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. 2021 Jul 22;11(8):379. doi: 10.1007/s13205-021-02919-0

Hemicellulosic biomass conversion by Moroccan hot spring Bacillus paralicheniformis CCMM B940 evidenced by glycoside hydrolase activities and whole genome sequencing

Soufiane Maski 1,4, Serigne Inssa Ngom 1,2, Bahia Rached 1,3, Taha Chouati 1, Mohamed Benabdelkhalek 4, Elmostafa El Fahime 5, Mohamed Amar 1,3, Christel Béra-Maillet 1,2,✉
PMCID: PMC8298745  PMID: 34447652

Abstract

Thermophilic bacteria, especially from the genus Bacillus, constitute a huge potential source of novel enzymes that could be relevant for biotechnological applications. In this work, we described the cellulose and hemicellulose-related enzymatic activities of the hot spring Bacillus aerius CCMM B940 from the Moroccan Coordinated Collections of Microorganisms (CCMM), and revealed its potential for hemicellulosic biomass utilization. Indeed, B940 was able to degrade complex polysaccharides such as xylan and lichenan and exhibited activity towards carboxymethylcellulose. The strain was also able to grow on agriculture waste such as orange and apple peels as the sole carbon source. Whole-genome sequencing allowed the reclassification of CCMM B940 previously known as B. aerius into Bacillus paralicheniformis since the former species name has been rejected. The draft genome reported here is composed of 38 contigs resulting in a genome of 4,315,004 bp and an average G + C content of 45.87%, and is an important resource for illuminating the molecular mechanisms of carbohydrate metabolism. The annotated genomic sequences evidenced more than 52 genes encoding glycoside hydrolases and pectate lyases belonging to 27 different families of CAZymes that are involved in the degradation of plant cell wall carbohydrates. Genomic predictions in addition to in vitro experiments have revealed broad hydrolytic capabilities of the strain, thus reinforcing its relevance for biotechnology applications.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02919-0.

Keywords: Bacillus paralicheniformis, Whole genome, Hemicellulose, Glycoside hydrolase, Lichenan, Xylan

Genome research reports

Lignocellulosic biomass, mainly composed of cellulose, hemicelluloses and lignin, is the most abundant complex biopolymer in the nature. It is of major interest for biotechnology as this feedstock is used for the production of renewable biofuels and other high-value chemicals, through an enzymatic saccharification process (Zhang et al. 2018). However, depolymerization of lignocellulose is challenging and it requires high-performance enzymes that act in synergy (Houfani et al. 2020). Therefore, enzymes produced by microorganisms, such as bacteria, are used for the deconstruction of the complex polysaccharides of cellulose and hemicelluloses which are tightly bound together in plant cell walls (Zeng et al. 2017).

Xylan is the main component of hemicelluloses and consists of a β-1,4-d-xylan backbone with short side chains of O-acetyl, β-l-arabinofuranosyl, d-α-glucuronic acid and phenolic acid (Biely et al. 2016). Other polysaccharides such as xyloglucan, lichenan and mannans are part of the hemicellulolytic fraction of plant cell walls. Xylanases, xyloglucanases, lichenases, and endoglucanases are necessary to completely degrade these polysaccharides into simple sugars to allow glycolytic fermentation to produce energy. These enzymes belong to one of the glycoside hydrolase (GH) families, a subgroup of the carbohydrate-active enzymes (CAZymes) (Lombard et al. 2014) and very often associated with carbohydrate-binding modules (CBM) that facilitate adhesion of enzymes to cellulose or hemicelluloses.

Most microorganisms living in extreme environmental habitats such as hot springs have been considered attractive producers of lignocellulolytic enzymes for industrial bioconversion processes (Thapa et al. 2020). Morocco has more than 20 hot springs distributed in different regions (Cidu and Bahaj 2000; Bouchaou et al. 2017) which are suitable habitats for thermophilic microorganisms growing optimally between 55 and 80 °C. In addition, thermophilic bacteria are known for their ability to produce thermostable enzymes of biotechnological interest (Knapik et al. 2019). Previously, Aanniz et al. (2015) investigated the diversity of thermophilic bacteria in 4 different hot springs in Morocco, which enabled the isolation of 79 bacterial strains belonging to the genus Bacillus (www.ccmm.ma). Some of these strains were identified as Bacillus aerius. They grow optimally at 55 °C and exhibit amylolytic, proteolytic or cellulolytic activity (Aanniz et al. 2015). However, strains belonging to B. aerius species should be reclassified as this species name has been rejected since 2015 (Dunlap 2015). For this reason, we chose the CCMM B940 strain as the representative of the B. aerius group isolated by Aanniz et al. (2015) from a hot spring for biological characterization of its potential hemicellulolytic and cellulolytic activities and whole-genome sequencing analysis.

