Cellulolytic Bacteria Screened from Qinling (China) for Biomass Degradation and Cellulases First Cloned from Bacillus methylotrophicus

Cellulosic biomass degradation still needed more studies while bioenergy is becoming mainly energy in future and more evaluate bacteria isolation laid a foundation of further study. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. The aim of this work was to find potential cellulolytic bacteria in depredating multiform carbon source cellulose substrate. In this study, 55 potential cellulolytic bacteria screened out and were identified. Based on the results of the investigation of cellulase activities and reducing sugar content via different carbon substrate effect, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further taken to hydrolyze wheat straw, corn stover and switchgrass, suggesting that B. methylotrophicus 1EJ7 was the most preponderant bacterium, obtaining highest sugar content (95mg/100mL) in switchgrass, wheat straw and corn stover. Scanning electron microscopy (SEM) and X-ray diffraction results of wheat straw surface and crystallinity indicated the hydrolyzation. By ascertaining the target sequence of cellulase for the cloning and expression in an economical and convenient manner, the genes of β-glucosidase (243 aa) and endoglucanase (499 aa) of B. methylotrophicus 1EJ7. Recombinant β-glucosidase from GH16 family and enzyme activity was 1670.15±18.94 U/mL. Endoglucanase consist of GH5 family catalytic domain and a carbohydrate-binding module belongs to CBM3 family and enzyme activity was 0.130±0.002 U/mL. Screened, identified the cellulolytic bacteria from rotten wood of Qinling Mountains and explored their ability in degrading different carbon source cellulose substrate, including

purified and natural carbon sources. Bacillus were the predominant species among the isolated strains, and Bacillus methylotrophicus 1EJ7 performant well on cellulose degradation. In the meantime, the β-glucosidase and endoglucanase were successfully cloned and expressed from Bacillus methylotrophicus for the first time. The strain and the recombinant enzyme have potential application in industrial production.

Background
Cellulosic biomass (composed of cellulose, lignin and hemicellulose) is one of the most abundant renewable resources. It is considered a potential and promising raw material for future energy production as well [1]. Cellulose is considered the critical component that can be converted into various value-added products: e.g. ethanol, 5-hydroxymethylfurfural (HMF), levulinic acid, butanol, alkanes, hexane, succinic acid, ethyl lactate, and other chemicals. In these procedures, cellulose should be firstly hydrolyzed to glucose, where after, various bio-or chemical processes can be carried out. Therefore, the degradation of cellulosic material has aroused huge attention to enrich reducing sugars to the greatest extent.
Many methods, including acid-activated montmorillonite catalysts, steam explosion, acid and alkaline, enzymatic hydrolysis and microbiological methods, have been developed to hydrolyze cellulose. From the perspective of environmental friendliness and energy saving, the enzymatic hydrolysis and microbiological method are prioritized to be practically applied, both of which are associated with microorganisms, such as fungi and bacteria [2]. It is true that fungi exhibit a strong ability to secret considerable extracellular enzymes including multi-cellulases. Given this, extensive literature have been made about cellulases producing fungi, such as Trichoderma reesei RUT-C30 [3], Trichoderma koningiopsis FCD3-1 [4], and Melanoporia sp. CCT 7736 [5]. Besides, it is also found that the culture and genetically modification of fungi are relatively more difficult to achieve than bacteria, seriously hindering the practical application of fungi and fungiproducing cellulases to hydrolyze celluloses [6,7]. In general, bacteria were commonly considered a powerful tool for functional modification or genomic operation, for instance, the cloning and expressing of single cellulase or recombinant cellulases. However, the library of bacteria exhibiting good ability to hydrolyze cellulose was not sufficient, thus requiring further enrichment. It has been reported that various of bacteria, such as  [14]. It is also generally considered that Qinling Mountains exhibits unique climate, plants, and microorganism resource. Besides, rotten woods originating from Qinling Mountains contains various of biomass degrading microorganisms, providing good materials for screening valuable bacteria to degrade lignocellulose. Thus, in the present study, bacteria exhibiting the capability of degrading cellulose were isolated and identified from Qinling Mountains rotten woods. Subsequently, cellulase activities were assayed and the strains were inoculated into the wheat straw, corn stover and switchgrass to assess the degraded extent of lignocellulosic biomass. Furthermore, by searching target sequence based on NCBI, genes of β-glucosidase and endoglucanase were successfully cloned and expressed on the pET-28a(+) plasmid in E.coli BL21 (DE3).

Results
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. And then, 55 cellulolytic strains were further screened by Congo red method (Fig. 1) 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.4).

