Temperature, a critical factor affecting the activities of microorganism and conversion rate of fermentation products, is significantly linked to the economic benefit of hydrogen technology. The effect of temperature on hydrogen production was investigated in the present study in the range of 25–42 °C at pH 6.0, becauseV. tritonius strain AM2 is capable of growing at temperature between 15 and 40 °C [27]. At 37 °C, the bacterium showed maximum hydrogen yield. It is also confirmed that the hydrogen yields of the strain at 37 °C below and above pH 6.0 were significant lowered than that at pH 6.0 (unpublished data). Growth rate of the strain AM2 at 37 °C was likely to be higher than that at 30 °C, this may cause the higher speed of hydrogen production.
High salt concentrations inhibit H2 production by preventing cell activity [9], [22], [23], [24] and [25]. The molar yield and production rate of H2 by E. aerogenes (1.6 mol H2/mol mannitol at maximum) markedly decreased to 1.2 and 0.8 mol H2/mol mannitol under 2% and 3% (v/w) NaCl, respectively [23]. There are only a few reports on H2 production under marine conditions; one report focused on Bacillus sp. B2 [40]. It is widely accepted that Na+ concentration has a strong inhibitory effect on anaerobic processes including H2 production [25], [41] and [42]. More recently, Pierra et al. [25] reported the presence of a moderately halophilic biohydrogen producing microbial population in a saline sediment of a lagoon collecting salt factory wastewaters, and the majority of the population might consist of Vibrionaceae bacteria. The family of Vibrionaceae thus becomes a dominant species under moderate halophilic conditions and exhibits the highest hydrogen yields (0.9 mol H2/mol Glc) at the highest NaCl concentration of 75 g/L [25]. As these vibrios have not been isolated yet, we were not able to compare the biohydrogen producing ability of V. tritonius AM2 directly to the lagoon vibrios. Vibrios are becoming important H2 producers in halophilic conditions today. Surprisingly, the maximum molar yield of H2 of the V. tritonius strain AM2 was recorded with 1.7 mol H2/mol mannitol (initial 2.25% (w/v) NaCl) at pH 6 and 37 °C. To the best of our knowledge, the maximum molar yield of H2 produced by strain AM2 was higher than that of any other wild-type strains of facultative anaerobes under saline conditions. Unfortunately, the effects of NaCl concentration on H2production by strain AM2 are unlikely to be fully evaluated in this study. Under aerobic conditions, strain AM2 is capable of growth at 0.5–8% (w/v) NaCl range with the optimum growth at 3% (w/v) [27]. Thus, further studies on the effects of NaCl on H2 production by strain AM2 are both worthwhile and necessary. Marine bacteria including vibrios possessed a variety of systems to use Na+ gradient to maintain stoichiometry and to power the cells on the basis of the whole genome sequencing of this strain (unpublished data). To understand how salts affect hydrogen production in the vibrios cells, global transcriptome studies may provide further insights.
Formate is a precursor of H2 produced by FHL in most H2-producing facultative anaerobes. Formate is either excreted by the cell through the formate channel (FocA) or metabolised to CO2 and H2 by FHL in enterobacteria [29], [30], [31] and [32]. In strain AM2, the molar yields of H2 and formate showed opposite trends with changes in the pH of the medium (Fig. 2), and the sum of the molar yields of H2 and formate, which is nearly equal to that of total formate produced by PFL, was also stable at the pH range 6–7.5 (Table 1). The dependence of the production dynamics of H2 and formate on pH indicates that strain AM2 mainly used FHL and produced H2 by the oxidation of formate under slightly acidic conditions (pH 5.5 and 6.0).
The H2-producing pathway using mannitol is elucidated by determining the balance of fermentative products at various levels of pH in the medium. Mannitol, which is a reduced form of hexose, can generate more molecules of NADH during glycolysis than glucose [32]. Generally, excess reducing equivalents, such as NADH, are consumed by the production of lactate, succinate, ethanol and 2.3-butanediol[32] and [43]. In strain AM2, the molar yield of ethanol of mannitol-grown cells was greater than of glucose-grown cells, and the molar yield of acetate showed the opposite trend (Fig. 2 and Table 1). The shift in carbon flux from ethanol to acetate also suggests that the mannitol-grown AM2 cells might be a physiologically reduced state. The molar yields of H2 also showed trends similar to those of ethanol with changes in the pH of the medium (Fig. 2). In most facultative anaerobes, which use only the FHL pathway, NADH is unlikely to be used in H2 production [30] and [31]. However, the enhanced H2 yield of the mannitol-grown cells suggests that there might be some effects on H2 production due to changes in reducing equivalents and carbon flux when mannitol metabolism is used in the strain.
