Application of hydrogen-producing consortia enriched from various organic substances for increasing the hydrogen yield from agro-industrial wastewater

As a part of new economic model of Thailand 4.0, reducing for the dependency on the utilization of fossil fuels is considered as the main awareness of Thailand government. The realization of Thailand 4.0 can be focused by transition to renewable energy which aims to environmental protection, which will create an economic system adjusted to climate change and a low carbon society. Because Thailand is one of the world export leaders for agricultural product, Thailand has massive and potential renewable biomass-based resources. This type of feedstocks is highly available, low cost, and organic substrate and it can be used as suitable feedstock to produce renewable energy. By utilizing the agro-industrial waste and wastewater to high-added value products (such as bioenergy), the concept of waste-to-energy can be achieved. This concept has advantages for “three problems can be solved at once”, management of waste, reduction of greenhouse gas emission, and production of energy. The waste-to-energy concept which emphasizes to 5R (reduce, reuse, recycle, recovery, and reclamation) is also a fundamental concept for establishing a circular economy system.

         Hydrogen is one of alternative energy (biogas) for substituting the traditional fossil fuels due to its renewable nature, clean, and high energy content. Compared to hydrocarbon fuels, hydrogen is considered clean fuel since its combustion product is only water vapor (instead of greenhouse gas CO₂ and other toxic gases). However, hydrogen gas is not naturally freely available. Hydrogen can be produced by various methods. Until now, fossil fuels are the main feedstock (96%) for conventional hydrogen production and 4% remaining is from water electrolysis and biomass. Conventional techniques to produce hydrogen, (steam reforming of hydrocarbons or autothermal reforming) are not cost-effective, energy-exhaustive, and less eco-friendly (CO₂ production) and do not meet the criteria for sustainable and circular economy concept. Hydrogen production via dark fermentation by utilization of organic waste and wastewater is emphasized for decreasing the required energy for its production, decreasing the cost of feedstocks, and reducing the carbon dioxide emission. Biological dark fermentation has double advantages for its efficient waste utilization and bioenergy production. In addition, the utilization of high availability and cost-effective organic waste or wastewater can boost the economic value of biohydrogen produced from the dark fermentation.

         Hydrogen is mostly produced from the carbohydrate-based wastes or wastewater. Hydrolysis of those substances results in various simple sugars, such as sucrose, glucose, xylose, or hexose, which are easily utilized by hydrogen-producing microorganisms. However, exploration for various feedstocks for hydrogen production from agro-industrial waste and wastewater is required to secure the availability and sustainability of feedstock. Lipid-rich, protein-rich and glycerol-rich wastewaters has potential for being used as feedstock for hydrogen production due to their high organic compounds and minerals. The main challenge of the utilization of these feedstocks for hydrogen production is hydrolysis step which is commonly considered as the limiting step. The efficiency of hydrogen production from agro-industrial wastewater is determined by the microbial community involved in the dark fermentation system. Several research reported that inoculum for biohydrogen reactor can be obtained from pure culture (such as Clostridium sp. And Klebsiella sp.) and mixed culture (pre-treated anaerobic sludge). Although pure culture can efficiently produce hydrogen in small scale, several problems arise when applying this microbial culture for producing hydrogen from wastewater, such as contamination issue, complicated preservation, and shift on metabolic pathways. Pre-treated anaerobic sludge can also be used as inoculum for biohydrogen reactor. However, the main problem for those pre-treated mixed microorganisms is the consistency and stability for their activity caused by pre-treatment steps (heat-shock or acid/base treatments). Since the limiting step of hydrogen production from wastewater is hydrolysis, microbial consortia consisting of several microbial groups can be alternative for boosting the hydrogen production. Hydrogen production involves hydrolysis and acidogenesis phase in which hydrolytic microorganism breaks down large organic molecule from complex substrates into smaller molecules which the hydrogen-producing microorganisms utilize them to produce hydrogen. Metabolic synergy between hydrolytic microorganisms and hydrogen producers is critical for utilizing the complex organic waste or wastewater and ensuring the economic viability of hydrogen production at large scale. Eco-biotechnological enrichment approach with complex organic substance can be conducted to obtain these microbial groups or microbial consortia. This approach emphasizes on the natural selection and competition by giving the selective stress in mixed microbial community based on the specific substrate and operational conditions. The microbial consortia enriched via eco-biotechnological approach are expected to have more robust characteristics and synergistic effect among microbes for hydrogen production from organic wastewater than single microbial culture or defined co-culture.

         The proposed research aims for investigating the applicability and efficacy of hydrogen-producing microbial consortia enriched by eco-biotechnological enrichment approach using various organic substances. The efficacy of the hydrogen-producing consortia will be determined for their performance for increasing hydrogen production from agro-industrial wastewater. To obtain highly active hydrogen-producing consortia, this research will use various organic substances, such starch (carbohydrate-based), vegetable oil (lipid-based), peptone (protein-based), and glycerol (alcohol-based), as specific substrates for eco-biotechnological enrichment. The utilizations of these various substrate are expected to result in unique and different microbial community in the consortia consisting of synergism between highly active hydrolytic microorganisms and hydrogen producers. The proposed research will be conducted into three main parts. At the first part, anaerobic sludge from the anaerobic reactors will be obtained and then pre-treated to remove methanogens. The suitability of inoculum for enrichment process is determined based on the hydrogen production activity and microbial community after pre-treatment step. The second part of proposed research is focused on the eco-biotechnological enrichment process for obtaining highly effective hydrogen-producing consortia using various organic substances. In this part, carbohydrate-, lipid-, protein, and glycerol-based will be used as selective carbon sources for the enrichment process. The microbial activity and their community dynamics during enrichment process will also be observed. Then, the third part of research will investigate the applicability and efficacy of hydrogen-producing consortia enriched from each selective substrate for hydrogen production from various agro-industrial wastewater. It is expected that this proposed research will contribute to more knowledge for enrichment strategy for highly active hydrogen-producing consortia which can be used for biohydrogen production from agro-industrial waste. Efficient hydrogen production from wastewater by highly active microbial consortia further contribute to efficient waste or wastewater management and clean bioenergy production.​​​​​​​