Improved bio-hydrogen production by overexpression of glucose-6-phosphate dehydrogenase and FeFe hydrogenase in Clostridium acetobutylicum

Highlights

• Overexpression of FeFe hydrogenase (hydA) improves the hydrogen production in C. acetobutylicum.

• Overexpression of glucose-6-phosphate dehydrogenase (zwf) improves the hydrogen production in C. acetobutylicum.

• Lactate accumulation was significantly decreased by overexpression of hydA in C. acetobutylicum.

Abstract

Clostridium acetobutylicum is an attractive industrial microorganism for biochemical production, but there have been few attempts for bio-hydrogen production based on metabolic engineering. In this study, metabolically engineered C. acetobutylicum carrying glucose-6-phosphate dehydrogenase (zwf) and FeFe hydrogenase (hydA) were constructed as recombinant strains CA-zwf(pIMP-zwf) and CA-hydA(pMTL-hydA), respectively, to improve hydrogen productivity. The results showed that the engineered strains produced 1.15 and 1.39-fold higher hydrogen yield, respectively, than the wild type. Furthermore, when pH and glucose concentration were optimized for the CA-hydA strain, enhanced hydrogen productivity of 25.8% was achieved in 7 L jar scale fermentation. This result provides an insight into the future direction for metabolic engineering of C. acetobutylicum for improved hydrogen production.

Introduction

The enormous use of fossil fuels over the past century has raised environmental concerns and led to the potential for an energy crisis. Hydrogen is considered an ideal clean energy material as it is converted to water after combustion without production of oxide pollutants, such as CO_x, NO_x, and SO_x [28]. Over the past half century, there has been interest in hydrogen as an alternative energy source because of demands for increases in energy and decreases in gas emissions associated with global warming [1]. Recently the global energy paradigm has been rapidly changing because of demand for environment friendly energy sources to counter abnormal global climate conditions, and hydrogen has become one of the major alternative energy sources for replacement of fossil fuels.

Hydrogen can be electrochemically or chemically produced as a by-product of coal or oil processing. Especially, approximately 96% of hydrogen is produced from natural gas or oil [6]. Also, biological hydrogen production has been studied since the 1800s and has recently drawn much attention [26]. Biological hydrogen production can be achieved by either photosynthesis, dark-fermentation, or microbial electrolysis cells, these processes are advantage to be more eco-friendly and less energy concentrated than physico-chemical processes [18]. Among these, dark-fermentation by anaerobic fermentative bacteria is one of the most studied techniques, and the main focus has been on the Clostridium species according to excellent hydrogen production rates and typical properties for industrial application [16]. Nevertheless, hydrogen yield per unit of substrate is still low, which poses a hindrance for industrialization of hydrogen production because the substrate is a major contributor to costs in production [26].

Clostridium species have a mechanism to convert hydrogen gas from reducing powers using hydrogenase, therefore, it can be archived by anaerobic fermentation to produce large amounts of hydrogen. Clostridium acetobutylicum is a well-known strain for acetone-butanol-ethanol (ABE) production, and there have been numerous studies on the fermentation process, strain development, genetic and metabolic engineering, etc. [14,31]. There has also been a focus on biological hydrogen production with Clostridium species, which produce hydrogen through the pyruvate:ferredoxin oxidoreductase (PFOR) pathway, a key intermediate in ATP production. In principle, NAD is reduced to NADH when the glucose metabolized into pyruvate through glycolysis. Pyruvate converts to acetyl-CoA through the PFOR pathway, carrying CO₂ and reduced ferredoxin. The reduced ferredoxin must be regenerated, and the electrons gained in that reaction can be formed molecular hydrogen with proton by a hydrogenase, or used to regenerate NAD⁺ by NADH-ferredoxin-oxidoreductase [4]. Consequently, the hydrogen production by FeFe hydrogenase promoted from reduced ferredoxin, and this led to various approaches involved in NADH rebalancing and FeFe hydrogenase expression based on metabolic engineering for hydrogen production.

In the pentose phosphate (PP) pathway, glucose-6-phosphate is oxidized by G-6-P dehydrogenase and generated NADH which supplies reducing power for intracellular biosynthesis [11]. In addition, it was demonstrated that high hydrogen yield is related with the NAD(P)H supply rate from glucose degradation through the PP pathway [11,15,20]. Therefore, metabolically engineered strains have been made to increase hydrogen yield by overexpression of G-6-P dehydrogenase (zwf) or deletion of phosphoglucose isomerase (pgi) [24,27,30].

Furthermore, it is well known that Clostridium species produces hydrogen gas during growth. Characterized hydrogenase and its information have been reported from several clostridia: hydrogenase of C. acetobutylicum P262, hydrogenase A of C. perfringens, and Fe-hydrogenase I of C. pasteurianum [9,21,22]. Enhanced hydrogen productivity by overexpression of hydrogenase was demonstrated in Clostridium species: C. paraputrificum M − 21 showed a 1.6-fold higher hydrogen yield than that of the wild type [19], while higher yield has also been proven in C. tyrobutyricum JM1 [7]. Meanwhile, for C. acetobutylicum it was reported that there was no effect on hydrogen yield by overexpression of hydA [12], and no further study was conducted.

In this study, we aim to evaluate hydrogen production yield by overexpression of zwf and hydA from C. acetobutylicum. Hydrogen production by recombinant strains carrying each gene was investigated in a range of initial pH and glucose concentration. Finally, hydrogen production by a recombinant strain was evaluated in 7 L jar scale fermentation.