To understand carbon flux during H2 production, the sum of the H2 and formate yields was compared between glucose- and mannitol-grown cells at each pH of the medium. The sums of the H2 and formate yields from glucose were significantly lower than that from mannitol (P < 0.05) at pH 5.5, whereas there were no significant differences between the glucose- and mannitol-grown cells at pH 6.0–7.5. Such results suggest that the carbon flux of strain AM2 during glucose metabolism might be less efficient at pH 5.5 than under the other culture conditions examined. In other words, the consistently high total sum of yields of H2and formate for mannitol-grown cells might indicate efficient mannitol metabolism by strain AM2. However, the amounts of mannitol consumed were lower than the amounts of glucose consumed at pH 5.5 and 6. However, both substrates (5% (w/v)) were consumed completely at pH 6.5 and 7.5, when gas production stopped (data not shown). These results indicate that there was a difference in the efficiency between H2production and sugar consumption of mannitol-grown cells. Therefore, more detailed analysis must focus on the metabolic and genetic control and/or feedback to understand the entire mannitol metabolism of strain AM2.
To enhance the molar yield of H2, the carbon flux of the upstream pathway of formate and acetyl-CoA are important factors to be considered. Based on the fermentation pathway, in particular that described in E. coli, the disruption of lactate- and succinate-producing pathways enhances the molar yield of H2. Inactivation of both the ldhA gene (lactate dehydrogenase gene) and the frdBC gene (fumarate reductase gene) enhanced the molar yields of H2 from 1.08 to 1.82 mol H2/mol glucose in E. coli [35] and [36]. The molar yield of lactate produced from pyruvate was remarkably low at all pH levels examined for strain AM2 (Table 1). Although succinate production could not be measured here (though the production was recently determined to be tiny (below 0.1 mol/mol sugar)), strain AM2 shows good capability in not wasting excess reducing equivalents through lactate and succinate.
Finally, we succeeded in achieving practical H2 production from powdered seaweed feedstock using a jar fermenter under pre-optimised culture conditions (Fig. 3). Our batch culture experiments demonstrate the advantages of strain AM2 as a high-yield H2 producer from mannitol, which is the primary reserve compound of brown macroalgae under saline conditions. The batch culture experiment was carried out in a 5 L jar fermenter, with 3 L marine broth added to 300 g powdered brown macroalgae (S. sculpera) at 31.1% (w/w). After 72 h of cultivation, all mannitol present in the seaweed feedstock was consumed ( Fig. 3). The results show that the use of seaweed feedstock for fermentation can be realised by simple pre-treatment, such as pulverisation. When seaweed feedstock was used as a substrate, the molar yields of H2 and ethanol could not be compared directly to the values observed in the in vitro culture with pure mannitol as a substrate, in which strain AM2 achieved 80% of the theoretical molar yield of H2 by facultative anaerobes. The gap in yields might be caused by scale-up of the working volume (data not shown) or by fermentation inhibition due to various compounds contained in seaweed feedstock.
These batch culture experiments also reveal two further challenges to H2 production from seaweed feedstock using strain AM2. One is the rather low H2 production rate of strain AM2 compared to those of enterobacteria such as E. coli [32] and E. aerogenes [23] and [37]. Improvement of the efficient culture method needs to be in order to increase the H2 productivity. Tanisho [23] reported that the molar yield and production rate of H2 in continuous culture are generally higher than in batch culture when the same strain is used. Designing gene-disrupted strains or the overexpression of genes responsible for H2 production are also effective strategies [35] and [36]. Therefore, the establishment of a continuous culture system is essential for technical advances. The other challenge is the further treatment of the residual organic fraction after fermentation of powdered seaweed, which contains mainly alginate, a component not consumed by strain AM2. Recently, we found that strain AM2 was capable of converting formate directly to bioH2 (unpublished data). Therefore, we need to increase H2 productivity in both the development of efficient utilization system of the residual fractions and the metabolic engineering of strain AM2.
