Comparative genomic and secretomic characterisation of endophytic Bacillus velezensis LC1 producing bioethanol from bamboo lignocellulose

doi:10.21203/rs.2.17974/v1

Background: Compared to physical and chemical methods, microbial lignocellulosic degradation is a green process. Bacillus is an excellent organic matter degrader, and it has exhibited various abilities required for lignocellulose degradation, including the degradation of cellulose, hemicellulose and lignin. Several B. velezensis strains encode lignocellulosases. However, their usefulness based on their ability to transform biomass has not been appreciated. Genomic, comprehensive comparative genomic and secretomic analyses were used to clarify the lignocellulose-degrading potential of these bacteria, which is necessary for their future application.

Results: In the present study, the complete genomes of 20 B. velezensis strains and an endophytic bacterium, B. velezensis LC1, were analysed to find common and unique genes encoding carbohydrate-active enzymes (CAZymes) and evaluate their potentials to degrade lignocellulose. By comparative whole genomic and CAZyomic analyses of all 21 strains, we identified genes of lignocellulolytic enzymes with the potential to degrade cellulose (GH5, GH30, GH4, GH1, GH16 and GH32) and hemicellulose (GH43, GH30, GH51, GH26 and GH53). Further identification of the secretome of B. velezensis LC1 by liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that a considerable number of proteins in the culture medium are involved in lignocellulose degradation, including endoglucanase, hemicellulases, and other related proteins. Moreover, assays of the activities of several lignocellulolytic enzymes including endoglucanase, exoglucanase, β-glucosidase, xylanase, lignin peroxidase, laccase, and manganese peroxidase show that these enzymes are more active in bamboo powder compared to glucose substrates. After a 6-day treatment, the degradation efficiencies of cellulose, hemicellulose and lignin from bamboo powder were 59.90%, 75.44% and 23.41%, respectively. The hydrolysate was subjected to ethanol fermentation with Saccharomyces cerevisiae and Escherichia coli KO11, yielding 10.44 g/L ethanol after 96 h.

Conclusions: These findings indicate that Bacillus velezensis strains have the potential to degrade a range of polysaccharides in lignocellulosic biomass. One strain, B. velezensis LC1, efficiently degrades bamboo lignocellulose components, allowing for subsequent ethanol production.

Biotechnology and Bioengineering

comparative genome

secretome

Bacillus velezensis LC1

bamboo lignocellulose

ethanol

Bamboo, a perennial woody grass with a high lignocellulose content, is considered an excellent raw material for the production of many valuable products, including ethanol and biogas [1-3]. Ethanol production is divided into the following processes: pretreatment of raw materials, saccharification of cellulose and hemicellulose, and fermentation of the hydrolysate. Lignocellulose degradation is a key link in the conversion of biomass into ethanol, and it remains a difficult problem to solve completely and effectively [4]. Much research has been devoted to the pretreatment of raw materials to improve saccharification efficiency [3,5]. Biological treatment, especially the microorganisms involved in degradation, has been mostly ignored in these research efforts, which have tended to concentrate on the unique mechanism and specific enzymes involved in lignocellulose degradation. Research on bacterial systems have tended to focus on strains with rapid growth, high adaptability, and those that are easy to genetically manipulate [6, 7].

Compared with physical and chemical methods, microbial lignocellulosic degradation is a green process[8]. Bacillus is generally an excellent degrader, with specific strains exhibiting various abilities for lignocellulose degradation, including the degradation of cellulose, hemicellulose, and lignin [9-13]. Several B. velezensis strains encode lignocellulosases [14-18]. They are often regarded as biological agents because of their capabilities in promoting growth and anti-pathogenic activity. However, their potential usefulness based on their ability to transform biomass has not been appreciated [19-24]. To prove their potential usefulness, genomic and comprehensive comparative genomic and secretomic analyses will be necessary to clarify their lignocellulose-degrading potential.

