Screening, identification, and optimization of fermentation conditions for high-yielding cellulase producing Bacillus subtilis
In recent years, with the unlimited use of fossil fuels, global oil prices have fluctuated dramatically, seriously affecting human daily life. People are increasingly concerned about the research and development of new alternative energy sources, and cellulose biomass fuel is currently recognized as the most valuable and promising renewable energy source. Cellulose biomass is the most widely distributed and abundant biomass on Earth, mainly composed of cellulose, hemicellulose, and lignin. However, cellulose, hemicellulose, and lignin are tightly bound through hydrogen and covalent bonds, possessing a stubborn biomass structure. We have only developed less than 2% of cellulose resources. The use of modern biotechnology to convert cellulose into liquid fuels such as ethanol has stable, renewable, and pollution-free yields, and has the potential to solve problems such as energy crisis, environmental pollution, and food crisis. But to solve this problem, the most crucial thing is to obtain efficient and low-cost cellulases.

Cellulase is a collective term for a group of complex enzyme systems that degrade cellulose to produce monosaccharides or polysaccharides, mainly including endoglucanase, exoglucanase, and β -1,4 glucosidase. These three enzymes work together to degrade cellulose substances. At present, research on cellulase producing bacteria mainly focuses on fungi such as Trichoderma, Penicillium, and Aspergillus. However, most of their cellulases bind to the cell membrane, which is inconvenient to use, and the cost of enzyme separation and extraction is too high. Moreover, fungal cellulases not only have a long production cycle, but most of the cellulases produced also need to be at higher temperatures to exert good efficiency. At room temperature or lower temperatures, enzyme activity is very low. Bacteria grow faster than fungi, can obtain higher expression levels of recombinant enzymes, and can produce more complex glycoside hydrolases for enzymatic hydrolysis synergistic reactions. Meanwhile, bacteria have a high natural diversity and are more likely to produce heat-resistant and alkali stable enzymes. So far, the bacteria that produce cellulase that have been extensively studied include Vibrio, Bacillus, Bacteroides, Clostridium, Erwinia, Ruminococcus, and Thermomonas.

So far, although a large amount of research has been conducted on the conversion of lignocellulose, the conversion of cellulose biomass into bioethanol still faces significant challenges due to the high commercial costs of hemicellulases and cellulases. Therefore, the development of new, efficient, and low-cost cellulases is urgent, and the breeding of high-yield cellulase strains is particularly important. Screening high-yielding cellulase producing bacterial strains from laboratory preserved strains, conducting identification and research on them, providing strain sources for cellulase preparation research, and laying a certain foundation for genetic improvement, genetic engineering strain construction, and other work.

 

In this study, a total of 5 strains of bacteria with high cellulase production were screened from laboratory preserved strains. After morphological, physiological, and biochemical characteristics, as well as 16S rDNA analysis, they were identified as Bacillus subtilis, 2 strains of Bacillus subtilis, and 3 strains of Bacillus amyloliquefaciens. Many fungi and bacteria in nature can produce various cellulases. At present, there are many studies on fungal cellulases, and the cellulases on the market mainly come from fungi. The cellulase producing bacteria have also attracted extreme attention due to their strong adaptability and other advantages, and they are likely to become a very important source of cellulase in industry. Among them, Bacillus subtilis can produce and secrete a large number of extracellular enzymes, making it the main bacteria studied. In addition, they can also form endospores and secondary metabolites under conditions of slow growth in cellulose matrix, giving them a competitive advantage. Among them, Bacillus subtilis is the most reported excellent cellulase producing bacterium, while Bacillus amyloliquefaciens is rarely reported.

The ratio of hydrolysis circle diameter to colony diameter on the Congo Red CMC plate in this article is greater than 4.5, with the minimum ZB6 being 4.61 and the maximum AF1 bacterial ratio reaching 8.04, far exceeding the high enzyme activity bacterial strains screened using the same method as reported in existing domestic and foreign literature. The highest enzyme activity reported in the literature was a strain PX19 isolated by Tang Hao et al. from the intestinal tract of the giant bamboo elephant. The ratio of hydrolysis circle diameter to colony diameter of the highest enzyme activity strain was only 4.83. After optimizing and screening the solid fermentation conditions for cellulase production by AF1 strain, its highest filter paper enzyme activity can reach 26.904U/g. In addition, the previous research team found that AF1 bacteria can also produce highly active amylase, protease, and broad-spectrum antibacterial substances. When used for feeding chicks, it can significantly improve feed conversion rate, inhibit pathogen growth, reduce disease occurrence, and lower mortality rate. It is a powerful strain with multiple functions and great potential for development.