B940 strain was grown twice on Luria–Bertani (LB) agar medium (BD Difco, Sparks, Maryland, USA) for 18 h at 37 °C under aerobic conditions. Individual colonies were then picked from LB plates and cultivated at 37 °C for 18 h in LB agar medium supplemented with 0.5% Icelandic moss Lichenan (Megazyme, Wicklow, Irlande), beechwood Xylan (Megazyme, Wicklow, Irlande), or medium viscosity Carboxymethylcellulose (CMC, Sigma, MO, USA), respectively. A second bacterial strain (CCMM B945) from the same B. aerius group, known for inability to degrade lichenan and xylan (data not shown) was used as a negative control for degradation of hemicellulose and following the same experimental protocol as for CCMM B940. Both strains grew well on LB agar medium supplemented with purified fibres (Fig. 1A).

Fig. 1.

Fig. 1

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Growth A and detection of lichenase (a), xylanase (b) and carboxymethylcellulase (c) activities B of Bacillus CCMM B940 and CCMM B945 strains cultivated in LB agar streak plates supplemented with 0.5% (w/v) lichenan, xylan or carboxymethylcellulose (CMC), respectively. Plates were incubated 18 h at 37 °C. Congo red staining was performed as described in Patrascu et al. (2017). Clear halos around the streaks indicate the degradation of the corresponding polysaccharide.

To reveal whether the strains were able to degrade the complex polysaccharides added into the medium, the plates were stained with 0.1% Congo red as previously described (Patrascu et al. 2017). Clear halos around the streaks indicated that B940 strain can degrade all three polysaccharides, and thus capable of producing the appropriate enzymes. As expected, lichenases and xylanases were only produced by the CCMM B940 strain (Fig. 1B). Strain CCMM B945 exhibited significant activity against CMC, indicating production of endoglucanases, similar to the CCMM B940 strain.

The Congo red test demonstrated that live cells of the B940 strain exhibited lichenase and xylanase activities and also produced endoglucanases for glucan-polymer degradation. However, to evaluate biomass utilization potential of this strain for biotechnology applications, we searched for the presence of those enzymes in the rather easily accessible B940 strain supernatants. For this purpose, the strain was cultivated in the basal salt solution (BSS), a chemically defined medium modified from Parab et al. (2017), with a lower salt concentration (NaCl 6 g L−1). The medium was supplemented with fruits peels (orange or apple) as the sole source of carbon and then sterilized 15 min at 121 °C. Fruit peels are a rich source of cellulose, hemicellulose, lignin and pectin (Rivas et al. 2008; Joglekar et al. 2019). Orange peel is rich in structural polysaccharides, including cellulose (18–20%), hemicellulose (14–16%), pectin (20–22%) and lignin (5–7%) (de la Torre et al. 2019). Apple residues typically contain 7.2–43.6% cellulose, 4.3–24.4% hemicellulose, 15.2–23.5% lignin and 3.5–14.32% pectin (Dhillon et al. 2013). Both substrates were chosen because of their abundance as industrial waste around the world (de la Torre et al. 2019).

A single colony of CCMM B940 and CCMM B945 from LB agar medium was picked to inoculate 5 mL of LB Broth (Difco) and incubated overnight at 37 °C (with continuous stirring at 170 rpm), followed by second culture in 5 mL of BSS medium supplemented with 0.5% apple or orange peels and inoculated at 1%. Before adding them in the medium, apple and orange peels were dried for 24 h at 60 °C and crumbled finely using an electric grinder. After 48 h of incubation at 37 °C with stirring (170 rpm), both strains grew well in the BSS supplemented with fruit peels representing agro-industrial wastes as shown in Figure S1. Additionally, this test confirmed the ability of CCMM B940 and CCMM B945 strains to break down fibres from the peels and use the degradation products.