←Fig. 4
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.4 (a) shows reducing sugar concentration in different carbon sources of each strain. B. subtilis 1AJ3 and B. methylotrophicus 1EJ7 showed strong potential in producing reducing sugar, especially in lignocellulosic biomass without pretreatment (wheat straw, corn stover, and switchgrass), then followed by B. subtilis 3BJ4 and B. subtilis 1AJ2. The strains showed similar FPase and CMCase activity ( Fig.4b and Fig.4c The reducing sugar content in all medium tended to be stable (Fig.5) 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 was also obtained by B. methylotrophicus 1EJ7, which also indicated that no synergistic effect was observed in the pretreatment of mixture.
SEM could help us to understand the process of the straw degradation 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 hydrolyzed by B. methylotrophicus 1EJ7. It was found that the epidermis (Fig. 5a) of the wheat straw were dramatically changed (Fig. 5b) after bacteria pretreatment. Specifically, the initial intact structure was destroyed to form some holes and lots of bacteria adhered on the surface. The sunken tiny holes showed that the bacteria could hydrolyze straw and destroy the surface structure of wheat straw, and the similar phenomenon was also observed of corn stover hydrolysis [16].
As the cellulose content affect the crystallinity in most plant biomass, the increase of crystallinity is also an indication of increase of cellulose content and can be used to evaluate the efficiency of the pretreatment [17]. X-ray diffraction was used to analyze crystallinity in wheat straw samples. The Cr I of wheat straw decreased from 41.57 to 40.52 (Fig.5c) before and after pretreatment, which suggested that the degradation of wheat straw could be realized the B. methylotrophicus 1EJ7.

Cellulases clone and expression
Two cellulases, β-glucosidase of 732 bp and endoglucanase of 1500 bp, were cloned respectively. Universal primer T7 was utilized to amplify the two recombinant plasmids, pET-28a-Bgl and pET-28a-Egl, and then tested the complete sequences.
The pET-28a-Bgl and the pET-28a-Egl recombinant plasmids were constructed and sequenced, after which heterologous expression in E. coli BL21 (DE3) was carried out to obtain the enzymes. SDS-PAGE showed that two cellulases were both successfully expressed in E.coli BL21 (DE3), and their Mws were 28.5 kDa and 56.3 kDa (Fig.6 Meanwhile, by blast from PDB protein database, the highest identification of Bgl was endobeta-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.
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 our study, and the predicted secondary structure didn't obviously affect by these differences. By comparison, the Bgl of B. subtilis 168 showed more differences with the proposed Bgl as 22 amino acids were different (Fig.7). The Egl sequence showed that it had a 96.6%

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 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. The computed pI was 7.26 and the negative GRAVY score (−0.616) suggested the protein to be hydrophilic. Egl showed instability index and aliphatic index of 29.60 and 73.69, respectively. Instability index less than 40 indicated that both the Bgl and Egl from B. methylotrophicus 1EJ7 was stable.

Discussions
The unique cellulolytic bacteria in rotten woods from the Qinling Mountains Microbial biodegradation has been employed as an environmental-friendly method in cellulosic materials to generate various valuable compounds. In the past decades, numerous cellulolytic microorganisms have been isolated and characterized. In this study, it was found that the strains isolated from Qinling rotten wood exhibited widely taxonomic coverage (e.g. Bacillus subtilis, Pseudomonas aeruginosa, Bacillus licheniformis, Bacillus methylotrophicus and Bacillus megaterium),, in which Bacillus subtilis was found as the most abundant species.
In our previous study, as enrich medium, LB was used to obtain strains from initial material. As expected, Bacillus strains was found as the dominant strain in rotten wood.
Since Bacillus subtilis strains are considered to exhibit a robust enzymes secretory system Hydrolysis capability of the strains and its cellulolytic enzymes On the whole, wild bacteria exhibit low enzyme production capacity and generally low enzyme activity. In this study, CMC-Na medium with different stains had the maximum reducing sugar content of 4.83 mg/100mL, while Avicel medium only achieved 1.61mg/100mL reducing content (Fig. 3), revealing that the hydrolysis capability of stains can be affected by the types of cellulose substrates. Also note that the sugar consumption for the strain's growth was also a cause of the low content of reduced sugar in cultivation broth. It was also reported in some literature that no reducing sugars were detected in the finial CMC medium when cultured with isolated bacteria [29].
It is noteworthy that different carbon substrates could induce different cellulases and further lead to different capabilities of reducing sugar production. When 8 strains were taken to hydrolyze different carbon source substrates, the strains were found to exhibit a better performance in reducing sugar production in CMC-Na medium than those in Avicel.
This phenomenon can be explained from two parts: 1. the different structure of cellulose substrates: Avicel was harder to hydrolyze than CMC-Na by cellulase for its unique microcrystalline structure [30]; 2. the different action modes of exocellulase and endocellulase: since the type of cellulolytic enzyme is the critical factor for the hydrolysis of different cellulose substrates, the efficiency in hydrolyzing CMC or Avicel was also significantly affected by the enzyme types [31, 32].
Moreover, it was also found that the strains performing good in Avicel and CMC-Na degradation did not show a well performance in hydrolyzing biomass, probably attributed to the complex network of lignin-hemicellulos-cellulose. FPase and CMCase of different strains ( Fig.4b and Fig.4c) exhibited similar cellulase activity in different mediums.
Furthermore, except for B. subtilis 1AJ3 and the B. subtilis 3BJ4, all the rest strains could produce avicelase in all mediums.Other strains could generate enzyme in limited carbon source medium. For instance, B. subtilis 1AJ2 could produce avicelase enzyme only in CMC-Na medium. Since the avicelase is an inducible enzyme, some carbon sources could induce strain to produce avicelase, whereas some could not. Similar study also revealed that crystalline cellulose or more complex structure were hard to hydrolyze [33].
For the three substrates in this study, the pretreatment of switchgrass, considered major  [43], α-amylase [44], lactosylfructoside [45], and xylanase [46]. However, there were extremely few reports on cellulases from B. methylotrophicus. Only two types of cellulase have been reported, 1,3-1,4-beta-glucanase [47] and carboxymethyl cellulase [28], both obtained by being purified from strain cultivation broth. In the meantime, cloning and expression of polypeptides or enzymes of B. methylotrophicus have been rarely discussed.
Accordingly, the molecular biology method to clone and express cellulase from bacteria B. methylotrophicus 1EJ7 were employed in this study. Fortunately, two cellulases were As β-glucosidase and endoglucanase had been expressed successfully, more work can be done to obtain stronger hydrolyze ability cellulase. For example, optimize expression conditions or genetic modification in the subsequent study. Higher enzyme activity can also be optimized by factors (e.g. pH, temperature, and metal ions) to achieved. Furthermore, mutations can be made by genetic engineering to increase enzyme activity. Therefore, it laid a foundation of enzymes characters for further study and hydrolyze mechanisms, and also provide a choice of industrial applications via two cellulases.