In the present study, the whole genome sequences of 21 B. velezensis strains, including the endophytic bacteria, B. velezensis LC1, were retrieved from the NCBI database to search for genes encoding carbohydrate-active enzymes (CAZymes). There are six categories of CAZymes; the GHs, PLs, CEs, and CBMs all play roles in the degradation of polysaccharides; AAs function in the depolymerisation of lignin and GTs [25, 26]. Then, a comparative genomic analysis was conducted to reveal common and unique characteristics among these CAZyme genes. We analysed the secretomes of B. velezensis LC1 grown in a medium containing alkali-pretreated bamboo powder and explored the changes in the chemical components of pretreated bamboo lignocellulose. Furthermore, we measured lignocellulolytic enzyme activities and analysed the bamboo lignocellulose degradation by B. velezensis LC1. Finally, ethanol productivity of bamboo was assessed by a continuous reaction consisting of B. velezensis LC1 hydrolysing lignocellulose, Saccharomyces cerevisiae fermenting glucose, and Escherichia coli KO11 fermenting xylose.

Comparative genomic analysis of CAZymes of B. velezensis strains

The assembled genome of B. velezensis LC1, which contains 44 GHs, 38 GTs, 30 CEs, 3 PLs, 6 AAs, and 15 CBMs, was compared to the genomes of other B. velezensis strains. The 20 B. velezensis strains consist of: B. velezensis 10075, B. velezensis 157, B. velezensis FZB42, B. velezensis 83, B. velezensis UCMB5036, B. velezensis LB002, B. velezensis S3-1, B. velezensis At1, B. velezensis BCSo1, B. velezensis JTYP2, B. velezensis BS-37, B. velezensis CN026, B. velezensis B25, B. velezensis DR-08, B. velezensis LPL-K103, B. velezensis GFP-2, B. velezensis T20E-257, B. velezensis GYL4, B. velezensis TB1501, and B. velezensis LS69. Their genomes were comparatively analysed with B. velezensis LC1. At the whole genome level, the B. velezensis LC1 resembles most B. velezensis DR-08 (Fig. 1), while at the CAZyome level, LC1 resembles most B. velezensis S3-1 (Fig. 2).

Genes encoding CAZymes were detected in 21 genomes (Table 1 and Table S1). Sequence analysis results reveal the common and unique features in these lignocellulose degradation genes (Table 1 and Table S1). For example, some cellulolytic enzyme genes such as GH30 and GH5 are common to 21 strains, that also often contain endoglucanases (EC 3.2.1.4). GH1, GH4, and GH16 contribute to cellulose degradation by potentially functioning as 6-phospho-β-galactosidase, 6-phospho-β-glucosidase, and β-1,3-1,4-glucanase, respectively; they frequently appeared in 21 genomes. In addition, common genes capable of hemicellulose degradation were obtained. Arabinan endo-1,5-α-L-arabinosidase, α-N-arabinofuranosidase, and glucuronoxylanase are critical for xylan degradation; they belong to GH43, GH51, and GH30. GH53 hydrolyses (1→4)-β-D-galactosidic linkages in type I arabinogalactans [27]. GH26 functions as β-mannosidase [28]. Some CEs with the potential to deacetylate xylans and xylooligosaccharides, for example, CE3 (acetyl xylan esterase) [29], and CE 7 (acetylxylan esterase) [30] were also found frequently. The coexistence of these genes suggests that they play important roles in the enzymatic degradation of cellulose and hemicellulose. We consider these degradation enzymes in B. velezensis to have potential use for biothanol production.

Two PL1 and one PL9 genes are found in 21 genomes and are known to play roles in pectin degradation. The action of pectatelyase on (1→4)-α-D-galacturonan results in the production of oligosaccharides [31]. CE4, which contain polysaccharide deacetylases that function in polysaccharide degradation, [32] was also detected in the genomes. Moreover, we also identified several non-catalytic modules with binding functions, including CBM3, which binds to cellulose, and CBM6, which binds to amorphous cellulose and β-1,4-xylan [33].

GH32 contains sucrose-6-phosphate hydrolase and levanase, and are thus expected to function in sucrose hydrolysis [34]. GH13 (α-amylase, α-glucosidase, α-glycosidase, and α-α-phosphorylase) and CBM50 (involved in starch hydrolysis) [35] were also detected. GH65 (maltose phosphorylase) was identified in several B. velezensis genomes; this sequence is associated with trehalose degradation [36].

