Metabolic flux network analysis of fermentative hydrogen production: Using Clostridium tyrobutyricum as an example
Introduction
Development of a clean and renewable energy source has become an urgent need in modern times because of the depletion of fossil fuel, and also the possible correlation between global climate change and its consumption (Jo et al., 2008). Hydrogen (H2), with the highest energy density and generating only water after combustion, is considered as one of the most promising alternatives (Das and Veziroglu, 2001, Fang et al., 2006). Besides its cleanliness, in terms of no greenhouse gas (GHG) production during combustion, hydrogen has many benefits, such as being used as the fuel for a conventional fuel cell, where electricity can be produced without combustion. Among hydrogen production processes, fermentative hydrogen production (FHP) is an attractive route since a variety of carbon sources can be used as the feedstock, such as starch (Liu and Shen, 2004), cellulose (Lay, 2001), sucrose (Chen et al., 2001), glucose (Lee et al., 2008, Li et al., 2010, Whang et al., 2011), xylose (Zhu and Yang, 2004), and mixture of biomass and organic wastes (Das and Veziroglu, 2001, Hawkes et al., 2008, Juang et al., 2011, Cheng et al., 2012).
It is widely known that various volatile fatty acids (VFAs) and/or alcohols are produced through dark fermentative hydrogen production. The theoretical H2 yield is 4 mol H2/mol glucose when acetate is the sole product while a maximum yield of 2 mol H2/mol glucose was obtained when butyrate as the end product. The maximum yield of 12 mol H2/mol glucose which should be achieved from the complete conversion of glucose to H2 and CO2 has never been attained in known biological system until now (Fang et al., 2006). The low conversion yield might presumably due to the natural consequence that microorganism tend to produce cell biomass evolutionarily instead of hydrogen, and to the competing reactions such as hydrogen consumption or division to other products (Hallenbeck and Benemann, 2002). In practice, H2 yield is related to the metabolic pathways and end-products, like when butyrate-to-acetate ratio (B/A ratio) and lactate yield have been proved to be related to H2 production. Therefore, it appears that there are some critical metabolic activities in H2 fermentation which may be ignored in the currently proposed metabolic reactions and equations (Cai et al., 2011) and the potential of FHP anaerobes has not totally explored and their metabolic network requires to be understood.
Possible fermentation pathways of H2 production by Clostridium sp. utilizing glucose have been reported in several researches (Sridhar and Eiteman, 2001, Zhu and Yang, 2004, Cai et al., 2010), it seems that Clostridium sp. share similar pathways. Clostridium tyrobutyricum is one of saccharolytic clostridia which is wildly incubated in butyric acid production (Zhu et al., 2002, Wu and Yang, 2003) and H2 fermentation (Jo et al., 2008, Whang et al., 2011) since it has many advantages over other species, including simple medium for cell growth and relatively high product purity and yield (Michel-Savin et al., 1990). However, the impact of incubation factors such as hydraulic retention time (HRT) or initial substrate concentration on metabolic flux distribution in the biological H2 fermentative system applying C. tyrobutyricum is still not well-known. The quantification of the pathway fluxes becomes important for both bioreactor operation and metabolic engineering in order to enhance H2 production. Metabolic flux analysis (MFA) has been considered as a useful methodology to calculate intracellular fluxes from extracellular fluxes, and would be helpful for the comprehension of flux regulation in the H2 fermentation (Manish et al., 2007, Cai et al., 2011).
In this study, the metabolic network model for the fermentative hydrogen production of C. tyrobutyricum was developed. The MFA methodology was employed to illustrate the possible mechanism and flux distribution during glucose and lactate/acetate metabolism of C. tyrobutyricum, and to study the effect of HRT and initial substrate concentration on the intracellular fluxes under a constant temperature and pH.
Section snippets
Bioreactor operation
Two continuous stirred tank reactor-type (CSTR) bioreactors, fed with glucose (GA) and lactate/acetate (LA) respectively, were conducted in the present study. For the GA bioreactor, an isolated pure culture of C. tyrobutyricum FYa102 from previous studies (Li et al., 2010, Whang et al., 2011) was applied as seeding sludge, and 12,000 mg/L of glucose, 1400 mg/L of ammonium chloride, and 360 mg/L of peptone were fed as a substrate. The seeding microorganism for LA bioreactor was obtained from a
Node analysis of GA bioreactor
For the GA bioreactor, the metabolic flux distribution under the HRT between 2 and 18 h are shown in Fig. 2. The errors of carbon mass balance of each run were less than 5%. In general, HRT presented a significant impact on H2 production. H2 production became unfavorable when HRT approached to 4 h which was the doubling time of C. tyrobutyricum, as found in batch experiments.
In order to further understand the detailed flux distribution, node analysis was applied. Table 1 and Fig. 2 summarize the
Conclusion
The metabolic fluxes network of C. tyrobutyricum in H2 production from glucose and lactate/acetate fermentation was evaluated in this study. MFA results indicate that HRT presents a significant impact on the metabolic flux of hydrogen production from glucose. At HRT between 4 and 18 h, increase of HRT increased hydrogen production but decreased lactate production, while at HRT below 4 h decrease of HRT increased hydrogen production but decreased lactate production. The flux for lactate, butyrate
Acknowledgements
The authors would like to acknowledge the financial support from the National Science Council of Taiwan under Grant NSC 98-3114-E-006-013, NSC 100-3113-E-006-017, and NSC 101-3113-E-006-016.
References (35)
- et al.
Metabolic flux network and analysis of fermentative hydrogen production
Biotechnol. Adv.
(2011) - et al.
Optimization of two-phase thermophilic anaerobic digestion of biowaste for hydrogen and methane production through reject water recirculation
Bioresour. Technol.
(2011) - et al.
A two-stage bioprocess for hydrogen and methane production from rice straw bioethanol residues
Bioresour. Technol.
(2012) - et al.
Hydrogen production by biological processes: a survey of literature
Int. J. Hydrogen Energy
(2001) - et al.
Acidophilic biohydrogen production from rice slurry
Int. J. Hydrogen Energy
(2006) - et al.
Biological hydrogen production; fundamentals and limiting processes
Int. J. Hydrogen Energy
(2002) - et al.
Fermentative production of hydrogen from a wheat flour industry co-product
Bioresour. Technol.
(2008) - et al.
Comparison of metabolic pathway for hydrogen production in wild-type and mutant Clostridium tyrobutyricum strain based on metabolic flux analysis
Int. J. Hydrogen Energy
(2013) - et al.
Biological hydrogen production by immobilized cells of Clostridium tyrobutyricum JM1 isolated from a food waste treatment process
Bioresour. Technol.
(2008) - et al.
Evaluation of bioenergy recovery processes treating organic residues from ethanol fermentation process
Bioresour. Technol.
(2011)