Cellulolytic bacteria isolation and identification
Each sample was broken into pieces, and 1 g was added into LB medium (10 g/L NaCl, 10 g/L tryptone and 5 g/L yeast extract), then incubated at 37 ℃ for 24 h with a constant shaking speed of 120 rpm. The bacteria suspension was respectively transferred to two selective media. The two selective media, CM and AM, were used CMC-Na and Avicel as single carbon source separately, contained of 2.0 g/L sodium carboxymethyl cellulose (CMC-Na) or Avicel (PH-101), and others of 2.0 g/L (NH 4 ) 2 SO 4 , 0.5 g/L MgSO 4 •7H 2 O, 1.0 g/L K 2 HPO 4 at natural pH of 7.20 were the same. The strains were cultured for 48 h at 37 ℃ with 120 rpm before being spread on the selective media agar plates with 0.4 g/L Congo red. Plates were incubated at 37 ℃ for 72 h, and then different colonies on the plates were picked.
The strains which probably could produce cellulolytic enzymes had a hydrolyzed circle around the colony. 10 μL broth of each isolated strains was dripped on the Congo red agar plates and the hydrolysis circle diameters of were measured to primarily evaluate the cellulolytic capability. The selected strains were shown in Fig.1.

←Fig. 1
The strains were cultured in broth for 48 h, then the cells were harvested and subjected to genome DNA extraction by a DNA extraction kit (Sangon Biotech, Shanghai, China). The universal primers of 27F and 1492R were utilized to amplify the 16S rRNA gene fragments.
Agarose gel electrophoresis was used to confirm target products and the PCR products were sequenced. The sequences were applied to BLAST on the NCBI database The gene encoding the β-glucosidase was amplified by PCR (94℃ for 5min, and then 35 cycles of 94℃ for 1min, 65℃ for 1min (-0.5℃/c), 72℃ 3min, and 72℃ for 10min) with a forward primer of 5′-CATGCCATGGGCATGTTTTATCGTATGAAACGAGTG (NcoI site was underlined) and a reverse primer 5′-CCGCTCGAGTTTTTTTGTATAGCGCACCCA (XhoI site was underlined) using a Takara ExTaqHS (Takara Bio, Shiga, Japan). The gene encoding the endoglucanase was amplified under the same PCR condition described above with a forward primer of 5′-CATGCCATGGGCATGAAACGGTCAATTTCTATTTTT (NcoI site was underlined) and a reverse primer of 5′-CCGCTCGAGATTGGGTTCTGTTCCCCAAA (XhoI site was underlined). The amplified genes were double digested with NcoI and XhoI, and inserted into the corresponding site of the pET-28a vector (Novagen) by T4 ligase.
Then, the constructed plasmid was transformed into E.coli BL21 (DE3) by hot hit under 42℃ for 90s and correct transformants were identified by PCR amplification and sequencing. The transformant was cultured in 1 L LB medium containing 1 mg/mL kanamycin at 37 °C until the absorbance at 600 nm reached 0.6. Then    Tables   Table 1 Isolated strains growth situation and clear zone size on Congo red plates     Reducing sugar and cellulase activities of eight strains in different carbon source medium.

Figure 5
Reducing sugar production by different strains in 7% wheat straw, corn stover and switchgrass for 72h. SEM of wheat straw before (5a) and after (5b) 72h fermentation by Bacillus methylotrophicus 1EJ7. X-ray of untreated wheat straw and fermented by Bacillus methylotrophicus 1EJ7 was showed in 5c.   Helix, strand and coil was showed on top of aa sequence.

Supplementary Files
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