Genome sequencing of gut symbiotic Bacillus velezensis LC1 for bioethanol production from bamboo shoots

Background Bamboo, a lignocellulosic feedstock, is considered as a potentially excellent raw material and evaluated for lignocellulose degradation and bioethanol production, with a focus on using physical and chemical pre-treatment. However, studies reporting the biodegradation of bamboo lignocellulose using microbes such as bacteria and fungi are scarce. Results In the present study, Bacillus velezensis LC1 was isolated from Cyrtotrachelus buqueti, in which the symbiotic bacteria exhibited lignocellulose degradation ability and cellulase activities. We performed genome sequencing of B. velezensis LC1, which has a 3929,782-bp ring chromosome and 46.5% GC content. The total gene length was 3,502,596 bp using gene prediction, and the GC contents were 47.29% and 40.04% in the gene and intergene regions, respectively. The genome contains 4018 coding DNA sequences, and all have been assigned predicted functions. Carbohydrate-active enzyme annotation identified 136 genes annotated to CAZy families, including GH, GTs, CEs, PLs, AAs and CBMs. Genes involved in lignocellulose degradation were identified. After a 6-day treatment, the bamboo shoot cellulose degradation efficiency reached 39.32%, and the hydrolysate was subjected to ethanol fermentation with Saccharomyces cerevisiae and Escherichia coli KO11, yielding 7.2 g/L of ethanol at 96 h. Conclusions These findings provide an insight for B. velezensis strains in converting lignocellulose into ethanol. B. velezensis LC1, a symbiotic bacteria, can potentially degrade bamboo lignocellulose components and further transformation to ethanol, and expand the bamboo lignocellulosic bioethanol production.


Background
Lignocellulose, a widely distributed, renewable and enormous biomass resource, is one of the most important raw materials for bioethanol production [1]. Bamboo, a lignocellulosic feedstock, is a regenerated biomass material with abundant resources, short growth cycle, high yield and similar chemical composition as wood, and it is considered as a potentially excellent raw material [2,3]. Many studies have evaluated bamboo lignocellulose degradation and bioethanol production, with a focus on using physical and chemical pre-treatment [4,5]. However, studies reporting the biodegradation of bamboo lignocellulose using microbes such as bacteria and fungi are scarce.
Lignocellulose hydrolysis, especially cellulose degradation, remains a considerable challenge in lignocellulosic bioethanol production [6]. In nature, numerous examples for lignocellulose degradation are present; of these, phytophagous insects are considered the most notable. In these insects, intestinal symbiotic microbes played important roles in lignocellulose degradation [7]. Therefore, the intestines of phytophagous insects were considered as important locations for isolating lignocellulolytic microbes [8].
Microbial degradation of lignocellulose is a green biological refining method with advantages over physical and chemical methods [9]. The bacterial genus Bacillus is an excellent degrader that exhibits various abilities for degrading lignocellulose biomass, including cellulose, hemicellulose and lignin [10,11]. Furthermore, genome sequencing, considered an efficient method for investigation of function, has been utilized in lignocellulose degradation research. However, the lignocellulose degradation of Bacillus is still unclear [12]. Dunlap et al. [13] reported that B. oryzicola and B. methylotrophicus, were classified into the B. velezensis group. Recently, the complete genome and genes associated with lignocellulose degradation of several B. velezensis strains were sequenced and are enriched in the genome [14][15][16]. However, its potential application in converting lignocellulose into bioethanol has received little attention.
In the present study, we isolated an endophytic bacteria from the gut of Cyrtotrachelus buqueti that showed a bamboo lignocellulose-degrading ability [17] and sequenced the whole genome of the bacteria B. velezensis LC1, determined the cellulase activities and analysed the ethanol production of bamboo shoot. CAZy genes involved in degradation of lignocellulose were identified through genomic analysis. The chemical changes of the cell wall components were investigated, as well as the hydrolytic and ethanol-fermenting properties of bamboo shoots.