There are few auxiliary activities (AA) genes in all strains. Only the AA10 family was annotated in all strains, while AA4, AA6, and AA7 were only found in strain LC1. AA4 includes vanillyl-alcohol oxidases that exert invertase activity on phenols [37]. In addition, AA7 can biotransform or detoxify lignocellulose [26]. Several Bacillus strains have been considered lignin degraders. For example, Bacillus sp. LD003 affects lignin fractions [38]; B. pumilus C6 and B. atrophaeus B7 have laccase activity that degrades kraft lignin and the dimer guaiacylglycerol-b-guaiacyl [10].

Effect of alkali pretreatment on chemical components of bamboo

Lignocellulose-derived ethanol is widely considered a clean liquid fuel [39]. However, raw lignocellulose has a recalcitrant structure that lowers bioconversion efficiency, making pretreatment necessary to make lignocellulose more vulnerable to enzymes or bacteria for the fermentation of ethanol and other products [40,41]. Bamboo lignocellulose pretreated by alkali has a higher enzymatic digestibility than the raw material [42-44]. In the present study, we used sodium hydroxide (NaOH) to pretreat the bamboo powders. Table 2 lists the chemical compositions of pretreated and unpretreated bamboo powder (solid fraction) and shows that dried bamboo contains cellulose, hemicellulose, lignin, ash, neutral detergent fibre, acid detergent fibre, and acid detergent lignin in approximate percentages of 22.13 ± 2.52, 44.94 ± 4.12, 18.71 ± 2.76, 1.94 ± 0.02, 87.72 ± 3.87, 65.59 ± 4.65 and 20.65 ± 1.77, respectively. After alkaline pretreatment, the lignin content decreased to 6.92% ± 0.84%, while the hemicellulose increased to 30.19% ± 2.61%. Cellulose remained stable before and after alkaline pretreatment, which is consistent with previous studies [45]. The results indicate that NaOH pretreament of bamboo efficiently removes lignin without significantly impacting the cellulose.

Identification of secretomes of B. velezensis LC1 grown on medium containing with bamboo powder

To further confirm the lignocellulase system in B. velezensis LC1 cultured in bamboo powder, we analysed the secretomes of B. velezensis LC1 grown on bamboo powder and glucose medium (control). First, the supernatants of a three-day treatment were precipitated with 12% (w/v) trichloroacetic aid (TCA) to obtain proteins. Secretomic protein was detected only in the bamboo powder medium (Fig. 3a). These proteins were analysed by 1D-PAGE (Fig. 3b) and LC–MS/MS. The basic characteristics of the proteins are shown in Fig. 3c. The pI values of most proteins are in the range of 5.0-10.0. A total of 142 proteins were identified; these include a lignocellulolytic enzyme, protease, and other proteins (Table S3). These proteins were then functionally annotated, resulting in the characterisation of 612, 69, 348, and 79 proteins as being enriched in biological processes, cell components, molecular functions, and KEGG pathway functions, respectively (Fig. 3d; Fig. S1; Fig. S2). In addition, degradation-related enzymes including hemicellulases, cellulases, and other GHs were abundant in bamboo powder medium (Table 3).

Hemicellulose is the second most abundant component in lignocellulose; it is hydrolysed into monosaccharides by multiple enzyme systems [46-48]. Arabinogalactan endo-beta-1,4-galactanase (EC 3.2.1.89), which belongs to GH 53, catalyses the hydrolysis of β-1,4-galactosidic bonds in arabinogalactan and galactan side chains [49]. Other GHs exhibit hemicellulose activities. For example, β-xylanases cleave the xylan backbone, while β-xylosidases release the xylose units from xylobiose and xylooligomers. Acetyl xylan esterase is an accessory enzyme that functions synergistically with other enzymes in removing side chain residues from the hemicellulose backbone [50,51]. In the present study, four hemicellulases were identified, consisting of one arabinogalactan endo-β-1,4-galactanase (GH53) (1384349889), one beta-xylanase (GH43) (1384351325), one glucuronoxylanase (GH30) (1384349284), and one acetylxylan esterase (CE1) (1384350660) (Table 3).

