Improved bio-hydrogen production by overexpression of glucose-6-phosphate dehydrogenase and FeFe hydrogenase in Clostridium acetobutylicum
Graphical abstract
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 COx, NOx, and SOx [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.
Section snippets
Bacterial strains, media and growth conditions
The strains used in this study are summarized in Table 1. Escherichia coli DH5α was used for plasmid cloning work and were grown aerobically in Lurea-Bertani (LB) medium at 37 °C. For the selection of recombinant strains, 100 μg/mL of ampicillin and 50 μg/mL of kanamycin were added to the medium. C. acetobutylicum strains used in this study were cultured in an anaerobic chamber (97% N2, 3% H2) at 37 °C in Reinforced Clostridial Medium (RCM; Difco, Kansa City, MO). The RCM medium composed of the
Engineering of C. acetobutylicum for production of hydrogen
It is known that C. acetobutylicum has two phases depends on the growth condition: an acidogenic phase where hydrogen and organic acids are produced, and a solventogenic phase in which butanol, ethanol and acetone are produced from the organic acids [17]. During the acidogenic phase, NAD(P)+ cofactor is reduced to NAD(P)H through the PFOR pathway, and it is oxidized by FeFe hydrogenase and organic acid production (Fig. 1). In these processes, NADPH can be generated through PP pathway under the
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by National Research Foundation of Korea grant funded by the Korea government (Ministry of Science and ICT, MSIT) (NRF-2019M3E6A1103839) and the Korea Institute of Energy Technology Evaluation and Planning and the Ministry of Trade, Industry and Energy of the Republic of Korea (20188550000540).
References (30)
- et al.
Hydrogen production, storage, transportation and key challenges with applications: a review
Energy Convers Manag
(2018) - et al.
The production of biohydrogen by a novel strain Clostridium sp YM1 in dark fermentation process
Int J Hydrogen Energy
(2014) - et al.
Energy conservation via electron bifurcating ferredoxin reduction and proton/Na+ translocating ferredoxin oxidation
Biochim Biophys Acta Bioenerg
(2013) - et al.
Metabolic flux analysis of hydrogen production network by Clostridium butyricum W5: effect of pH and glucose concentrations
Int J Hydrogen Energy
(2010) - et al.
The role of hydrogen in low carbon energy futures-A review of existing perspectives
Renew Sustain Energy Rev
(2018) - et al.
Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1
Int J Hydrogen Energy
(2010) - et al.
The effects of pH on carbon material and energy balances in hydrogen-producing Clostridium tyrobutyricum JM1
Bioresour Technol
(2008) - et al.
The hydA gene encoding the H-2-evolving hydrogenase of Clostridium perfringens: molecular characterization and expression of the gene
FEMS Microbiol Lett
(1999) - et al.
Influence of hydrogenase overexpression on hydrogen production of Clostridium acetobutylicum DSM 792
Enzym Microb Technol
(2010) - et al.
Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production
Curr Opin Biotechnol
(2011)
Metabolic flux analysis of biological hydrogen production by Escherichia coli
Int J Hydrogen Energy
Outlook of fermentative hydrogen production techniques: an overview of dark, photo and integrated dark-photo fermentative approach to biomass
Energy Strategy Rev
Overexpression of a hydrogenase gene in Clostridium paraputrificum to enhance hydrogen gas production
FEMS Microbiol Lett
Metabolic-flux analysis of hydrogen production pathway in Citrobacter amalonaticus Y19
Int J Hydrogen Energy
21st Century's energy: hydrogen energy system
Energy Convers Manag
Cited by (16)
Recent advances in fermentative biohydrogen production
2024, International Journal of Hydrogen EnergyEnhancement of biohydrogen production in Clostridium acetobutylicum ATCC 824 by overexpression of glyceraldehyde-3-phosphate dehydrogenase gene
2023, Enzyme and Microbial TechnologyRecent progress and challenges in biotechnological valorization of lignocellulosic materials: Towards sustainable biofuels and platform chemicals synthesis
2023, Science of the Total EnvironmentCitation Excerpt :Besides these studies, other researchers also aimed at genetically engineering and redirecting metabolic pathways of H2 producers to facilitate H2 yield. For example, Son et al. (2021) reported an increased H2 yield by overexpressing glucose-6-phosphate dehydrogenase and FeFe hydrogenase in C. acetobutylicum. The metabolically engineered C. acetobutylicum strains, CA-zwf(pIMP-zwf) and CA-hydA(pMTL-hydA), produced 1.2- and 1.4-fold higher H2 yields than the wild-type, respectively.
Wastewater-derived biohydrogen: Critical analysis of related enzymatic processes at the research and large scales
2022, Science of the Total Environment
- 1
These authors contributed equally to this work.