Results and discussion
Identification and cellulose-degrading potential of Bacillus velezensis LC1 Five cellulolytic strains, including PX9, PX10, PX11, PX12 and PX13, which produced clear zones around the colonies after Congo red staining, were isolated from the intestine of C. buqueti on CMC agar. Among the five strains, PX12 exhibited the highest cellulose hydrolysis capacity, with a higher hydrolysis capacity ratio (HCR: 4.71) than PX9 (HCR: 2.41), PX10 (HCR: 1.92), PX11 (HCR: 2.12) or PX13 (HCR: 2.56), as determined using the cellulose hydrolysis assay (Fig. 1a, b; Additional file 1: Figure S1). Based on the HCR ratio, many potent cellulolytic bacteria were previously screened from various regions, such as Geobacillus sp. from a hot spring and Paenibacillus lautus BHU3 from a landfill site [18,19]. Similarly, PX12 was considered as a good cellulolytic bacterium and was used for further study.
Several B. velezensis strains have been noted for their lignocellulose-degrading abilities [14][15][16]. To investigate the cellulose-degrading ability, B. velezensis LC1 was cultured on CMC agar to determine cellulase activities by the dinitrosalicylic acid spectrophotometric (DNS) method for 6 days ( Fig. 1e-g) [20]. The cellulase activities of strain LC1 were then determined. The endoglucanase activity was 0.689 ± 0.011 U/ml at day 1 and increased to 0.752 ± 0.013 U/ml at day 6, which was in accordance with the exoglucanase activity (from 0.359 ± 0.016 U/ml to 0.385 ± 0.022 U/ml), whereas the β-glucosidase activity decreased from day 6 to day 1. Previous studies have reported the cellulase activities of other lignocellulolytic Bacillus strains. For example, Bacillus sp. 275, Bacillus sp. R2, B. velezensis 157 and B. velezensis ZY-1-1 [10,11,21,22] showed similar results as those achieved in our study. This indicated that the strain played a potential role in cellulose degradation.

Genome sequencing and assembly of Bacillus velezensis LC1
The genome contributes to a clear understanding of bacterial decomposition mechanisms of cellulose; thus, the genome of B. velezensis LC1 was analysed to decipher the genetic code involved in cellulose degradation. The complete genome sequence of B. velezensis LC1 was assembled into a ring chromosome with 3,929,782 bp and had a GC content of 46.5% (Fig. 2). A length of 3,502,596-bp genes was found based on gene prediction, and the ratio of gene length/genome was 89.13%. The intergene region/ whole genome ratio was 7.19%, and the GC contents of the gene and intergene region were 47.29% and 40.04%, respectively. Furthermore, 4018 CDSs were contained in the genome, and all were assigned functions. CDSs were further annotated in NR, Swiss-Prot, COGs, KEGGs, GO and Pfam, and their numbers were 4018, 3520, 2996, 2186, 2718 and 3315, respectively (Table 1).

COGs involved in carbohydrate metabolism
In total, 3046 genes were classified into 2996 COGs, of which carbohydrate transport and metabolism, amino acid transport and metabolism and transcription were the most enriched COGs, which represented 9.46%, 7.62% and 7.29%, respectively (Fig. 3a). To elucidate the function of B. velezensis LC1 in cellulose degradation at the genetic level, specific COGs involved in carbohydrate metabolism were analysed. A total of 222 genes were annotated into carbohydrate metabolism, including 130 COGs, of which the most abundant COGs were COG0366 (alpha-amylase), COG0524 (pfkb domain protein), COG2814 (Major facilitator), COG0726 (4-amino-4-deoxy-alpha-l-arabinopyranosyl undecaprenyl phosphate biosynthetic process), COG1263 (PTS System), COG0477 (major facilitator superfamily), COG1455 (pts system), COG1940 (ROK family) and COG2723 (beta-glucosidase) (Additional file 2: Table S1). COG366 encodes an alpha-amylase that acts on a bond between starch and glycogen, hydrolysing polysaccharides into glucose and maltose [23]. COG2814 is involved in cellular transport of some complexes, such as carbohydrates and amino acids. COG0477, a secondary active transporter, helps to catalyse the transport of various substrates [24,25]. Moreover, other important COGs in carbohydrate metabolism were annotated, e.g. COG0395 was reported to participate in carbohydrate uptake [26] and COG1109 catalysed the conversion of glucosamine-6-phosphate [27]. The high diversity of function annotations indicated that B. velezensis LC1 had a potent capability in lignocellulose degradation.