Protein samples from B940 and B945 strains cultivated for 48 h at 37 °C in 30 mL BSS medium supplemented with 0.5% orange and apple peels were prepared to detect and quantify hemicellulolytic and cellulolytic activities in the extracellular fluids. Briefly, cultures were centrifuged for 15 min at 5000×g and 4 °C, supernatants were filtered through a 0.22 μm Millipore filter, and then concentrated 25-fold in a Spin-X UF10K concentrator following the supplier’s protocol (Corning B.V., Amsterdam, The Netherlands).

Agar-well plate enzymatic assays were performed to detect xylanase, lichenase and endoglucanase activities using concentrated extracellular proteins. Concentrated extracellular proteins from CCMM B940 and control strain CCMM B945 were prepared in the same way. The samples were loaded (60 μL/well) in Petri dishes containing BSS medium supplemented with either 0.1% chromogenic AZCL-Xylan (Megazyme, Wicklow, Ireland), 0.5% non-chromogenic CMC (Sigma-Aldrich, MO, USA) or lichenan (Megazyme) polysaccharides. The plates were incubated overnight at 37 °C and those with non-chromogenic polysaccharide were stained with Congo red as described above. Clear and blue halos around the wells indicated that the CCMM B940 cultivated with apple peels was able to produce lichenase (Fig. 2A), endoglucanase (Fig. 2C, although weakly in the tested conditions), and xylanase (Fig. 2B). The same results were obtained with orange peels (not shown).

Fig. 2.

Fig. 2

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Detection of lichenase A, xylanase B and carboxymethylcellulase C activities of Bacillus CCMM B940 and B945 strains, using agar-well plate assays with concentrated extracellular proteins of BSS cultures with 0.5% apple. Agar plate BSS medium was supplemented with 0.5% (w/v) lichenan, carboxymethylcellulose (CMC), or 0.1% AZCL beechwood xylans. Plates were incubated overnight at 37 °C. Clear and blue halos around the wells indicate a positive sample for the corresponding glycoside hydrolase activity

The CCMM B940 strain is able to grow at 55 °C in Tryptic Soy broth (Aanniz et al. 2015) and we also observed that the strain is capable of growing in BSS medium supplemented with lichenan and xylan at the same temperature (data not shown), thus reinforcing the interest of its characteristics for industrial applications requiring thermostable enzymes.

In contrast to CCMM B940 strain results, the control strain CCMM B945 was not able to produce xylanase or lichenase when cultivated in the presence of apple or orange peels (Fig. 2).

Moreover, extracellular hemicellulolytic activities of the strain CCMM B940 were quantified using the dinitrosalicylic acid (DNS) spectrophotometric method for reducing sugars as described by Miller (1959). Protein concentration was determined by the Bradford protocol (Bradford 1976) with bovine serum albumin (BSA) as the standard. The amount of protein samples and the incubation time were chosen to provide assay conditions in which the measured activity was proportional to these two parameters. The enzymatic assays were all carried out at 55 °C to quantify the activities under conditions close to those of industrial processes using thermostable enzymes. Reducing sugars that were released after enzyme–substrate incubations were quantified in triplicates using glucose or xylose as a standard. One international unit (IU) was defined as the amount of enzyme, which produced 1 µmol of reducing sugars in 1 min. The xylanase and lichenase activities of the extracellular proteins from BSS cultures with 0.5% apple and orange peels are listed in Table 1. Strain CCMM B945 was again used as a control. No reducing sugars was detectable for strain CCMM B945 proteins incubated with xylan or lichenan. The lichenase activity of CCMM B940 was approximately two times higher than the xylanase activity in the cultures with natural peels. Both activities were higher in apple peel cultures than in orange peels, suggesting that the produced enzymes were either more numerous or more active in metabolizing carbohydrates in apple peels. This could be due to the nature of fibres present in the peel (sugar composition and types of glycosidic bonds) and/or to differential induction of glycoside hydrolases encoding gene expression. Strain CCMM B940 exhibited xylanase activities approximately twofold and over tenfold higher in apple peel cultures than thermophilic strains B. licheniformis 2D55 (Kazeem et al. 2017) and Bacillus sp. 275 (Gong et al. 2017), respectively.

Table 1.

