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. Meanwhile, based on “diameters ratio between clear zone and strain” during the investigation by Congo red method (Supplementary 1) and the growth of strains in the process of subculture, 55 cellulolytic strains were finally selected for the further study. In addition, it needed to be mentioned that strains named as B. subtilis 1CJ1 and Bacillus sp. 1CJ4 had the largest diameters of clear zone more than 25 mm, and the largest value of “diameters ratio between clear zone and strain” was 3.71 belonged to Bacillus sp. 3AJ7 (Supplementary 2).
The isolated strains were identified according to their 16S rRNA gene, after which phylogenetic tree was established as shown in Fig 1. Results indicated that the strains could be classified into Bacillus subtilisa, Bacillus sp., Pseudomonas aeruginosa, Bacillus licheniformis, Bacillus methylotrophicus and Bacillus megaterium, which suggested that the Bacillus might be the predominant strains possessing the cellulose degradation activity in the rotten wood.
Cellulase activities and hydrolysis capability
The isolated strains were inoculated into sole carbon source medium for 48 h at 37 ℃ under 120 rpm. Reducing sugar concentration and cellulase activities were investigated and shown in Fig. 2 as a heat map which obviously indicated the relationship between bacteria and the cellulase activity as well as the production of cellulose degradation (reducing sugar content). In addition, the detail results were also supplied in the Supplementary 3.
The crude enzyme extracts were collected to determine reducing sugar content and cellulase activities. The maximum reducing sugar concentration was observed in CMC-Na medium by B. subtilis 1AJ3 of 4.83 mg/100mL, followed by B. subtilis 1BJ4 of 4.54 mg/100mL and B. subtilis 1BJ6 of 4.47 mg/100mL. Compared with CMC-Na medium, a maximum production of reducing sugar content (1.61 mg/100 mL) was obtained by B. subtilis 3BJ7 in Avicel medium. The results showed that strains selected from CMC-Na medium had a higher enzyme activity than which selected from Avicel. In addition, B. subtilis 1BJ4 had both the highest FPase activity (0.0133 U/mL) and CMCase activity (0.0368 U/mL), while B. licheniformis 3EJ7 had the highest Avicelase activity of 0.010 U/mL. For the majority of strains, the results were coincided with the general sense that strains selected from CMC-Na always had a high CMCase activity, while that from Avicel had a high Avicelase activity. It was also interesting to find that strains processing both CMCase activities and Avicelase activities did not appear in present study. For example, B. subtilis 1BJ4 had the highest CMCase activity, but it didn’t exhibit Avicelase activity, which was possibly explained by the fact that different strains might produce different cellulases under the same or different carbon source [20]. Interestingly, although B. subtilis 3CJ6 had the highest Avicelase activity, and both other two enzyme activities were tested, no reducing sugar was detected.
From the heat map, it found that the difference of intergeneric impacted the cultivation process and which led to the differences in reducing sugar production and cellulase activities. As the results shown, B. subtilis strains possessed the advantages in secreting of cellulases, which led to the high reducing sugar content, FPase and CMCase activity. Meanwhile, B. methylotrophicus and B. licheniformis performed relatively well just in the reducing sugar content. In addition, bacteria isolated from CMC-Na as solo carbon source medium had the higher FPase and CMCase activities than that from Avicel medium.
According to the reducing sugar content and cellulase activities, eight strains were further selected as: B. subtilis 1AJ2, B. subtilis 1AJ3, B. subtilis 1BJ4, B. methylotrophicus 1EJ7, B. subtilis 3BJ4, B. subtilis 3CJ6, B. subtilis 3CJ8 and B. methylotrophicus 3EJ7, which would be applied to carbon sources to evaluate the cellulose degradation activity.
Reducing sugar production and cellulase activities in different carbon sources
The selected eight strains were cultured with different carbon sources: wheat straw, corn stover, switchgrass, Avicel and CMC-Na (Fig.3).