GO terms annotations
To explain the relevance of the genome of B. velezensis LC1, GO analysis was used to categorize genes into three categories according to matches with known sequences. In three categories, molecular function contained most numerous GO terms and gene number (3730), followed by biological process (Gene number: 3025) and cellular component (Gene number: 1637) (Fig. 3b). In molecular function, the most five pathway was ATP binding (GO:0005524; 314 genes), DNA binding (GO:0003677; 254 genes), transcription factor activity (GO:0003700; 115 genes), metal ion binding (GO:0046872; 114 genes) and hydrolase activity (GO:0016787; 84 genes). Oxidation-reduction process (GO:0055114) and regulation  of transcription (GO:0006355) were most pathways in the biological process, and integral component of membrane (GO:0016021), cytoplasm (GO:0005737) and plasma membrane (GO:0005886) pathways in cellular component. Furthermore, we analysed the GOs that are associated with carbohydrate metabolism. We identified 114 GO items associated with carbohydrate metabolism, including GO:0004553 (hydrolase activity that hydrolyses O-glycosyl compounds), GO:0005975 (carbohydrate metabolic processes) and GO:0016787 (hydrolase activity) (Additional file 3: Table S2).

KEGG annotations
The CDSs of B. velezensis LC1 were submitted to KAAS and KEGG pathways to identify metabolism pathways (Additional file 4: Table S3). As shown in Fig. 3c, of the six classification of KEGG pathways, metabolism contained the most numbers of genes, followed by environmental information processing. In KEGG metabolism annotations of B. velezensis LC1, carbohydrate metabolism and amino acid metabolism, which are considered its main functions, contained 392 and 285 genes, respectively. For these metabolisms, some pathways were dominant, such as sucrose and starch metabolism (ko00500), glycolysis/gluconeogenesis (ko00010), and amino and nucleotide sugar metabolism (ko00520). Forty-one genes were related to ko00500, and common enzyme endoglucanase (EC.3.2.1.4), present in ko00500, was involved in cellulose degradation (Fig. 3c). Starch and sucrose metabolic pathways occurred in B. velezensis LC1, indicating that cellulose could be hydrolysed into cellobiose and ultimately, β-d-glucose. In the genome, 39 genes were found in ko00010, in which d-glucose was phosphorylated into d-glucose-6-phosphate. In addition, ko00010 was linked with other pathways. For example, d-glucose-6-phosphate could be converted to pyruvate, which can be oxidized to acetyl-CoA, having an ability to enter the citrate cycle. Furthermore, ko00520 indicated that glucose from the ko00010 finally entered other pathways under various catalytic reactions. α-d-galactose can be transferred and isomerized in ko00520 and then entered into the ascorbate and aldarate metabolism pathways. Additionally, fructose, 1,4-β-d-xylan, and extracellular mannose were metabolized in ko00520.
The annotation involved in the degradation of lignin or aromatic compounds has also been identified. We identified various enzymes associated with lignin degradation, including oxidoreductase, reductases, dehydrogenases, esterases, thioesterases, transferases and hydrolases. Moreover, 13 monooxygenases, 12 dioxygenases, 2 peroxidases (including one DyP-type peroxidase) and 1 laccase were been identified (Additional file 5: Table S4).
CEs contributing to the decomposition of xylans were also identified in the genome, including two CE3s, one CE7s, three CE10s, and seven CE4s. CE3 as a potential acetyl xylan esterase enhanced xylan solubilization [36].
The acetylxylan esterase CE7 was considered as a capable xylan-degrading enzyme [37]. CE10 previously exhibited carboxylesterase and xylanase activities involved in hemicellulose degradation [38]. Polysaccharide deacetylases, which play a role in degrading polysaccharides and are classified as a CE4, were also identified. CE4 contained not only highly specific acetylxylan esterases, but also peptidoglycan N-deacetylates involved in chitin degradation [39].

Comparative genomic analysis of CAZymes with other B. velezensis strains
The assembled genome of B. velezensis LC1 was com-  Table 4). 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 bioethanol production.

