Understanding the regulation of the metabolic network associated with fermentative hydrogen production in Clostridium butyricum.

Abstract

Hydrogen is an environmentally friendly and high energy caffier that could be a potential replacement for depleting fossil fuels. Fermentative hydrogen production (FHP) has received great interest in recent decades, as it offers a potential means of producing H₂ from a variety of renewable and waste resources via a low energy process. However, the commercial application of the FHP process has been hampered by its low yields. It becomes important to obtain a better understanding of metabolic pathways and their regulation in H₂-producing microorganisms. The aim of this work was to improve fundamental knowledge of the central metabolic flux network associated with FHP using Clostridium butyricum, and to restructure metabolic pathways to enhance hydrogen production yield. The metabolic network model was firstly constructed for C. butyricum and metabolic flux analysis (MFA) using this model was applied to predict metabolic flux distribution under varying initial glucose concentrations and operational pHs when the specific growth rate was chosen as the objective function. MFA results suggested that pH has a more significant effect on hydrogen production yield compare to the initial glucose concentration. These results also suggested that the phosphoenolpymvate (PEP), pyruvate and Acetyl CoA (AcCoA) nodes are not rigid nodes. MFA was found to be a useful tool to provide valuable information for optimization of the fermentative hydrogen production process and for future design of metabolic engineering strategies. The butyrate formation pathway was blocked using a shuttle plasmid pMTL007 containing a group II intron designed for targeting hbd, which encodes β-hydroxybuffil-CoA dehydrogenase in the C. butyricum 'W5 genome. A method for transforming the plasmid into C. butyricum was developed. Fermentation studies showed that the resulting hbd-deficient strain M3-1 performed less H₂ production with a substantial increase in ethanol production compared.to the wild type strain W5; while under nitrogen sparging conditions, M3-1 exhibited increased H₂ production with the simultaneouS decrease of ethanol production. These results indicated that H₂ production by C. butyricum may compete for reduced nicotinamide adenine dinucleotide (NADH) with the ethanol formation pathway. Homologs of all three subunits of a bifurcating hydrogenase from Thermotoga maritime were amplified from the strain W5, indicating that W5 may possess a potential bifurcating hydrogenase which utilized reduced ferredoxin and NADH simultaneously to produce H₂. The ethanol formation pathway was blocked by disrupting the acetaldehyde CoA dehydrogenase (ACDH) domain on aad (encoding a bifunctional aldehyde-alcohol dehydrogenase) using pMTL007C-E2. Fermentation studies showed that the aad-deficient strain M6 produced 484% more lactate, 32% more acetate, 9% less butyrate and 78% less H₂ than the wild type strain W5; while with the addition of sodium acetate (NaAc) to the culture of M6, carbon flux to the lactate formation pathway was redirected to the butyrate formation pathway, resulting in the increase of final H₂ yield from 0.94 mol/mol glucose to 1.65 mol/mol glucose. The results from this study have provided a better understanding of the metabolic flux network associated with hydrogen production by C. butyricum, and developed a genetic and metabolic approach to the enhancement of hydrogen production yield.