Each strain was separately inoculated into the medium with five different carbon sources (wheat straw, corn stover, switchgrass, Avicel, and CMC-Na) for 48 h with 6% seed inoculation. Fig.3 (a) shows the reducing sugar concentration obtained from different carbon sources by the treatment with each strain. B. subtilis 1AJ3 and B. methylotrophicus 1EJ7 showed strong potential in the producing of reducing sugar, even in the lignocellulosic biomass without any other pretreatments (wheat straw, corn stover, and switchgrass), which then followed by B. subtilis 3BJ4 and B. subtilis 1AJ2. It also found that the strains showed similar FPase and CMCase activities (Fig.3b and Fig.3c) in different carbon sources. Specifically, only B. subtilis 1AJ3 and B. subtilis 3BJ4 produced Avicelase in all medium, and B. subtilis 1AJ2 only produced Avicelase in CMC-Na medium. Meanwhile, other strains produced Avicelase in three or more carbon sources. Accordingly, based on the reducing sugar content, cellulase activities and carbon source type, three strains (B. subtilis 1AJ3, B. methylotrophicus 1EJ7, and B. subtilis 3BJ4) were selected for the further study.
Pretreatment of lignocellulosic biomass
Three strains of B. subtilis 1AJ3, B. methylotrophicus 1EJ7 and B. subtilis 3BJ4 were used to pretreat wheat straw, switchgrass and corn stover separately or mixed-up. After sterilization at 121 ℃ for 20 min, the initial reducing sugars concentration were 136.34 mg/100mL, 109.46 mg/100mL, and 39.16 mg/100mL in the medium of corn stover, switchgrass and wheat straw, respectively.
The reducing sugar content in all samples tended to be stable (Fig.4) after culturing with 36 h, and the highest sugar content of 95 mg/100 mL was obtained by B. methylotrophicus 1EJ7 in switchgrass. Meanwhile, 73mg/100 mL in wheat straw and 72 mg/mL in corn stover were also obtained by B. methylotrophicus 1EJ7, which also indicated that no synergistic effect was observed in the pretreatment of the mixture.
SEM test was benefit for the understanding of the degradation process of the straw degradation caused by the proposed strains. As one of the major agricultural waste in China, wheat straw has a relatively denser lignocellulosic structure, and which was selected as the sample to be investigated after the hydrolyzation by B. methylotrophicus 1EJ7. It was found that the surface (Fig. 4a) of the wheat straw particles was dramatically changed (Fig. 4b) after bacteria pretreatment. Specifically, it was obviously found that the smooth surface of wheat straw particles was destroyed to form numerous holes and lots of bacteria were observed as adhering on the surface. Therefore, the sunken tiny holes suggested that the bacteria processed the cellulase activity and which initially destroyed the surface structure of wheat straw. In addition, the similar phenomenon was also observed in corn stover hydrolysis [21].
As the cellulose content affected the degree of crystallinity in various of plant biomass, the decrease of crystallinity is also an index of the decrease of cellulose content or the destruction of the cellulose structure, therefore, which was also used to evaluate the efficiency of the pretreatment [22]. Hence, X-ray diffraction was used to analyze crystallinity of wheat straw samples in the present study. The results indicated that the Cr I of wheat straw decreased from 41.57 to 40.52 (Fig. 4c) before and after the pretreatment, which also verified the degradation of wheat straw caused by the pretreatment of B. methylotrophicus 1EJ7.
Cellulases clone and expression
The target genes of β-glucosidase and endoglucanase were 732 bp and 1500 bp, respectively, and which were successfully cloned. In addition, universal primer T7 was utilized to amplify the two recombinant plasmids (pET-28a-Bgl and pET-28a-Egl), and then that were tested for the complete sequences. Thereafter, heterologous expressions of the proposed two genes in E. coli BL21 (DE3) were carried out to obtain the enzymes. As shown in SDS-PAGE, two cellulases were both successfully expressed in E.coli BL21 (DE3), and the Mws were tested as 28.5 kDa and 56.3 kDa (Fig.5), respectively. The results of crude cellulase activities showed that the activity of crude Bgl and Egl were 1670.15±18.94 U/mL and 0.130±0.002 U/mL, respectively (Supplementary 4).
