Abstract
BackgroundCellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria.ResultsIn present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase β-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15 ± 18.94 U/mL and 0.130 ± 0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that β-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family.ConclusionsBased on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and B. methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the β-glucosidase and endoglucanase were successfully cloned and expressed from B. methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.
Highlights
Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed
It has been found that the culture and genetically modification of fungi were relatively more difficult to achieve than bacteria, which seriously hindered the practical application of fungi and fungi-producing cellulases to celluloses hydrolyzation [11, 12]
Isolation and identification of cellulolytic bacteria A total of 81 strains were isolated from five rotten wood samples, in which 8, 17, 19, 15 and 22 isolates were obtained from weed tree, red birch, poplar, alpine rhododendron and willow, respectively
Summary
Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Cellulosic biomass (composed of cellulose, lignin and hemicellulose) is one of the most abundant renewable resources, which is considered as a potential and promising raw material for future energy production [1]. It has been found that the culture and genetically modification of fungi were relatively more difficult to achieve than bacteria, which seriously hindered the practical application of fungi and fungi-producing cellulases to celluloses hydrolyzation [11, 12]. The library of bacteria that possessed powerful activity to hydrolyze cellulose was not sufficient, which partly limited the study and application of the cellulolytic bacteria. BS-5 [13], Bacillus licheniformis 2D55 [14], Bacillus subtilis BY4 [15], Paenibacillus chitinolyticus CKS1 [16], Ochrobactrum sp K38 [17], and Clostridium thermocellum [18], which suggested that various species of bacteria from different origins should be screened and highlighted
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