Cellulose is hydrolysed synergistically by different enzymes, that is, endoglucanases randomly break internal bonds, then, exoglucanases remove cellobiose, and finally β-glucosidases release glucose from cellobiose [52-55]. β-Glucanases catalyse the degradation of β-glucans and are classified into β-1,3-1,4-glucanases (EC 3.2.1.73), β-1,4-glucanases (EC 3.2.1.4), β-1,3-glucanases (EC 3.2.1.39), and β-1,3(4)-glucanases (EC 3.2.1.6) [56-58]. Microbial β-1,3-1,4-glucanases have been identified as GH 16 and are mainly produced by Bacillus [59-62]. Two different enzymes, an endoglucanase (GH5) (1384349288) and a β-1,3-1,4-glucanase (GH16) (1384351103), which probably participates in cellulose degradation, were identified (Table 3).

AAs play a vital role in lignin degradation [26]. In this study, two AA6s (1384350268 and 1384350569), one AA7 (1384350476), and one AA10 (1384349337) protein were identified. Moreover, other enzymes involved in starch degradation, plant cell wall modifications, and protein degradation (proteases) were detected (Table 3).

Hydrolysis of bamboo powder by B. velezensis LC1 grown on medium with or without bamboo powder

The following B. velezensis strains have attracted attention due to their applications in agriculture and biotechnology: B. velezensis GH1-13 [17], B. velezensis S3-1 [19], B. velezensis FZB42 [20], B. velezensis M75 [21], B. velezensis LS69 [18], B. velezensis 9912D [22], and B. velezensis S499 [23]. B. velezensis LC1, which has shown bamboo lignocellulose degrading ability, was isolated from the intestinal microbiome of Cyrtotrachelus buqueti. The lignocellulose degrading ability of B. velezensis LC1 was demonstrated through bamboo degrading experiments.

Enzymes play critical roles in lignocellulose degradation. For example, lignin peroxidase and manganese peroxidase are important and necessary for the degradation of lignin and laccase, respectively [63]. Endoglucanases, exoglucanases, and β-glucosidases synergistically act to degrade cellulose [64], while xylanases are essential components of the hemicellulose degrading system [65]. Then we measured cellulase (endoglucanase, β-glucosidase and exoglucanase), hemicellulase (xylanase), and ligninase (laccase, lignin peroxidase and manganese peroxidase) activities, the activities of xylanase, lignin peroxidase, and laccase were significantly higher for bamboo powder than those for glucose (Fig. 4). Endoglucanase, β-glucosidase, and manganese peroxidase activities for bamboo powder were significantly higher than those for glucose after 3 d of treatment. However, exoglucanase activity was higher after 4 d of treatment. These results indicate that higher lignocellulolytic enzyme activities are involved in bamboo lignocellulose degradation.

Then, we measured lignocellulose degradation efficiencies using pretreated bamboo powder. After 6 d of culturing, the degradation products were analysed. The degradation efficiencies of cellulose, hemicellulose, and lignin in bamboo powder were 59.90%, 75.44%, and 23.41%, respectively (Fig. 5a). Several B. velezensis strains have lignocellulose degrading ability [14-18], which supports our finding that B. velezensis LC1 can efficiently degrade bamboo lignocellulose components.

Hydrolysate fermentation

Bamboo used for mushroom cultivation contains abundant cellulose, which is considered suitable for bioethanol production. Much of the research devoted to bamboo lignocellulose bioethanol production have focused on the physical and chemical pretreaments [3,5]. In our study, the ethanol productivity of bamboo was assessed by a continuous reaction of B. velezensis LC1 hydrolysing lignocellulose, Saccharomyces cerevisiae fermenting glucose, and Escherichia coli KO11 fermenting xylose. The bamboo-based ethanol production process is shown in Fig. 5b; its ethanol yield reached 10.44 g/L at 96 h, while the amount of reducing sugar decreased. This indicates that B. velezensis LC1 played an important role in the bioconversion of bamboo lignocellulose into ethanol.

The genomes of 21 strains of B. velezensis were compared and their CAZyomes were analysed. Genes with common and unique lignocellulolases were identified. The bamboo powder secretome of B. velezensis LC1 contains proteins related to lignocellulose degradation. The cellulose, hemicellulose, and lignin degradation of bamboo powder by strain LC1 were 59.90%, 75.44% and 23.41%, respectively. The hydrolysate was subjected to ethanol fermentation, resulting in an ethanol yield of 10.44 g/L at 96 h. These findings indicate that B. velezensis LC1 efficiently degrades bamboo lignocellulose components that then yield ethanol.

