Eco-biotechnological Approach for Enrichment of Highly-Effective Hydrogen-Producing Microbial Consortia from Lipid and Glycerol-Rich Wastewaters

Thailand government focus their awareness to reduce the dependency on fossil fuels and focus more to renewable energy policies as a part of new economic model of Thailand 4.0. Transition to renewable energy is a decisive phase for realization of Thailand 4.0 aiming to environmental protection, which will create an economic system adjusted to climate change and a low carbon society. Since 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 agroindustrial 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. (Nielsen, 2017; Sariatli, 2017).

     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, consisted of 49% from natural gas, 29% from liquid hydrocarbons, and 18% from coal. While, 4% remaining is from water electrolysis and biomass (Baeyens และคณะ, 2020). Conventional techniques to produce hydrogen, such steam reforming of hydrocarbons, autothermal reforming, etc., are not economical (expensive), 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 and also reduction of carbon dioxide emission. Biological dark fermentation has double advantages for its efficient waste utilization and bioenergy production. In addition, the utilization of organic waste, such as agroindustrial wastewater, can increase the economic value of biohydrogen from dark fermentation.

     In dark fermentation, hydrogen is mostly produced from polysaccharides-based agroindustrial waste and wastewater, such as starch and lignocellulosic-rich waste and wastewater (Kim และคณะ, 2012). Hydrolysis of those material results in various simple sugars, such as sucrose, glucose, xylose, or hexose, which are easily utilized by hydrogen-producing microorganisms (Yasin และคณะ, 2013). However, exploration for more and various feedstock for hydrogen production from agroindustrial waste and wastewater is required in order to secure the availability and sustainability of feedstock. Lipid- or glycerol-rich wastes or 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 lipid- or glycerol-rich feedstock is to enhance the hydrolysis step which is commonly considered as the limiting step (Kim และคณะ, 2012; Yasin และคณะ, 2013). Applying the suitable and highly-efficient microbial consortia can be conducted to solve the limitation of hydrolysis step of lipid- or glycerol-rich feedstock.

     Mixed microorganisms or microbial consortia, which consist of several microbial groups, is more preferred than pure culture for degrading the complexity of organic compounds waste or wastewater to hydrogen. Several drawbacks have been reported for the utilization of pure microbial culture for hydrogen productions, such as contamination issue, complicated preservation, and shift on metabolic pathways (Ntaikou และคณะ, 2010). Several research reported the hydrogen production from agroindustrial wastewater using pre-treated mixed microorganisms, such as heat treated-anaerobic sludge, digested sludge, and activated sludge (Badiei และคณะ, 2012; Varrone และคณะ, 2013; Tapia-Venegas และคณะ, 2015; Norfadilah และคณะ, 2016). However, the main problem for those pre-treated mixed microorganisms is the consistency and stability for their activity. Moreover, less research investigates the utilization of enriched microbial consortia from the specific environment for being utilized as inoculum for hydrogen production.

     In this research, eco-biotechnological approach will be used to enrich the highly-active hydrogen-producing microbial consortia. The eco-biotechnological approach for microbial enrichment emphasizes on the natural selection and competition by giving the selective stress in mixed microbial community based on the specific substrate and operational conditions. This approach does not modify or bioengineer the microbes, but their ecosystem (Rajesh Banu และคณะ, 2020). By using this approach, the enriched microbial consortia are expected to have robust characteristics and synergistic effect between microbes for hydrogen production compared to single microbial culture or defined co-culture. As a result, the hydrogen as bioenergy can be produced from low-cost and high availability of lipid- and glycerol-rich wastewater.

     For initial step of this proposed research, the potential microbial sources for enrichment study from different environments is explored using 16S rDNA sequencing analysis to determine the presence of existed biodiversity of hydrogen producers in those environments. Then, the eco-biotechnological enrichment approach is conducted by creating the strict conditions for enhanced-natural selection and interaction between targeted microbial consortia. Feeding strategy for abrupt applying lipid/glycerol as carbon source will be compared with the strategy of gradual decrease of glucose and lipid/glycerol ratio for their effectivities to produce high-efficient microbial consortia with high activity and the specific microbial community. The shift of microbial community during enrichment process is determined using 16S rDNA sequencing analysis in order to investigate the main microbial community involved in hydrogen production. After enrichment process, the activity, efficacy and performance of enriched microbial consortia will be compared with the commercial hydrogen producers and heat-treated anaerobic mixed sludge to produce hydrogen from lipid or glycerol-based substrate. It is expected that the microbial consortia from enrichment process can show synergistic effect for efficiently degrading lipid- or glycerol-based substrate to hydrogen.