed that proper inoculum size to influent substrate (glucose) enhanced the bacterial productivity of hydrogen. Further increased of inoculum size was shown to affect on negative effect on bacterial productivity of hydrogen suggested that available substrate restrict the metabolism of initial inoculum size (acid or solvent: for Clostidium). Concluded that proper inoculum size should be used for maximum bacterial production of any products (here hydrogen).
Hydrogen evolution by Clostridium is primarily a growth-associated
process and therefore it is expected that the highest rates of evolution
be attained during the growth phase. Optimum inoculum size with substrate
concentration in fermentation medium gave a better chance for bacteria
to meet the available substrate and maximize its production. This implied
that for the same concentration of substrate, an initially high substrate
to cell ratio would prolong the growth phase and facilitate a longer duration
of high rates of H2 production by the growing cells and that
would result in high H2 production but not the hydrogen yield.
This however disagrees with the findings of Lay (2001) which showed that
a low substrate (cellulose) to cell density facilitated high H2
generation by a mixed hydrogen producing culture.
Effect of substrate concentration: Production of hydrogen in anaerobic
fermentation process is accompanied by the breakdown of an organic substrate
like glucose in the present study. So, the initial glucose concentration
plays an important role in the volume and the rate of hydrogen production
during the course of fermentation. The results of earlier study were obtained
using 5 g L-1 glucose and inoculum size of 10%. The results
shown in Table 7 and 8 shown that
H2 production decreased with increasing glucose concentration.
Although the highest H2 production volume was obtained in 30
g L-1 glucose supplied, but the H2 yield reduced
by 73% from 347 mL g-1 in 1 g L-1 glucose supplied
to 97 mL g-1 in 30 g L-1 glucose supplied. Whereas
hydrogen yield per glucose utilized decreased with increasing of influent
glucose from 365 mL g-1 in 1 g L-1 to 324 mL g-1
in 10 g L-1 with maximum yield of 391 mL g-1 glucose
utilized using 5 g L-1. Furthermore the percentage of consumed
glucose of more than 10 g L-1 cannot be detected as shown in
Fig. 3b. The sharp decreased in hydrogen production
for further increased in influent glucose was useful for bacterial growth
with maximum biomass concentration of 1.91 g L-1 at 30 g L-1
of supplied glucose as shown in Fig. 3d. Table
7 and 8 shows all results obtained for the effect
of substrate (glucose) concentration on hydrogen production by C. acetobutylicum
NCIMB13357.
| Table 7: | Results of substrate concentration on glucose consumption and H2P (mL/L/h) by C.acetobutylicum NCIMB13357 |
| Inoculum size 10% (v/v) I pH 7.0, Temp. 30 °C | |
| Table 8: | Results of substrate concentration on H2 production by C. acetobutylicum NCIMB13357 |
| Y1P/S: (H2 mL g-1 glucose supplied) (mL g-1), Y2P/S (mL g-1) (Utilized): (H2 mL g-1 glucose utilized), [Biomass] (g L-1): Biomass production g per L culture, YP/X (mL/g/L): (H2 mL g-1 Biomass L-1), YX/S: (Biomass production per g glucose supplied), YH2 /s: (conversion of H2 (mL) to H2 (g) per g glucose utilized) Inoculum size 10% (v/v), initial pH. 7.0 Temp. 30 °C | |
The rate of hydrogen production varied with the variation of glucose content in the feed of the bioreactor. At relatively low initial glucose concentrations of 1-3 g L-1, the rate of fermentation is also low, according to the law of mass action (Fabiano and Perego, 2002). The results shown in Fig. 3a indicated that at initial glucose concentration of 1 to 10 g L-1, the highest hydrogen yield of 391 mg L-1 was the maximum by using 5 g L-1 compared with further concentration and the batch process was finished earlier than by using higher glucose concentration and that mainly due to the exhausted of available substrate. At 1 to 5 g L-1 the results indicated that at these concentrations as shown in Fig. 3c the bacterial productivity with maximum of 97 mL/L/h at 1 g L-1 was better than higher substrate concentration and that due to higher inoculum size to glucose concentration, then started too decreased to 77.5 mL/L/h at 5 g L-1. Furthermore, increasing influent glucose gave lower hydrogen productivity and that due to increasing in bacterial production of acids which increased as the glucose substrate increased and that clear from the final pH of the culture medium as shown in Table 7.