The results of domains analysis showed that Bgl belonged to GH16 family (Supplementary 5), and Egl contained two domains as catalytic domain (CD) and carbohydrate-binding module (CBM), which belonged to GH5 family and CBM3 family, respectively (Supplementary 6).
Meanwhile, by blast from PDB protein database, the highest identification of Bgl was endo-beta-1,3-1,4 glucanase (PDB id 3O5S_A) from Bacillus subtilis 168 with a similarity of 93.55%, and Egl was 94.92% similarity with endo-1,4-beta-glucanase (PDB id 3PZT_A) and 90.41% with CBM3 lacking the calcium-binding site (PDB id 2L8A_A) from B. subtilis 168. In addition, compared with Bgl sequence of Bacillus velezensis JTYP2, it was found that only four amino acids (70M→V, 96V→A, 156A→K, 204N→T) were different with the Bgl in present study, and the predicted secondary structure didn’t obviously affect by these differences. It also indicated the Bgl of B. subtilis 168 showed more differences with the proposed Bgl as 22 amino acids were different (Supplementary 7). For the Egl, the sequence analysis showed that it had a 96.6% similarity with the Egl of Bacillus velezensis JTYP2, and there are 51 different bases between the two sequences led to 17 different amino acids (27A→T, 31G→E, 52Q→R, 199P→I, 238S→F, 285K→N, 316S→T, 331S→G, 332N→T, 334S→L, 339A→G, 364S→R, 382T→A, 404F→V, 411I→M, 414S→G, 440K→T), in which most changes appeared on the carbohydrate-binding module (CBM) and the linked peptide [23], and all the changes of the amino acids didn’t significantly influent the secondary structure (Supplementary 8).
Bioinformatics analysis and homology modeling
The recombined Bgl contains 251 amino acids included a His-tag with a molecular weight of 28.47kDa. The computed pI was 6.79, and the negative GRAVY score (−0.491) suggested that the protein might be hydrophilic. Bgl showed instability index and aliphatic index of 16.14 and 60.24, respectively. Correspondingly, the recombined Egl contained 507 amino acids including his-tag with a predicted molecular weight of 56.32 kDa, and the computed pI was 7.26 and the negative GRAVY score (−0.616) suggested the Egl was a hydrophilic protein. In addition, Egl showed instability index and aliphatic index of 29.60 and 73.69, respectively, and instability index less than 40 also indicated that both the Bgl and Egl from B. methylotrophicus 1EJ7 was stable.
Bgl and Egl homology structural models were obtained by the I-TASSER. The information about the active site was obtained through superimposing 3D model structure of the Bgl with the template structure of cellulase from Paenibacillus macerans hybrid endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase (PDB id 2AYH) [24], which provided accuracy of homology between two structures and also contributed to find the conserved active site residues. Active site of Bgl was represented by 6 amino acid residues as Leu103, Phe105, Thr175, Asp179, Tyr188 and Asp236. The 35th to 242th amino acid domain of Bgl included a classical sandwich-like beta-jelly roll fold, and formed by two main, closely packed and curved antiparallel beta sheets, which led to a deep channel harboring the catalytic machinery. Bgl was found to be a catalytic sequence motif similar with GH16 family [23, 25], E-[ILV]-D-[IVAF]-[VILMF] (0, 1)-E, which was formed by amino acid 134th to 138th (EIDIE). The structural analysis of Egl showed that domains of CD and CBM, in which the catalytic domain had the critical TIM-barrel fold structure of GH5 family, consisting of 8 β-strands surrounded by 8 α-helices with the active site located at the cleft.