Cellulose degradation efficiency and fermentation efficiency of bamboo shoots by B. velezensis LC1
Several previously reported genomes of B. velezensis strains contained genes encoding enzymes having lignocellulose-degrading potential [14][15][16]. However, lignocellulose-degrading abilities have not been completely verified in B. velezensis [12]. B. velezensis LC1, isolated from the intestine of C. buqueti, which was reported to be a microflora with lignocellulose-degrading ability, was prepared in a bamboo shoot powder (BSP) degradation assay [17]. We first determined the cellulose degradation efficiency to be 39.32% (Fig. 5a). Furthermore, we determined the glucose and xylose content in the degradation products to be 55.30 ± 1.40 mg/L and 488.81 ± 45.06 mg/L, respectively (Fig. 5b). The reducing sugars in the culture medium was mainly derived from the hydrolysis of cellulose and hemicellulose in BSPs, the reducing sugar content was determined to reflect the degree of conversion of lignocellulose. It indicated that the cellulose and hemicellulose of BSP were degraded with incubation with B. velezensis LC1. Shimokawa et al. [43] reported that bamboo shoot was an excellent biomass stock for ethanol production

Insect sample, isolation and identification of cellulolytic bacteria
Cyrtotrachelus buqueti specimens were sampled from the Muchuan County (E 103° 98′, N 28° 96′), China. Gut was extracted from individual insects and stored at 4 °C for isolation of bacteria. The gut was blended, homogenized and serially diluted (10 −1 to 10 −9 ), and inoculums of 10 −7 to 10 −9 dilution were plated on carboxymethyl cellulose (CMC) agar [44] for cellulolytic bacteria screening. Congo red dye was used to screen the cellulose-degrading bacteria as described by Teather and Wood [45]. Hydrolysis zone = clearance zone/colony diameter.

Molecular characterization of bacterial isolate
The 16sRNA V3-V4 region was considered for amplification for bacterial identification and amplified using bacterial primers (27F 5′-AGA GTT TGATCMTGG CTC AG-3′ and 1492R 5′-TAC GGY TAC CTT GTA CGA CTT-3′); moreover, F 5′-GCC CAT ATT TCC ATT TCT CC-3′ and R 5′-GTG GTC GTT ATG GAA ATA AAGG-3′ were selected for amplification of the house-keeping gene rpoB. Thermocycling conditions were as follows: initial denaturation at 94 °C (2 min), followed by 30 cycles of denaturation at 94 °C (30 s), annealing at 55 °C (30 s) and extension at 72 °C (100 s), ultimately extending at 72 °C (2 min). The amplicons were checked by electrophoresis on a 1% agarose gel. MEGA5 was used to establish phylogenetic relationships among the obtained sequence and reference genes that were retrieved in NCBI GenBank through the neighbour-joining method.

Function annotation of B. velezensis LC1
Glimmer 3.02 was used to predict coding DNA sequences (CDSs). A BLAST search was then conducted for CDSs in some widely used databases: NCBI non-redundant (NR) database, Swiss-Prot, COGs, Gene Ontology (GO) and KEGGs [48,49]. GO, an important bioinformatics tool, unified expressions of gene and genetic products in all species [50]. KEGGs, a database resource to understand high-level functions, also could analyse metabolic pathways. Additionally, the CAZymes were identified, classified and annotated using CAZymes database (CAZyDB: http://www.cazy.org/).

Quantitative real-time PCR
The primers used for qRT-PCR in this study were performed in Additional file 7: Table S6. PCR was performed under following conditions: 10 min initial denaturation at 95 °C, 45 cycles of 5 s denaturation at 95 °C, 50-65 °C anneal for 30 s, and 30 s extension at 55 °C, finally 10 s extension at 95 °C. All experiments were performed three times and analysed by 2 − ΔΔCT Method. 16S rRNA was used as reference gene.

Bamboo shoots degradation by B. velezensis LC1
To obtain fermentable sugar from bamboo shoots, B. velezensis LC1 was used to degrade the bamboo shoot powder (BSP), which was prepared as described by Luo et al. [17]. B. velezensis LC1 was cultured in liquid medium at a pH of 7.2, temperature of 37 °C and 200 rpm for 6 days. The culture medium comprised BSP 10 g/L, (NH4) 2 SO 4 2 g/L, K 2 HPO 4 1 g/L, KH 2 PO 4 1 g/L, MgSO 4 0.2 g/L, CaCl 2 0.1 g/L, FeSO 4 ·7H 2 O 0.05 g/L and MnSO 4 ·H 2 O 0.02 g/L. The reaction mixture was incubated at 100 °C for 30 min to terminate the reaction and centrifuged at 13,000 rpm for 10 min, and the hydrolysate-containing supernatant and deposit were collected separately. The obtained deposit was dried and weighed to determine the cellulose levels using the Van Soest method [51]. The hydrolysate-containing supernatant was used to determine the glucose and xylose contents according to the NREL methods [52].