Mining of homologous genes related to lignocellulose degradation

Genomic data of 21 B. velezensis strains were retrieved from the GenBank DNA database, including nucleotide, amino acid, and putative coding sequences (Suppl Table S2). These sequences were analysed by dbCAN (http://csbl.bmb.uga.edu/dbCAN/annotate.php) and dbCAN HMMs version 4.0 [66]. CAZymes were identified by the CAZy database [25], and homologous families were analysed by OrthMCL software http://orthomcl.org/orthomcl/and annotated manually.

Phylogenetic analysis

Whole genome phylogenomic analysis of LC1 was performed by aligning the whole genomes of LC1 and other retrieved representatives of B. velezensis. Mugsy was used to align the genomic data, and the bx-python tool kit (https://bitbucket.org/james_taylor/bx-python) was used to concatenate homologous blocks. RAxML version 7.2.8 (24) and FigTree version 1.3.1 (http://tree.bio.ed.ac.uk /software/fifigtree/) was used to construct and visualise phylogenetic trees from concatenated blocks using 1000 bootstrap replicates and the maximum-likelihood method. The CAZyome phylogenomic analysis was performed on homologous CAZy genes.

Pretreament of the raw bamboo materials

The bamboo materials were sampled from the bamboo botanical garden of the Bamboo Diseases and Pests Control and Resources Development Key Laboratory of Sichuan Province, China. The bamboo powders were prepared as described by Luo et al. [67]. BP samples were treated with 3% (w/w) Tween-80 to enhance alkali pretreatment efficiency. Then, dried BPs were added in 1M NaOH at a concentration of 10% w/v, followed by autoclaving at 121 ℃ for 60 min. Afterwards, compounds were filtered and centrifuged (10,000 × g for 10 min). Precipitates were neutralised by 1M H₂SO₄and recentrifuged to remove salts formed during neutralisation. Finally, treated samples were washed by distilled water and dried to constant weight at 60℃.

Secretome preparation and analysis

The B. velezensis LC1 was cultured in liquid medium at pH 7.2, 37 °C and 200 rpm. The medium consists of pretreated bamboo shoot powder (10 g/L), (NH4)₂SO₄ (2 g/L), K₂HPO₄ (1 g/L), KH₂PO₄ (1 g/L), MgSO₄ (0.2 g/L), CaCl₂ (0.1 g/L), FeSO₄•7H₂O (0.05 g/L), and MnSO₄•H₂O (0.02 g/L). Cultures of B. velezensis LC1 growing in bamboo powder media were harvested on the 3rd day. First, the culture was filtered, then the supernatant was obtained by centrifugation (5000 rpm, 10 min, 4 ℃), followed by another round of filtration (0.2 µm membranes). The filtrate was precipitated by acetone to obtain total proteins [68]. Secretome proteins were harvested by precipitating the filtrate using 12% (w/v) trichloroacetic acid overnight at 4 ℃, then centrifuging (12,000 rpm, 30 min, 4 ℃), and finally washing for three times with 96% ethanol (v/v). The dried secretome protein pellets were resuspended in a solution containing 8 M urea and 4% (w/v) 3-[(3-cholamidopropyl) dithiothreitol.

LC–MS/MS analysis was implemented at Shanghai OE Biotech. Co., Ltd. as previously described [69]. Measurements were performed by nanoflow reversed-phase C18 liquid chromatography (EASY nLC, Thermo Scientific) coupled online to a 7-Tesla linear ion trap Fourier-Transform ion cyclotron resonance mass spectrometer (LTQ FT Ultra, Thermo Scientific). MaxQuant software was used to identify and quantify proteomic spectra, and then map the peptides to the annotated B. velezensis LC1 genome (data not shown) using the default settings [70].

