Isolation and characterization of cellulolytic bacteria for improving biogas production from crop residues through biological pretreatment and codigestion

Abstract

Biofuel production from renewable resources is a rising concern due to depletion of fossil fuels, increasing fuel prices and green house gas emissions in the world. Zimbabwe is renowned for its agricultural industry, which generates millions of tonnes of crop residues, such as straw, husks, cobs, stover and hulls. Crop residues contain large amounts of organic matter that can be converted into biofuels. Anaerobic digestion (AD) is a technology which combines biogas production and sustainable waste management in a circular bioeconomy. Biogas is a clean renewable energy carrier with numerous benefits. The main objective of the study was to isolate, identify and characterize cellulolytic bacteria that can be used to enhance biogas production from crop residues through biological pretreatment and codigestion. The recalcitrant nature of crop residues makes pretreatment an essential step towards sustainable biogas production. In order to select the most suitable pretreatment strategy for crop residues, a systematic study was conducted using the PRISMA method. Biological pretreatment was found to be the best-fit option for improving the hydrolysis of crop residues. It was regarded as an ecofriendly technology with low capital and energy needs, and no generation of toxic compounds. As a result, there was need to promote biological pretreatment as a technology that enhances biogas production from crop residues. The focus was on isolating cellulolytic bacteria from hot springs for potential pretreatment agents. Of all the strains screened from hot springs, only three strains designated as LB-4, LB-6 and LB-8 had high cellulolytic activity. The strains were preliminarily identified as rod shaped, Gram positive and appeared to belong to a group of motile bacillus through morphological and biochemical identification. Homology analysis against the NCBI GenBank showed strains LB-4, LB-6 and LB-8 to be 99.13%, 98.26% and 98.91% related to Bacillus subtilis, Bacillus sp., and Bacillus licheniformis, respectively. Using submerged fermentation, the optimum cellulase activity of the strains were observed after 24 hours at pH 7 and 40ºC while utilizing 1% CMC as a carbon source and 1% yeast extract as a nitrogen source. Comprehensive analysis of maize stover, wheat straw and soybean straw for proximate composition showed a significant variability among the three crop residues. All the crop residues were reported to contain more than 30% cellulose and revealed high potential for biogas production. Cellulose, hemicelluloses and lignin content in crop residues ranged from 34.6 - 37.8%, 19.7 - 28.2% and 16.2 - 23.5%, respectively. Wheat straw had a higher cellulose (37.8%) and hemicellulose content (28.2%) than the other crop residues. Soybean straw reported the highest lignin content of 23.5%. However, the acidic nature (pH 5.3 - 5.5) and high total nitrogen content (3.1 - 8.2%) of the crop residues highlighted the need for codigestion with other organic substrates. Strains B. subtilis LB-4, Bacillus sp. LB-6 and B. licheniformis LB-8 were used to construct a hot spring cellulolytic microbial consortium (HSCMC). The HSCMC consortium was applied for biological pretreatment of crop residues. The pretreated feedstocks were characterized for total reducing sugar, ash, total solids (TS) and volatile solids (VS). Significant variation between pretreated crop residues and control conditions was observed in relation to chemical iii characteristics. The highest decline in VS (69.2%) content was reported from maize stover, whereas wheat straw showed maximum reduction of 83.9% in TS content. The TRS concentrations for pretreated hydrolysates of maize stover, wheat straw and soybean straw were significantly enhanced by 60.9, 96.3 and 84.7%, respectively. The biomethane potential assay was performed using batch fermentation to evaluate the feasibility of pretreating crop residues using the HSCMC consortium. Results established that pretreatment of crop residues using HSCMC can significantly improve the cumulative methane yield of maize stover, wheat straw and soybean straw by 50.2%, 50.6% and 56.6%, respectively. Cattle manure with pH, ash, VS and TS content of 7.12, 15.10%, 47.20% and 51.34%, respectively, was selected as a cosubstrate for this study. Codigestion of pretreated crop residues with cattle manure increased methane yield in the range of 13.3 - 25.1% compared to unpretreated groups. In conclusion, bacteria with high cellulolytic ability were successfully isolated from hot springs and an effective microbial consortium, HSCMC was developed. The HSCMC consortium enhanced methane yield of crop residues after pretreatment and codigestion with cattle manure. This study provides useful information and original contribution that could validate the upscaling of bench-scale findings to pilot- and demonstration-scale towards commercialization