Conclusions
There is still a lack of literature on hydrogen production from marine bacteria. In an attempt to fill the gap, hydrogen production by a marine derived bacterial strain was investigated with the concurrent objectives of optimising culture condition which produce the most H2. Metabolically versatile Vibrionaceae spp. [26]. are phylogenetically related to Enterobacteriaceae
Temperature, a critical factor affecting the activities of microorganism and conversion rate of fermentation products, is significantly linked to the economic benefit of hydrogen technology. The effect of temperature on hydrogen production was investigated in the present study in the range of 25–42 °C at pH 6.0, becauseV. tritonius strain AM2 is capable of growing at temperature between 15 and 40 °C [27]. At 37 °C, the bacterium showed maximum hydrogen yield. It is also confirmed that the hydrogen yields of the strain at 37 °C below and above pH 6.0 were significant lowered than that at pH 6.0 (unpublished data). Growth rate of the strain AM2 at 37 °C was likely to be higher than that at 30 °C, this may cause the higher speed of hydrogen production.
High salt concentrations inhibit H2 production by preventing cell activity [9], [22], [23], [24] and [25]. The molar yield and production rate of H2 by E. aerogenes (1.6 mol H2/mol mannitol at maximum) markedly decreased to 1.2 and 0.8 mol H2/mol mannitol under 2% and 3% (v/w) NaCl, respectively [23]. There are only a few reports on H2 production under marine conditions; one report focused on Bacillus sp. B2 [40]. It is widely accepted that Na+ concentration has a strong inhibitory effect on anaerobic processes including H2 production [25], [41] and [42]. More recently, Pierra et al. [25] reported the presence of a moderately halophilic biohydrogen producing microbial population in a saline sediment of a lagoon collecting salt factory wastewaters, and the majority of the population might consist of Vibrionaceae bacteria. The family of Vibrionaceae thus becomes a dominant species under moderate halophilic conditions and exhibits the highest hydrogen yields (0.9 mol H2/mol Glc) at the highest NaCl concentration of 75 g/L [25]. As these vibrios have not been isolated yet, we were not able to compare the biohydrogen producing ability of V. tritonius AM2 directly to the lagoon vibrios. Vibrios are becoming important H2 producers in halophilic conditions today. Surprisingly, the maximum molar yield of H2 of the V. tritonius strain AM2 was recorded with 1.7 mol H2/mol mannitol (initial 2.25% (w/v) NaCl) at pH 6 and 37 °C. To the best of our knowledge, the maximum molar yield of H2 produced by strain AM2 was higher than that of any other wild-type strains of facultative anaerobes under saline conditions. Unfortunately, the effects of NaCl concentration on H2production by strain AM2 are unlikely to be fully evaluated in this study. Under aerobic conditions, strain AM2 is capable of growth at 0.5–8% (w/v) NaCl range with the optimum growth at 3% (w/v) [27]. Thus, further studies on the effects of NaCl on H2 production by strain AM2 are both worthwhile and necessary. Marine bacteria including vibrios possessed a variety of systems to use Na+ gradient to maintain stoichiometry and to power the cells on the basis of the whole genome sequencing of this strain (unpublished data). To understand how salts affect hydrogen production in the vibrios cells, global transcriptome studies may provide further insights.
Formate is a precursor of H2 produced by FHL in most H2-producing facultative anaerobes. Formate is either excreted by the cell through the formate channel (FocA) or metabolised to CO2 and H2 by FHL in enterobacteria [29], [30], [31] and [32]. In strain AM2, the molar yields of H2 and formate showed opposite trends with changes in the pH of the medium (Fig. 2), and the sum of the molar yields of H2 and formate, which is nearly equal to that of total formate produced by PFL, was also stable at the pH range 6–7.5 (Table 1). The dependence of the production dynamics of H2 and formate on pH indicates that strain AM2 mainly used FHL and produced H2 by the oxidation of formate under slightly acidic conditions (pH 5.5 and 6.0).
The H2-producing pathway using mannitol is elucidated by determining the balance of fermentative products at various levels of pH in the medium. Mannitol, which is a reduced form of hexose, can generate more molecules of NADH during glycolysis than glucose [32]. Generally, excess reducing equivalents, such as NADH, are consumed by the production of lactate, succinate, ethanol and 2.3-butanediol[32] and [43]. In strain AM2, the molar yield of ethanol of mannitol-grown cells was greater than of glucose-grown cells, and the molar yield of acetate showed the opposite trend (Fig. 2 and Table 1). The shift in carbon flux from ethanol to acetate also suggests that the mannitol-grown AM2 cells might be a physiologically reduced state. The molar yields of H2 also showed trends similar to those of ethanol with changes in the pH of the medium (Fig. 2). In most facultative anaerobes, which use only the FHL pathway, NADH is unlikely to be used in H2 production [30] and [31]. However, the enhanced H2 yield of the mannitol-grown cells suggests that there might be some effects on H2 production due to changes in reducing equivalents and carbon flux when mannitol metabolism is used in the strain.