Pretreated bamboo powder degradation by B. velezensis LC1

To obtain fermentable sugar from bamboo, B. velezensis LC1 was used to degrade the bamboo powder. The B. velezensis LC1 was cultured as described above for 6 d. Growth was terminated by heating at 100 °C for 30 min. After centrifuging (13,000 rpm, 10 min), the supernatant hydrolysate and pellet were collected. The pellet was dried and weighed to determine the levels of cellulose using the Van Soest method [71]. The glucose and xylose contents of the superficial hydrolysate were measured according to the NREL methods [72]. The bamboo shoot supernatant hydrolysate was sterilised and stored at -20 ℃ until it was used for ethanol fermentation.

Enzyme assays

Two millilitres of supernatant hydrolysate were obtained daily from 6-d-cultures for assays performed to measure lignocellulolase activities as described by Luo et al. [73]. Briefly, carboxymethyl cellulose (CMC), microcrystalline cellulose (MCC), and salicin were used as substrates to measure the activities of endoglucanase, exoglucanase, and β-glucosidase, respectively. Veratryl alcohol (VA), 2,2′-azino-bis (ABTS), and xylan were used as substrates to determine LiP, laccase, and xylanase activities, respectively. Manganese peroxidase (MnP) activity was measured spectrophotometrically by monitoring the oxidation of 2,6-DMP at 469 nm (ε469 = 49,600 mol⁻¹cm⁻¹). All the experiments were repeated five times.

Fermentation

The glucose-fermenting Saccharomyces cerevisiae was pre-cultured in YPD at 30 ℃ for 24 h, while Escherichia coli KO11, a xylose-fermenting strain, was pre-cultured in LB at 37 ℃ for 24 h. S. cerevisiae (50 g/L) and E. coli KO11 (100 g/L) cells were prepared after centrifugation of the pre-cultured cells. The initial cell concentrations were 0.33 g/L (S. cerevisiae) and 1.0 g/L (E. coli KO11) at the beginning of fermentation. One-hundred millilitres of hydrolysate was used for ethanol fermentation in 250-ml serum bottles under anaerobic conditions at 37 ℃. The fermentation system was shaken at 200 rpm for 96 h. Starting from 48 h of fermentation, ethanol production was monitored every 12 h. The ethanol concentration was determined via High Performance Liquid Chromatography (HPLC). All reactions were repeated three times.

B. velezensis: Bacillus velezensis; GH: Glycoside Hydrolase; GT: glycosyltransferase; CE: carbohydrate esterase; CBM: carbohydrate binding domain; PL: polysaccharide lyase; AA: Auxiliary Activitie; CAZyme: carbohydrate-active enzyme; NCBI: the national center for biotechnology information; ABTS: [2,2’-Azino-bis (3-ethylbenzothiazoline-6- sulfonic acid)]; BP: bamboo powder; YPD: Yeast Extract Peptone Dextrose Medium; DNS: 3,5-dinitrosalicylic acid; CDS: Sequence coding for amino acids in protein; NCBI: National Centre for Biotechnology Information.

Ethical approval and consent to participate

Not applicable.

Consent for publication

The authors have consented for publication.

Availability of data and material

The sequence reads from this article have been deposited at the NCBI Sequence Read Archive under the accession PRJNA574012. The assembly data set supporting the results of this article has been deposited at GenBank under the accession CP044349. The version described in this paper is CP044349.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Sichuan science and technology program (2019YFG0139) and Scientific Research Foundation of Leshan Normal University (XJR19001).

Authors’ contributions

CBL designed and performed the experiments; HT and YQL wrote the manuscript; LZ, LL, YQL, XWY and HT analyzed the data. All authors read and approved the final manuscript.

Acknowledgements

The data were analyzed on the free online platform of Majorbio Cloud Platform (www.majorbio.com).

Authors’ information

¹College of life science, Leshan Normal University, Leshan, 614000, Sichuan, China.