Lichenase and xylanase specific activities of extracellular proteins from Bacillus CCMM B940 and CCMM B945 cultivated in BSS medium supplemented with 0.5% apple and orange peels

Strains Apple Orange
CCMM B940 CCMM B945 CCMM B940 CCMM B945
Lichenase activity (IU/mg) 4.06 ± 1.1 nd 1.6 ± 0.2 nd
Xylanase activity (IU/mg) 2.39 ± 0.29 nd 0.98 ± 0.2 nd
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Specific activities are given as µmol of glucose (incubation with lichenan) or xylose (incubation with xylans) equivalent produced per min and per mg of protein. Each value is the mean of three different assays (± SD)

nd not detectable

To further investigate the putative functions of the CCMM B940 strain and to achieve its reclassification within the genus Bacillus, we sequenced and examined the whole genome of this strain. Thus, genomic DNA was extracted with Wizard® Genomic DNA Purification Kit (Promega, Charbonnières-les-Bains, France). The concentration and quality were determined using Nanodrop and Qubit spectrophotometers. Genomic DNA was sequenced at Eurofins Genomics (Cologne, Germany) using Illumina HiSeq technology. The library was prepared with 2 × 150-bp paired-end read length, including DNA fragmentation, adapter ligation, amplification, and size selection. All the steps were performed according to Eurofins Genomics protocols, producing 7,612,190 reads.

The raw reads were then trimmed using Trimmomatic v0.39 (Bolger et al. 2014) with the following parameters: SLIDINGWINDOW:4:20; ILLUMINACLIP: TruSeq3-PE.fa:2:30:20:2: LEADING:3, TRAILING:3 and MINLEN:60. We verified the presence/absence of phix sequences using bowtie2 (Langmead and Salzberg 2012) with the following parameters: –trim-to 80 –end-to-end –no-unal –no-sq –no-head –p 12. Processed reads were then assembled using the SPAdes de novo assembler v3.13.1 (Bankevich et al. 2012) testing assemblies with k-mer values: 21,33,55,77 using the parameter: –careful. Depending on the size of insert, scaffolds smaller than 350 bp were filtered out. Assembly quality was assessed using in-house script and QUAST v5.0.0 (Gurevich et al. 2013). We estimated genome completeness at 99.59% and checked that no contamination was detected using CheckM v1.1.3 (Parks et al. 2015).

After eliminating low coverage and small contigs (< 350 bp), we obtained a total of 38 contigs and 34 scaffolds resulting in a genome of 4,315,004 bp (NCBI accession number JADRJL000000000) and an average G + C content of 45.87% which is close to other genomes from the Bacillus subtilis group (Rey et al. 2004; Du et al. 2019).The N50 of the draft genome is 440,121 bp, with an average of 126,912 bp and a notable four gap region (Fig. 3). Species identity was confirmed by comparing the genome reference sequences of B. licheniformis and Bacillus paralicheniformis available in the NCBI database (https://www.ncbi.nlm.nih.gov) using the BLAST service deployed on local servers with the genome obtained sequences and fastANI (Jain et al. 2018). The draft genome was most closely related to B. paralicheniformis Bac84 (accession number ASM299392v1) living in sea environment (Othoum et al. 2019) with an ANI value of 98.94%, and shared only 94.36% identity with B. licheniformis DSM 13T (NC_006270.3). B. aerius CCMM B940 was, therefore, reclassified as B. paralicheniformis CCMM B940, placing it in the B. subtilis group; a group known for its wide array of uses in biotechnology mainly through species like B. subtilis and B. licheniformis (Fan et al. 2017). Taxonomic identity of the CCMM B940 strain was also confirmed using the GTDB-Tk tool for genome classification (Chaumeil et al. 2020) with 98.95% identity to the B. paralicheniformis KJ-16 genome (accession number ASM104248v2). The genome was then annotated using the default parameters of Prokka 1.14 (Seemann 2014) showing 4259 protein-coding genes, 51 tRNAs and 4 complete rRNAs.

Fig. 3.

