Metabolic flux analysis (MFA) is a technique for the assessment of intracellular metabolic fluxes in a biological system with a central importance to maximize the product yield or to analyze in priori, the effect of targeted genetic modification on the product formation (Nissen et al., 1997, Manish et al., 2007). If the measured fluxes are not sufficient to determine all intracellular fluxes, optimization approaches are applied.
This method was mainly established by Edwards et al. (2002) and was named “flux balance analysis” (FBA). The analysis of possible metabolic routes falls into “metabolic network analysis” (MNA). MFA technique has been applied to optimize production of lysine (Vallino et al., 1993), acetate (Delgado et al., 1997), ethanol (Tunahan et al., 2004) etc.
In order to achieve high H2 production yield, an extensive analysis and understanding of the metabolic pathways in the H2-producing microorganisms is required which may further aim to redesign or redirect the metabolic pathways towards maximum product formation. The intracellular metabolic fluxes could be calculated by using mass balances across metabolites, stoichiometric reaction models as well as thermodynamics. Metabolic flux analysis (MFA), has been extensively applied in most research over the past decade in predicting changes in the fluxes and rate limiting steps of the specific pathway (Table 1.6).
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Table 1.6. Summary of literature on metabolic flux analysis of biohydrogen production utilizing different substrates and microbial strains
Microorganism Substrate Salient features Reference
Clostridium tyrobutyricum
Glucose and lactate/acetate
MFA methodology was applied to study the flux distribution during glucose and lactate/acetate metabolism and effect of HRT and initial substrate concentration. HRT showed significant impact on H2 production. Increase of HRT increased H2 production and reduced lactate production
Cheng et al., 2013
Rhodobacter capsulatus
Acetate, lactate, malate and CO2
Study the flux distribution in the photoautotrophic metabolism of R.
capsulatus for several substrates. Prediction of knockouts mainly by blockade of Calvin cycle and reduction of formate leading to increase H2
yield using the constructed flux model.
Golomysova et al., 2011
Klebsiella
pneumoniae ECU-15
Glucose MFA method was used to estimate the effects of various culture conditions (temp, pH, initial glucose conc.) on production and uptake of hydrogen flux.
Higher temperature reduced the uptake hydrogen and enhanced H2
production. pH 7.0-7.5 was optimal for both the H2 flux and the producing H2
flux was maximum at 5g/L of initial glucose.
Niu et. al., 2011
Clostridium butyricum W5
Glucose Metabolic flux analysis (MFA) of fermentative hydrogen with variations in initial glucose concentration and operational pH. The results suggest that pH has more significant effect on H2 production compared to the glucose concentration.
Cai et al., 2010
Synechocystis sp.
PCC6803
Glucose Metabolic model was implemented for analysis of hydrogen production under three conditions (heterotrophic, autotrophic and mixotrophic) in terms of O2 production and CO2 fixation. Two conditions anoxic maximum and anoxic photoreduction
Navarro et al., 2009
INTRODUCTION AND LITERATURE
35 Table 1.6. (Continued…)
Citrobacter amalonaticus Y19
Glucose MetaFluxNet was employed for flux analysis of H2 production with varying glucose concn. High H2 yield of 8.7 mol H2/mol glucose was possible if glucose metabolism is directed to the PP pathway and NAD(P)-linked hydrogenase is used to produce H2.
Oh et al., 2008
Klebsiella pneumonia
Glycerol MFA of anaerobic glycerol metabolism for production of 1,3-propanediol.
Flux distribution revealed that branch points of glycerol and dihydroxy acetone phosphate were rigid compared to the flexible node of pyruvate and acetyl CoA to various environmental conditions.
Zhang et al., 2008
Escherichia coli Glucose Evaluation and comparison of metabolic network of wild and mutant (lacking lactate dehydrogenase) E.coli starin for H2 production. Ethanol and acetate play significant role in H2 production while lactate and succinate are not necessary.
Manish et al., 2007
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Although MFA has been applied for few case studies of Clostridium sp. to study the effect of environmental conditions on H2 yield (Table 1.6), to the best of our knowledge, investigation of metabolic pathway fluxes of fermentative hydrogen production using Clostridium pasteurianum was not found in the literature.
Flux balance analysis (FBA) is a mathematical approach for analyzing the flow of metabolites through a metabolic network. A metabolic network contains all of the known metabolic reactions in an organism and the genes that encode each enzyme. FBA calculates the flow of metabolites through this network, thereby making it possible to predict the growth rate of an organism or the rate of production of an important metabolite. Many reports have been published on FBA analysis of hydrogen production by Clostridium thermosuccinogenese (Sridhar et al., 2001); Clostridium butyricum W5 (Cai et al., 2010, Cai et al., 2011); Clostridium acetobutylicum (Senger et al., 2008). Oh et al., 2008 described flux analysis of hydrogen pathway in Citrobacter amalonaticus Y19 and concluded that a high H2 production yield of 8.7 mol H2/mol glucose is possible if glucose metabolism is directed to the PP pathway. Similarly Cheng et al., 2013 reported MFA for hydrogen production using Clostridium tyrobutyricum and concluded that HRT presents a significant impact on the metabolite flux of H2production from glucose. In another report, MFA analysis of biohydrogen by Clostridium butyricum W5 indicated that operational pH has a significant effect on H2 yield as well as metabolic flux distribution while initial glucose concentration had less impact on H2 yield.