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Table 1. Annotated common genes encoding lignocellulose-degrading enzymes of 21 B.

velezensis strains

Classification	CAZy	Predicted function	EC number
Cellulose-related	GH5	endo-1,4-β-glucanase	EC 3.2.1.4
	GH30	glucan endo-1,6-β-glucosidase	EC 3.2.1.-
	GH4	6-phospho-β-glucosidase	EC 3.2.1.86
	CH4	6-phospho-α-glucosidase	EC 3.2.1-
	GH4	alpha-glucosidase/alpha-galactosidase	EC:3.2.1.20
	GH4	6-phospho-β-glucosidase	EC 3.2.1.86
	GH1	6-phospho-beta-galactosidase	EC:3.2.1.85
	GH1	aryl-phospho-beta-D-glucosidase	EC:3.2.1.86
	GH1	6-phospho-β-galactosidase	EC 3.2.1.85
	GH16	β-glucanase	EC 3.2.1.-
	GH32	sucrose-6-phosphate hydrolase	EC 2.4.1.-
	GH32	levanase	EC 3.2.1.65
	GH13	α-glucosidase	EC 3.2.1.20
	PL1	pectate lyase	EC 4.2.2.-
	PL9	pectate lyase	EC 4.2.2.2
Hemicellulose-related	GH43	arabinan endo-1,5-α-L-arabinosidase	EC 3.2.1.-
	GH43	arabinoxylan arabinofuranohydrolase	EC 3.2.1.-
	GH43	1,4-β-xylosidase	EC 3.2.1.37
	GH51	α-N-arabinofuranosidase	EC 3.2.1.55
	GH30	glucuronoxylanase	EC 3.2.1.-
	CE 7	acetylxylan esterase	EC 3.1.1.72
	CE 3	acetylxylan esterase	EC 3.1.1.72
	GH53	arabinogalactan endo-1,4-β-galactosidase	EC 3.2.1.89

Table 2. Chemical components of bamboo powder (BP) in raw and alkali-pretreated materials

Components	Raw material	Alkali pretreament
NDF(%)	87.72 ± 3.87	90.11 ± 3.18
ADF(%)	65.59 ± 4.65	59.92 ± 5.97
ADL(%)	20.65 ± 1.77	16.58 ± 2.51
ASH(%)	1.94 ± 0.02	9.66 ± 0.53
Hemicellulose(%)	22.13 ± 2.52	30.19 ± 2.61
Cellulose(%)	44.94 ± 4.12	43.34 ± 2.72
Lignin(%)	18.71 ± 2.76	6.92 ± 0.84

Table 3. Identification of the main CAZy proteins in the secretome of B. velezensis LC1 in bamboo powder (BP) culture

Locus tag	Description	Peptides	Unique Peptides	Protein mass (kDa)	Isoelectric point	Score
1384350573	Bifunctional penicillin-binding protein 1C	2	2	88.3	9.88	3.92
1384350643	Putative multimodular carbohydrate-active enzyme	3	3	92.9	6.65	8.73
1384351304	ATP synthase subunit alpha	4	4	56	5.15	12.9
1384351201	Glycosyltransferase 2	3	3	55	6.58	4.67
1384349288	Endoglucanase	3	3	55.2	8.1	2.87
1384349889	Arabinogalactan endo-beta-1,4-galactanase	8	8	41.3	8.56	12.32
1384351325	Beta-xylanase	5	5	42.2	6.98	11.07
1384347997	Sucrose-6-phosphate hydrolase	10	10	56.1	5.69	54.68
1384351193	Sucrose-6-phosphate hydrolase	4	4	66.2	7.46	2.18
1384349284	Glucuronoxylanase	10	10	47.8	8.29	28.43
1384351103	Endo-beta-1,3-1,4-glucanase	5	5	26.7	6.92	15.06
1384348333	Alpha-glycosidase	12	12	119	4.88	38.21
1384347992	Alpha-amylase	7	7	72.4	6.49	29.61
1384351150	6-phospho-beta-glucosidase	5	5	48.6	5.4	4.16
1384350269	Beta-amylase	2	2	55.1	5.48	2.73
1384348933	Serine aminopeptidase	9	9	8.4	4.96	2.75
1384350660	Acetylxylan esterase	2	2	35.5	6.11	4.83
1384351298	Serine hydroxymethyltransferase	2	2	45	6.21	5.5
1384350476	FAD-binding oxidoreductase	2	2	50.1	9	4.16
1384350268	NAD(P)H-dependent oxidoreductase	4	4	25.6	5.97	9.25
1384350569	NAD(P)-dependent oxidoreductase	2	2	31.2	5.05	3.01

Comparative genomic and secretomic characterisation of endophytic Bacillus velezensis LC1 producing bioethanol from bamboo lignocellulose

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