Fig. 3

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Circular representation of the B. paralicheniformis CCMM B940 chromosome using PATRIC based on 34 assembled scaffolds. From outer to inner ring—contigs (scale—x1Mbp), CDS on the forward strand, CDS on the reverse strand, RNA genes, homologous CDS to known antimicrobial resistance (AMR) genes, CDS with homology to known virulence factors (VF), GC content and GC skew

We further used eggNOG-mapper v2.0.1 (Huerta-Cepas et al. 2017) to assign proteins sequences into functional categories. More than 76% of the predicted proteins were attributed to functional subsystems. Proportions of genes with known functions (3047 CDS) were assigned to each category as presented in Fig. 4. Interestingly, a high proportion of CCMM B940 genes was assigned to the carbohydrate transport and metabolism (11.3%) as well as amino-acid transport and metabolism (12.5%) proteins. In a study conducted on Bacillus velenzis, Bacillus safensis and Bacillus altitudinis genomes, only 6.1% of the predicted proteins were assigned to carbohydrate transport and metabolism (Datta et al. 2020), while in Bacillus amyloliquefaciens and Bacillus siamensis, it represents a maximum of 8% in the pan and the core genomes (Chun et al. 2019). B. paralicheniformis has not been thoroughly studied for carbohydrate metabolism yet, but is phylogenetically very close to the B. licheniformis that has been widely used in the fermentation industry for production of enzymes, antibiotics and other chemicals (Madslien et al. 2012).

Fig. 4.

Fig. 4

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Distribution of biological functions of B. paralicheniformis CCMM B940 based on eggNOG analysis. Colors show different gene features categories and their proportions. Percentages of genes with known functions are given within parentheses

Hereafter, we focused on CCMM B940 glycoside hydrolases related to hemicellulose, cellulose and pectin degradation, comparing their protein sequences to the sequences of GH and pectate lyase (PL) of seven strains of B. paralicheniformis (A4-3, ATCC 9945a, BL-09, CBMAI 1303, FA6, MDJK30, ZAP17) available in the CAZY database (Lombard et al. 2014; database release 22 November 2020). Blast results was obtained using default parameters. Best hits with almost 90% amino-acid identity and 50% length alignment were selected and illustrated in Table 2 (the corresponding sequences are provided in Fig S2). Among 74 GH and PL sequences from CCMM B940 showed homology to other strains of B. paralicheniformis, 52 are dedicated to the degradation of carbohydrates in plant cell walls, representing 27 different CAZymes families according to the classification of B. Henrissat (Lombard et al. 2014). Thirty-two sequences of GH1, GH3, GH5, GH8, GH11, GH16, GH26, GH30, GH31, GH32, GH42, GH43, GH51, GH53, GH68, GH73 were linked to the degradation of hemicellulose, 4 sequences of GH5, GH9, GH12, GH48 for cellulose metabolism, 1 sequence (GH5) either for hemicellulose or cellulose degradation, and 11 sequences of GH (GH28, GH105) and PL (PL1, PL3, PL9, PL11, PL2) were attributed to pectin degradation (Table 2). Four sequences corresponded to enzymes (GH1, GH4) involved in glycolytic pathways. The remaining sequences were mainly related to the hydrolysis of starch and peptidoglycan (not shown).

Table 2.

Annotated genes encoding lignocellulose-degrading enzymes in B. paralicheniformis CCMM B940