The cell concentration was found to increase with increase in glucose concentration in the feed. But the rate of production of hydrogen was maximum by using 0.5% (w/v) glucose concentration in formulated medium, while it decreased with further increase in glucose concentration presumably because of inhibition due to excess substrate. Therefore, Oh et al. (2003) attributed that lower H2 production rate at higher substrate concentration to that the carbon flux at high glucose concentrations is more directed to the production of reduced by-products such as ethanol and organic acids. The trend between the substrate concentration and the H2 productivity was in an inverse pattern.
The highest productivity of 97 and 11 mL/L/h was obtained in 1 and 30 g L-1, respectively. The reductions in the H2 yield and bacterial productivity of hydrogen in a concentration more than 5 g L-1 in this study were agreed with the suggestion of (Van Ginkel et al., 2001) they attributed that to the shift from the acidogenic to the solventogenic pathways where, H2 was consumed to reduce the acids to alcohols and with that of a steady decrease in the H2 yield with increasing substrate concentration observed for starch in starch wastewater (Zhang et al., 2003) and for cellulose (Lay, 2001), whereas disagree with Wu and Lin (2004) they observed a similar trend between the yield and molasses concentration, in which the highest yield was found at a substrate concentration of 40 g COD/L. above finding suggested that hydrogen production by fermentative bacteria depend in both microorganism (s) used and substrate used.
The results shown in Table 8 were used to calculate different yields like YP/X (mL/g/L): (H2 mL g-1 Biomass L-1), YX/S: (Biomass production per g glucose supplied) and YH2/s (conversion of H2 (mL) to H2 (g) per g glucose utilized). These results showed that as it is worth mentioned in above about the effect of inoculum size, the effect was same and both sections underscore the proper initial substrate concentration to inoculum size would enhanced the bacterial productivity of hydrogen. Further increased of substrate was shown to enforce the bacteria to make shift from acid phase to solvent phase and stop the first metabolites production and that clear from the final pH of different glucose concentration and total hydrogen produced, suggested that lower glucose concentration was necessary for maximum bacterial productivity of hydrogen with the inverse at higher concentration but the yield per glucose was maximum at proper concentration.
It was noted that investigators have reported H2 yield as mol H2 per mol substrate, mol H2 per gram substrate or H2 produced (mL) per gram substrate; hence, for ease of comparison with values reported, the H2 yields were all converted to H2 produced (ml) per gram substrate utilized. On that basis, it was found that the maximum yield of 3.13 molH2/mol-glucose utilized (391 mL g-1) (2.72 mol/ mol glucose supplied) obtained in this study was higher than other reported values even with different substrate. Present results regarding the yield was higher than the reported values of 132 mL g-1 glucose supplied (Oh et al., 2003), 92 mL g-1 starch supplied in starch wastewater (Zhang et al., 2003), 193 mL g-1 cellulose supplied (Ueno et al., 2001), 134 mL g-1 molasses supplied (Logan et al., 2002), 126 mL g-1 sucrose supplied (Lee et al., 2002) and than 314 mL g-1 sucrose supplied reported by Lin and Lay (2004). This indicated that C. acetobutylicum NCIMB13357 is one of the most efficient hydrogen producers.
DISCUSSION
Unlike other H2-forming species, such as green algae, production of hydrogen in anaerobic fermentation is accompanied by the breakdown of organic substrate, here glucose. So, the initial glucose concentration plays an important role in the yield and overall production rate of hydrogen during the course of fermentation. It has already been reported that substrate inhibition gets predominant at higher glucose concentration because this modifies the metabolic pathways (Fabiano and Perego, 2002).
