Beyond the Theoretical Yields of Dark-Fermentative Biohydrogen

Abstract

Theoretical hydrogen (H₂) yield by dark fermentative route is 12 mol/mol of glucose. Biological H₂ production yields of 3.8 mol/mol of glucose by microbes have been reported. Transient gene inactivation in combination with adaptive laboratory evolution strategy has enabled the H₂ yield to exceed the stoichiometric production values.

Fossil fuel reservoirs are depleting faster than they can be replenished. An associated limitation on the usage of these non-renewable fuels is their high pollution causing capacity. The need is to search for novel fuels, which can be based on renewable resources [1]. In addition, it provides an opportunity to identify an ideal fuel. Here, bioalcohol, biodiesel, biohydrogen and methane are the most competitive and ecofriendly fuels [2–11]. Each of them has its own merits and demerits. Biohydrogen has been assessed to be most energy efficient and non-polluting fuels, easy to transport, and can be converted into other forms of energy [1]. Different H₂ production methods include: (i) Thermochemical, (ii) Electrolytic, (iii) Photolytic and (iv) Biological. Biologically, H₂ can be produced by dark- and photo-synthetic microbes from pure sugars and biowastes [2]. Theoretically, 12 mol of H₂ can be generated via the complete oxidation of 1 mol of hexose sugar [12]. However, stoichiometrically, a maximum to 4 mol H₂ per mole of hexose sugar can be achieved. Under dark-fermentative conditions, maximum of 4 and 2 mol of H₂ can be produced depending upon the acetate and butyrate as fermentative byproducts, respectively (Eqs. 1 and 2).

$$\text{Hexose}+2{\text{H}}_2\text{O}\to 2\text{Acetate}+4{\text{H}}_2+2{\text{CO}}_2$$ 1

$$\text{Hexose}\to \text{Butyrate}+2{\text{H}}_2+2{\text{CO}}_2$$ 2

Overall, dark-fermentative H₂ production rates are significantly higher than those achieved through photo-fermentative process. In the past three decades, limited number of H₂ producers with little significant improvement in the biological H₂ yields have been reported. The dark-fermentative H₂ production by pure cultures such as: Bacillus, Caldicellulosiruptor, Clostridium, Thermotoga, and Enterobacter has been reported to be up to 3.80 mol/mol of hexose [12–14]. To achieve higher H₂ yields, various strategies have been adopted, including: (i) identification of novel producers, (ii) optimization of process parameters, and (iii) genetic engineering of native producers. In addition, different approaches have been suggested to improve the overall efficiency of the process by integrating various processes such as: dark- and photo-fermentations, polyhydroxyalkanoate production and biomethanation [15–19].

After a few decades of dedicated efforts, it has now been shown that we can go beyond the physiologic yields of dark-fermentative biohydrogen. It is remarkable that genetically modified extremophile Thermotoga maritima under dark-fermentation process showed a 1.9-fold higher H₂ yield of 11.54 mol/mol of maltose compared to the wild type [20]. Here, the strategy of transient gene inactivation to disrupt lactate dehydrogenase (ldh) to block lactate production was combined with adaptive laboratory evolution. After a few passage, strain Tma200 was evolved, which was slow growing and consumed maltose at a lower rate but could oxidize sugar more efficiency. It was found to be very effective in improving the H₂ yield to 5.77 mol/mol of hexose by competing the needs for cell biomass synthesis with metabolite (H₂) formation using maltose as feed. This novel strategy can be extended to other H₂ producers to improve the overall efficiency of the process using biowaste as an economical feed for the sustainable development.