Enzymes Accession number Activity CAZyme family Reference genes Identity (%) Subject sequence length (bp) Alignment length (bp)
Accession number Species
Hemicellulose related EOOONOAK_01749 Aryl-phospho-beta-d-glucosidase BglC GH1 ARA87766.1 Bacillus paralicheniformis MDJK30 99.78 472 472
EOOONOAK_03335 Aryl-phospho-beta-d-glucosidase BglH GH1 AGN35231.1 Bacillus paralicheniformis ATCC 9945a 100 100 100
EOOONOAK_03002 Aryl-phospho-beta-d-glucosidase BglC GH1 AJO16530.1 Bacillus paralicheniformis BL-09 100 491 478
EOOONOAK_01773 Aryl-phospho-beta-d-glucosidase BglH GH1 QEO05069.1 Bacillus paralicheniformis A4-3 99.57 469 469
EOOONOAK_03715 Endo-1,4-beta-xylanase A GH11 QEO05734.1 Bacillus paralicheniformis A4-3 100 213 213
EOOONOAK_02553 Beta-glucanase GH16 ARA86799.1 Bacillus paralicheniformis MDJK30 99.59 243 243
EOOONOAK_03322 Mannan endo-1,4-beta-mannosidase GH26 QFY39913.1 Bacillus paralicheniformis FA6 98.89 360 360
EOOONOAK_03205 Beta-hexosaminidase GH3 QEO05378.1 Bacillus paralicheniformis A4-3 99.69 644 643
EOOONOAK_02186 Glucuronoxylanase XynC GH30 AJO20016.1 Bacillus paralicheniformis BL-09 100 420 420
EOOONOAK_01418 Oligosaccharide 4-alpha-d-glucosyltransferase GH31 QFY38704.1 Bacillus paralicheniformis FA6 99.75 802 802
EOOONOAK_02225 Alpha-xylosidase GH31 AJO19971.1 Bacillus paralicheniformis BL-09 99.61 769 769
EOOONOAK_02112 Levanbiose-producing levanase GH32 ARA87316.1 Bacillus paralicheniformis MDJK30 99.8 515 495
EOOONOAK_01584 Sucrose-6-phosphate hydrolase GH32 AJO20434.1 Bacillus paralicheniformis BL-09 99.37 478 478
EOOONOAK_01729 Sucrose-6-phosphate hydrolase GH32 AJO20595.1 Bacillus paralicheniformis BL-09 98.98 492 492
EOOONOAK_00085 Levanase GH32CBM66 QFY38018.1 Bacillus paralicheniformis FA6 100 677 677
EOOONOAK_02224 Hypothetical protein GH3CBM6 AJO19972.1 Bacillus paralicheniformis BL-09 100 980 980
EOOONOAK_03904 Beta-galactosidase BglY GH42 ARA84401.1 Bacillus paralicheniformis MDJK30 99.42 690 690
EOOONOAK_04173 Beta-galactosidase YesZ GH42 QFY39293.1 Bacillus paralicheniformis FA6 100 665 665
EOOONOAK_01837 Beta-galactosidase GanA GH42 QFY40673.1 Bacillus paralicheniformis FA6 99.56 684 684
EOOONOAK_03858 Beta-xylosidase GH43 ARA84797.1 Bacillus paralicheniformis MDJK30 99.62 532 532
EOOONOAK_03859 Non-reducing end alpha-l-arabinofuranosidase GH43 ARA84798.1 Bacillus paralicheniformis MDJK30 99.03 515 515
EOOONOAK_03489 Extracellular endo-alpha-(1- > 5)-l-arabinanase 1 GH43 ARA85201.1 Bacillus paralicheniformis MDJK30 100 316 316
EOOONOAK_00089 Beta-xylosidase GH43 ARA86580.1 Bacillus paralicheniformis MDJK30 100 533 533
EOOONOAK_01777 Extracellular endo-alpha-(1- > 5)-l-arabinanase 2 GH43 ARA88110.1 Bacillus paralicheniformis MDJK30 99.79 469 469
EOOONOAK_02586 Extracellular endo-alpha-(1- > 5)-l-arabinanase 1 GH43 QFY37786.1 Bacillus paralicheniformis FA6 99.69 320 320
EOOONOAK_02185 Arabinoxylan arabinofuranohydrolase GH43CBM6 QFY37223.1 Bacillus paralicheniformis FA6 100 515 515
EOOONOAK_01157 hypothetical protein GH5 ARA85656.1 Bacillus paralicheniformis MDJK30 99.82 560 560