To understand carbon flux during H2 production, the sum of the H2 and formate yields was compared between glucose- and mannitol-grown cells at each pH of the medium. The sums of the H2 and formate yields from glucose were significantly lower than that from mannitol (P < 0.05) at pH 5.5, whereas there were no significant differences between the glucose- and mannitol-grown cells at pH 6.0–7.5. Such results suggest that the carbon flux of strain AM2 during glucose metabolism might be less efficient at pH 5.5 than under the other culture conditions examined. In other words, the consistently high total sum of yields of H2and formate for mannitol-grown cells might indicate efficient mannitol metabolism by strain AM2. However, the amounts of mannitol consumed were lower than the amounts of glucose consumed at pH 5.5 and 6. However, both substrates (5% (w/v)) were consumed completely at pH 6.5 and 7.5, when gas production stopped (data not shown). These results indicate that there was a difference in the efficiency between H2production and sugar consumption of mannitol-grown cells. Therefore, more detailed analysis must focus on the metabolic and genetic control and/or feedback to understand the entire mannitol metabolism of strain AM2.
To enhance the molar yield of H2, the carbon flux of the upstream pathway of formate and acetyl-CoA are important factors to be considered. Based on the fermentation pathway, in particular that described in E. coli, the disruption of lactate- and succinate-producing pathways enhances the molar yield of H2. Inactivation of both the ldhA gene (lactate dehydrogenase gene) and the frdBC gene (fumarate reductase gene) enhanced the molar yields of H2 from 1.08 to 1.82 mol H2/mol glucose in E. coli [35] and [36]. The molar yield of lactate produced from pyruvate was remarkably low at all pH levels examined for strain AM2 (Table 1). Although succinate production could not be measured here (though the production was recently determined to be tiny (below 0.1 mol/mol sugar)), strain AM2 shows good capability in not wasting excess reducing equivalents through lactate and succinate.
Finally, we succeeded in achieving practical H2 production from powdered seaweed feedstock using a jar fermenter under pre-optimised culture conditions (Fig. 3). Our batch culture experiments demonstrate the advantages of strain AM2 as a high-yield H2 producer from mannitol, which is the primary reserve compound of brown macroalgae under saline conditions. The batch culture experiment was carried out in a 5 L jar fermenter, with 3 L marine broth added to 300 g powdered brown macroalgae (S. sculpera) at 31.1% (w/w). After 72 h of cultivation, all mannitol present in the seaweed feedstock was consumed ( Fig. 3). The results show that the use of seaweed feedstock for fermentation can be realised by simple pre-treatment, such as pulverisation. When seaweed feedstock was used as a substrate, the molar yields of H2 and ethanol could not be compared directly to the values observed in the in vitro culture with pure mannitol as a substrate, in which strain AM2 achieved 80% of the theoretical molar yield of H2 by facultative anaerobes. The gap in yields might be caused by scale-up of the working volume (data not shown) or by fermentation inhibition due to various compounds contained in seaweed feedstock.
These batch culture experiments also reveal two further challenges to H2 production from seaweed feedstock using strain AM2. One is the rather low H2 production rate of strain AM2 compared to those of enterobacteria such as E. coli [32] and E. aerogenes [23] and [37]. Improvement of the efficient culture method needs to be in order to increase the H2 productivity. Tanisho [23] reported that the molar yield and production rate of H2 in continuous culture are generally higher than in batch culture when the same strain is used. Designing gene-disrupted strains or the overexpression of genes responsible for H2 production are also effective strategies [35] and [36]. Therefore, the establishment of a continuous culture system is essential for technical advances. The other challenge is the further treatment of the residual organic fraction after fermentation of powdered seaweed, which contains mainly alginate, a component not consumed by strain AM2. Recently, we found that strain AM2 was capable of converting formate directly to bioH2 (unpublished data). Therefore, we need to increase H2 productivity in both the development of efficient utilization system of the residual fractions and the metabolic engineering of strain AM2.
Conclusions
There is still a lack of literature on hydrogen production from marine bacteria. In an attempt to fill the gap, hydrogen production by a marine derived bacterial strain was investigated with the concurrent objectives of optimising culture condition which produce the most H2. Metabolically versatile Vibrionaceae spp. [26]. are phylogenetically related to Enterobacteriaceae
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