Acceleration and enhancement of anaerobic digestion of lignocellulosic biomass for biogas production: Microbial screening, enrichment and identification of effective anaerobic microbial community (Phase II)

Agricultural residues and agro-industrial wastes in Thailand were annually increased due to the increase of population resulting in high demand of food and bioresources that generating a huge number of solid residues and biowastes. Thailand is an agricultural and agro-industrial country generated 116 million tons of agricultural residue in year 2018. The residue composes of 55.33% sugarcane residues, 25.75% rice straw, 9.05% cassava pulp and stalk, 5.62% oil palm residues, and 3.16% maize straw. The idea of this research is to develop the way to treat and utilization these wastes with high efficiency and mitigation of environmental problem including generated high valued product for sustainable bioeconomy concept. Anaerobic digestion is a popular technology for the concept of waste treatment and utilization by biodegradation/ treatment of waste along with production of renewable energy. There are two main benefits of anaerobic digestion, they can have a positive impact on the environment (pollution, smell, pathogens, weed seeds etc.) and they can provide direct financial returns (capture methane, fertilizer/ plant nutrients). The production of biogas from biowastes consists of a four-step process in the absence of oxygen as follows: a) hydrolysis, b) acidogenesis, c) acetogenesis, and d) methanogenesis. In the case of hydrolysis of lignocellulosic biomass in anaerobic digestion, this step is the bottleneck and also be the rate-limiting step for biogas production. Due to its complex structure of cell wall, cellulose fibrils embedded in lignin and hemicellulose including crystalline cellulose made it is more strengthen and hardly biodegrade.

        Therefore, the lignocellulolytic enzymes need to be used for breaking down the structural compounds of lignocelluloses into a smaller molecular weight of products. The enhancement of enzymatic hydrolysis by a lignocellulolytic microorganism in anaerobic digestion is one major concern to accommodate. Therefore, it is necessary to open the structural compounds of lignocelluloses and to facilitate easy access of enzymes to hydrolyze lignocellulosic compounds. Pretreatments of lignocellulosic biomass were widely used for increasing the accessibility of the biomass polymers for the following enzymatic hydrolysis and fermentation. Pretreatment of lignocellulosic biomass by physical, chemical, or biological methods has been applied for anaerobic digestion. Pretreatments of lignocellulosic biomass were widely used for increasing the accessibility of the biomass polymers for the following enzymatic hydrolysis and fermentation. Physical pretreatment is increased in accessible surface area and pore size, nevertheless most of them cannot remove lignin and required high energy. Chemical pretreatment is decreased in cellulose crystallinity and partial or complete hydrolysis of hemicellulose, in contrast, there are a chemical requirement and harsh condition. In addition, a side effect of the pretreatment under acidic condition is the formation of lignocellulose derived by-products such as the common aliphatic carboxylic acid and furfural that inhibit on the fermentative microorganism and enzymatic biocatalysts. Therefore, biological pretreatment using fungi, bacteria and actinomycetes, becomes interested in biogas production. Due to it is mild environmental conditions, low energy and no chemical requirement, even a low rate of biodegradation. Biological pretreatment seems an eco-friendly process and no inhibitor generation during the process.

Biological treatment requires to be used for breaking down the structural compounds of lignocelluloses into a smaller molecular weight of products. The enhancement of enzymatic hydrolysis by lignocellulolytic microorganisms in anaerobic digestion is one major concern to accommodate. Anaerobic microorganisms especially anaerobic bacteria and anaerobic fungi are considered for this study under anaerobic conditions. Although anaerobic fungi are slower growing compared to anaerobic bacteria, however they can produce the enzyme complex called “multi-enzyme complex” to degrade cellulose and hemicellulose in lignocellulosic biomass. Another benefit, anaerobic fungal rhizoid can break the plant cell wall and get through to degrade cellulose and hemicellulose to monosaccharide into their cell to converse to acid, ethanol, hydrogen, carbon dioxide and acetate. In addition, the multi-enzyme complex from anaerobic fungi can convert hexose and pentose from hemicellulose. Less information about anaerobic lignocellulolytic microorganisms was reported. The specialty of anaerobic bacteria and anaerobic fungi for degrading plant cell walls is interesting to enrich and use for biogas production in part hydrolysis.

To achieve the efficacy of ALMC, screening, selection, and enrichment are still the important techniques in order to increase lignocellulosic biomass degradation in sustainable and economically competitive production processes. Several molecular approaches are applied to study the microbial community in samples from an ecosystem or habitat. Automated ribosomal intergenic spacer analysis (ARISA) acts as a high resolution, highly reproducible, and robust method for discriminating between microbial communities. To establish effective lignocellulolytic microorganisms for start-up bioreactor with high biogas production, molecular approaches analysis and functional characterization of the biogas microbiome have been used to explain their function and predict the microbial community action from gene potential. Biolog EcoPlates (Biolog Inc., CA, USA) provides the potential metabolic information of bacteria involved in the carbon cycle. Biolog EcoPlate™ has been used to evaluate the metabolic abilities of the microbial community before start-up in an anaerobic reactor for enhancing the degradation of organics materials. In addition, microbial function from Biolog EcoPlate™ has followed 16S rRNA gene sequence analysis of genetic diversity to determine the microbe responsible for carbon consumption of hydrolysis. Due to the complexity and diversity of anaerobic microbial consortium for methane production in anaerobic digestion processes, the study of anaerobic microbial communities has been developed with high-throughput sequencing technologies. A suitable bioinformatic analysis approach and analysis of high throughput sequencing data plays a critical role in the investigation of the microbial community. Metagenomic data analysis with computational approaches overcome the challenges of both assembly-based and mapping-based metagenomic profiling, especially high complex samples containing similar sequencing of the genome sequencing. Therefore, in this study for the first year (2022), techniques of screening, selection, and enrichment of lignocellulolytic microorganisms will be used to obtain the efficacy and stability of enriched ALMC with high methane production. Four different sources of seed inocula will be used to screen and select ALMC of high potential for methane production. Rice straw will be used as feedstock for biogas production during enrichment. B-ARISA will be applied to detect microbial community shifts during enrichment. After that in the second year (2023), not only the performance of effective ALMC will be determined by biochemical methane potential (BMP) but studies of metabolic diversity by EcoPlate, microbial community by 16S rRNA gene sequencing, and evaluation of microbial function, gene potential, and genetic diversity by shotgun metagenomics sequencing of community will also undertake to understand the interaction and predicate the microbial community in this consortium of lignocellulolytic inoculum. Moreover, the optimization of the obtained ALMC and AF will be done for biogas production from lignocellulosic biomass.