EOOONOAK_02594 Intracellular exo-alpha-(1- > 5)-l-arabinofuranosidase GH51 ARA86758.1 Bacillus paralicheniformis MDJK30 99.80 502 502
EOOONOAK_01836 Arabinogalactan endo-beta-1,4-galactanase GH53 AGN38579.1 Bacillus paralicheniformis ATCC 9945a 98.58 437 424
EOOONOAK_02113 Levansucrase GH68 ARA87315.1 Bacillus paralicheniformis MDJK30 100 481 481
EOOONOAK_04229 Hypothetical protein GH73 QEO06161.1 Bacillus paralicheniformis A4-3 98.95 570 570
EOOONOAK_03432 Reducing-end xylose-releasing exo-oligoxylanase Rex8A xylanase probable GH8 QFY39429.1 Bacillus paralicheniformis FA6 99.77 434 434
Cellulose-related EOOONOAK_03622 Endoglucanase S GH12 ARA86997.1 Bacillus paralicheniformis MDJK30 100 261 261
EOOONOAK_01156 Exoglucanase-2 GH48 AJO18128.1 Bacillus paralicheniformis BL-09 100 717 704
EOOONOAK_01385 Endoglucanase GH5, CBM3 AJO18373.1 Bacillus paralicheniformis BL-09 99.81 518 518
EOOONOAK_01155 Endoglucanase A GH9CBM3 AJO18127.1 Bacillus paralicheniformis BL-09 100 653 653
Glucose metabolism EOOONOAK_00372 6-Phospho-beta-glucosidase GmuD GH1 AJO18923.1 Bacillus paralicheniformis BL-09 100 471 471
EOOONOAK_01723 6-Phospho-beta-glucosidase GmuD GH1 AJO20590.1 Bacillus paralicheniformis BL-09 99.16 478 478
EOOONOAK_03054 Putative 6-phospho-beta-glucosidase GH4 ARA84298.1 0 Bacillus paralicheniformis MDJK30 99.55 444 444
Hemicellulose or cellulose related EOOONOAK_01655 Putative 6-phospho-beta-glucosidase GH4 ARA87677.1 Bacillus paralicheniformis MDJK30 100 442 442
EOOONOAK_01158 Hypothetical protein GH5 QEO07050.1 Bacillus paralicheniformis A4-3 100 395 395
Pectin degradation EOOONOAK_04163 Unsaturated rhamnogalacturonyl hydrolase YesR GH105 AJO17709.1 Bacillus paralicheniformis BL-09 99.71 344 344
EOOONOAK_02457 Unsaturated rhamnogalacturonyl hydrolase YteR GH105 AJO19596.1 Bacillus paralicheniformis BL-09 99.73 373 373
EOOONOAK_02256 Exo-poly-alpha-d-galacturonosidase GH28 QFY37288.1 Bacillus paralicheniformis FA6 100 436 434
EOOONOAK_02563 Hypothetical protein PL1 QFY37765.1 Bacillus paralicheniformis FA6 100 494 494
EOOONOAK_04252 Pectate lyase PL1 AJO17744.1 Bacillus paralicheniformis BL-09 99.53 428 428
EOOONOAK_01685 Pectate trisaccharide-lyase PL1 QEO04986.1] Bacillus paralicheniformis A4-3 99.71 341 341
EOOONOAK_04170 Rhamnogalacturonan exolyase YesX PL11 QEO06607.1 Bacillus paralicheniformis A4-3 100 628 628
EOOONOAK_04168 Rhamnogalacturonan endolyase YesW PL11 QEO08401.1 Bacillus paralicheniformis A4-3 100 622 622
EOOONOAK_04174 Hypothetical protein PL26 QEO06610.1 Bacillus paralicheniformis A4-3 99.07 886 863
EOOONOAK_02080 Pectate lyase C PL3 ARA87346.1 Bacillus paralicheniformis MDJK30 99.55 222 221
EOOONOAK_01392 Hypothetical protein PL9 QEO07258.1 Bacillus paralicheniformis A4-3 100 468 468
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The accession numbers correspond to the CDS sequences annotated with Prokka as indicated in the manuscript. The corresponding sequences are provided in Figure S2