pH is one of the important controlling factors of anaerobic fermentation processes. It has a profound effect on evolution rate of hydrogen. A major factor affecting H2 or solvent production is the pH. Jones and Woods (1986) reported that optimum pH for H2 production is 5.5, while the optimum pH for solvent production is ~ 4.5. The pH can affect the form of the acids produced during hydrogen production. Mizuno et al. (2000) indicated that during fermentation some organic acids were produced as metabolic products. Accumulation of these acids causes a sharp drop of culture pH and subsequent inhibition of bacterial hydrogen production. It was shown from their results that each species have optimum initial pH to initiate and get maximum production. Nath et al. (2006) found that the optimum initial pH for hydrogen production was 6.5 using Enterobacter cloacae DM11 and Cane Molasses as Feedstock. Mizuno et al. (2000) stated that poor hydrogen production rate at low pH; lower than 5.5, could be due to the increased formation of acidic or alcoholic metabolites, which destroys the cell`s ability to maintain internal pH. Bowles and Ellefson (1985) suggested that, at low pH it might have resulted in lowering of intracellular level of ATP, thereby inhibiting glucose uptake. Gottschal and Morris (1981) reported that solvent production was also not observed when C. acetobutylicum was grown in continuous culture with glucose or nitrogen limitation at pH value of 6.5 or 5.7. A detailed description of the mechanisms of acid inhibition can be found in Jones and Woods (1986). Van Ginkel and Logan (2005b) reported that the concentrations of the undissociated forms of acetic or butryic acid are ten times greater at a pH of 4.5 than at pH 5.5 and thus higher amounts of the undissociated form are present at the lower pH to cause inhibition. Undissociated acid concentrations can also be increased by increasing the substrate concentration from which the acids are produced. However, cultures fed high sugar concentrations are susceptible to both substrate and product inhibition. The substrates most widely used in biohydrogen research are sugars and the products are primarily H2 and CO2 gases and acetic and butyric acids. Many researchers have studied the effects of these acids on solvent production in traditional Acetone-Butanol-Ethanol (ABE) fermentation (Monot et al., 1984). However, the hydrogen fermentation was found to change into a solvent forming reaction once the undissociated acid concentration reached a critical threshold, but the threshold cannot be well predicted as it varies substantially over the range of ~2-30 mM (undissociated acid concentration) (Grupe and Gottschalk, 1992). It was reported by Bowles and Ellefson (1985), that butanol at higher concentration would inhibit the C. acetobutylicum growth, would destroy the ability of the cell to maintain internal pH, lowered the intracellular level of ATP and finally to inhibit the glucose uptake.
Van Ginkel and Logan (2005a) reported that inhibition is caused by nonpolar undissociated acids which being able to cross the cell membrane at a low pH, that then dissociate in the cell at the higher internal pH releasing a proton inside the cell. The uptake of protons in this way uncouples the proton motive force which causes an increase in maintenance energy requirements. The uptake of acid also causes a decrease in the available coenzyme A and phosphate pools which decreases the flux of glucose through glycolysis.
Obtained results show that, at higher substrate concentration the quantity of produced gas was higher but the yield per glucose consumed was lower. Produced liquid (acid and solvent mainly butanol) and gas affect inversely on the bacterial metabolism and its growth. It was reported by Ruzicka (1996), that under high gas pressure, the hydrogen gas would dissolved in fermentation medium and that would affect inversely on bacterial metabolism and its growth beside the glucose cleavage. Lee and Zinder (1988) stated that continuous H2-synthesis requires the partial pressure of H2 to be <50 kPa at 60 °C. At pH2 <50 kPa, H2 inhibits NADH reoxidation and electron flow via ferredoxin becomes thermodynamically unfavorable. Thus, high pH2 may be a principle reason for decrease in H2 synthesis rates under carbon-excess conditions. Thus the results obtained, also emphasize the need to optimize simultaneously the effect of initial glucose concentration with inoculum size with optimum initial pH for maximizing hydrogen productivity.
CONCLUSION
The results of this study suggested that proper culture pH of 7.0, inoculum size of 10% (v/v) and glucose concentration of 5 g L-1 showed to enhance the bacterial production of hydrogen as well as final biomass. Therefore this study demonstrate that hydrogen production can be maximized by optimize both inoculum size and substrate concentration with initial pH.
ACKNOWLEDGMENTS
The author would like the thank Universiti Kebangsaan Malaysia for financial assistance under grant No. UKM-OUP-BTK-14/2007.