Isolation and characterization of Bacillus sp. 275 exhibiting lignocellulolytic activities

Abstract

The objective of this research is to isolate lignocellulose-degrading bacteria from environment and characterize their lignocellulolytic activities. In addition, a study on the lignocellulolytic properties and genomic analysis of selected isolates, Bacillus sp. 275 were performed. In this study, various environmental soil samples and isolation media were used to acquire isolates. The cell growth, enzyme activities, and the degradation products of cellulose and xylan were analyzed. First, bacterial isolation from environmental soils and investigation of their cellulolytic, xylanolytic and lignolytic activities and taxonomic distribution were conducted. Finally, 89 bacterial strains were isolated. Lignocellulolytic bacteria were isolated from not only lignocellulose-rich soils but also lignocellulose-lack soil samples. All the isolates were included in 4 phyla: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. Bacillus and Streptomyces occupied over 70 % of bacteria showing multienzymatic activities and the most abundant genus with only lignolytic activity was Burkholderia. Lignin-containing medium was effective to isolating bacteria with lignolytic activity only, but not favorable to isolating bacteria exhibiting multienzymatic activities. Second, candidate bacterial isolate exhibiting high lignocellulolytic activities was selected comparing growth and enzyme activity. Bacillus sp. 275 was grown in both solid and liquid alkali lignin media, and showed relatively high cellulase, xylanase and peroxidase activities compared to isolates, strain 123 (i.e., Bacillus aryabhattai) and 414 (i.e., Streptomyces durhamensis). As a result, Bacillus sp. 275 was selected as final candidate for degradation of lignocellulose among 89 isolates. Third, whole genome sequencing of Bacillus sp. 275 was conducted and genomic analysis using complete genome sequence was investigated. A lot of genes concerning to the degradation of lignocellulose in the genome of Bacillus sp. 275 were detected. Endoglucanase, β-glucanase, glucohydrolase, glucosidase, 1,4-β-xylosidase, arabinoxylan arabinofuranohydrolase, glucuronoxylanase, dye decolorizing peroxidase and laccase were present in the genome of Bacillus sp. 275. Besides, the genes related to degradation of starch, mannan, galactoside and arabinan were also found. Finally, the characteristics of lignocellulolytic properties of Bacillus sp. 275 were investigated. The degradation products of cellulose (i.e., cellobiose and cellotriose) and xylan (i.e., xylose, xylobiose, xylotriose, xylotetraose and xylopentaose) were observed by extracellular proteins secreted by Bacillus sp. 275. In addition, filter paper degradation occurred actively by Bacillus sp. 275 for 11 days. Regarding lignin utilization, the cell growth with alkali lignin added to the culture medium (LB medium) was increased by 78% compared to that without alkali lignin. Moreover, the lag phase was shortened from 12 hours to 6 hours in the case of lignin-added culture medium. In this research, the isolation condition such as the sampling locations and the isolation medium was significant factors to efficiently isolating bacteria with lignocellulolytic activities. Bacillus sp. 275 showed cellulolytic, xylanolytic and lignolytic activities by extracellularly produced enzymes. In addition, Bacillus sp. 275 is potentially capable of degrading wide ranges of polysaccharides in lignocellulose. Considering only a few reports of bacteria using all major three component of lignocellulose (i.e., cellulose, hemicellulose and lignin) were published, Bacillus sp. 275 is a promising candidate for degrading or utilizing lignocellulosic biomass.