These predictions first confirmed the experimental observations in this study for the degradation of xylan, lichenan and CMC as the CCMM B940 genome carries glycoside hydrolases from GH5, GH9, GH11 and GH16 families of CAZYmes known to target these substrates. They also provided additional information on the carbohydrate metabolism of CCMM B940 such as pectinase activity as well as the presence of carbohydrate-binding modules (CBM3, CBM6, CBM66) associated with GHs helping them to bind to cellulose or hemicellulose polysaccharides to improve their efficiency. A few other studies (Wang et al. 2017; Chen et al. 2018) examined the genomic sequences of B. paralicheniformis and predicted their CAZymes, but without the experimental validation. To our knowledge, this is the first report that combines in silico genomic analysis and in vitro experiments to study the carbohydrate metabolism of B. paralicheniformis. These combined analyses reinforce the practical and potential use of CCMM B940 strain for industrial purposes. Indeed, Bacillus species are known to produce 60% of commercially available enzymes, mostly being homologous proteins that are naturally secreted in the growth medium (Westers et al. 2004). The strain CCMM B940 exhibits cellulolytic and hemicellulolytic activities at high temperature and produces extracellular thermostable enzymes.

Based on whole-genome sequencing, we demonstrated that the B. aerius strain CCMM B940 could be reclassified as B. paralicheniformis. Genomic analysis confirmed the fibrolytic potential of the strain CCMM B940 observed in vitro using purified and natural substrates and provided information on its ability to degrade pectins. This statement reinforces the strain's interest especially in agro-waste bioconversion. To our knowledge, this is the first report of lichenase and xylanase activities by a thermophilic strain of B. paralicheniformis isolated from a hot spring. The CCMM B940 strain is part of the large Coordinated Moroccan Collections of Microorganisms (CCMM), which comprise more than 1800 isolates of bacteria, yeasts and fungi. Many of these microorganisms have also been isolated from extremophile environments (Berrada et al. 2012; Aanniz et al. 2015) and they may harbor beneficial attributes such as production of heat-resistance enzymes as already observed in B. amyloliquefaciens (Beladjal et al. 2018) and B. subtilis (den Besten et al. 2017) strains. For this purpose, complete genome sequencing not only enables accurate identification and reclassification of CCMM isolates, i.e. the thermophilic strains of the B. aerius group, but also opens the door to the field of biotechnological applications such as production of second- and third-generation biofuels from raw or pre-treated biomass, medical and nutraceutical fields and food processing (Benedetti et al. 2019), in the favor of the fibrolytic potential (CAZymes) of these strains. Enzymatic activities of these strains should be further studied to determine the most suitable markets in biotechnology (agro-waste bioconversion, depollution, health, pharmaceuticals, etc.…).

Accession numbers

The assembled genome and all relevant sequences were deposited in NCBI's GenBank on November 24, 2020 under the following accession numbers, BioProject PRJNA680612; BioSample SAMN16895636; Genome JADRJL000000000.

Supplementary Information

Below is the link to the electronic supplementary material.

13205_2021_2919_MOESM1_ESM.txt (4.6MB, txt)

Fig. S1 Cultures of the CCMM B940 and CCMM B945 Bacillus strains in BSS medium supplemented with 0.5% orange or apple peels for 48h at 37°C. a. negative control without bacteria; b. growth (turbidity) is observed for cultures on both substrates (DOCX 1356 kb)

13205_2021_2919_MOESM2_ESM.docx (1.3MB, docx)

Fig.S2 CDS annotation of the genome of B.paralicheniformis CCMM B940 for lignocellulose-degrading enzymes (TXT 4743 kb)

Acknowledgements

Funding for this research was provided by Belgian Cooperation FRAB/CCMM2, the National Center for Scientific and Technical Research (CNRST) and the National Research Institute for Agriculture, Food and Environment (INRAE). Maski Soufiane is a PhD student at Faculty of Science, Mohammed V University, Rabat, Morocco. Serigne Inssa Ngom is a recipient of a grant for doctoral research allocated by the French ministry of higher education and research (doctoral school 569, University of Paris-Saclay). The authors would like to thank Mrs Zehra Esra Ilhan for English improvement of the manuscript.

Author contributions

MA, EE, MB and CBM conceived the study. SM, SIN, MA, EE, CBM designed the experiments. SM, SIN did genomic and enzymatic analyses, respectively. SIN, BR, TC prepared the samples and extracted DNA. SM performed nucleic Blast, the assembly and the annotation of the genome, and performed the map of functional categories. SIN performed all the enzymatic assays. MA, EE, MB, BR, CBM analyzed the data. SM, SIN, CBM, MA wrote the manuscript with the help of all authors. All authors have read and approved the final manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Contributor Information

Soufiane Maski, Email: soufianemaski1@gmail.com.

Serigne Inssa Ngom, Email: serigneinssa.ngom@inrae.fr.

Bahia Rached, Email: rached@cnrst.ma.

Taha Chouati, Email: chouati@cnrst.ma.

Elmostafa El Fahime, Email: elfahime@cnrst.ma.

Mohamed Amar, Email: m.amar.cnrst@gmail.com.

Christel Béra-Maillet, Email: christel.maillet@inrae.fr.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13205_2021_2919_MOESM1_ESM.txt (4.6MB, txt)

Fig. S1 Cultures of the CCMM B940 and CCMM B945 Bacillus strains in BSS medium supplemented with 0.5% orange or apple peels for 48h at 37°C. a. negative control without bacteria; b. growth (turbidity) is observed for cultures on both substrates (DOCX 1356 kb)

13205_2021_2919_MOESM2_ESM.docx (1.3MB, docx)

Fig.S2 CDS annotation of the genome of B.paralicheniformis CCMM B940 for lignocellulose-degrading enzymes (TXT 4743 kb)

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