Genomic Annotation and Validation of Bacterial Consortium NDMC-1 for Enhanced Degradation of Sugarcane Bagasse

Annals of Microbiology (2019) 69:695–711 https://doi.org/10.1007/s13213-019-01462-x

ORIGINAL ARTICLE

Genomic annotation and validation of bacterial consortium NDMC-1 for enhanced degradation of sugarcane bagasse

Varsha Bohra1 & Nishant A. Dafale1 & Zubeen Hathi1 & Hemant J. Purohit1

Received: 3 January 2019 /Accepted: 5 March 2019 /Published online: 22 March 2019 # Università degli studi di Milano 2019

Abstract Purpose This study aims at designing a consortium using rumen bacterial isolates for enhancing the hydrolysis of sugarcane bagasse (SB) for efficient biofuel formation. Methods The microbial population was screened through biochemical and molecular tools along with enzymatic activity to obtain potential isolates for diverse cellulolytic and hemicellulolytic carbohydrate active enzyme (CAZyme). Five strains (Paenibacillus, Bacillus, Enterobacter,andMicrobacterium) were selected for designing the consortium NDMC-1. The hydrolytic efficiency of NDMC-1 was determined based on cellulase production with simultaneous rise in monosaccharides, oligo- saccharides, and soluble chemical oxygen demand (sCOD) concentration. Cellulolytic machinery of these isolates was further explored using genome sequencing. Result The isolates selected for consortia NDMC-1 interacted synergistically leading to enhanced cellulase production. Maximal endoglucanase (1.67 μmol ml−1 min−1), exoglucanase (0.69 μmol ml−1 min−1), and β-glucosidase (2.03 μmol ml−1 min−1) activity were achieved with SB as a sole carbon source after 48 h of incubation. Enhancement in SB hydrolysis employing NDMC-1 was evident by the increase in sCOD from 609 to 2589 mg/l and release of 1295 mg/l reducing sugar, comprising 59.8%, 8.23%, and 6.16% of glucose, cellobiose, and cellotriose, respectively, which resulted in 5.5-fold rise in biogas production. On genome annotation, total 472 contigs from glycoside hydrolase family: 84 from Microbacterium arborescens ND21, 72 from Enterobacter cloacae ND22, 61 from Bacillus subtilis ND23, 116 from Paenibacillus polymyxa ND24, and 140 from Paenibacillus polymyxa ND25 were identified. On further analysis, total 33 cellulases, 59 hemicellulases, and 48 esterases were annotated in the reported genomes. Conclusion This work proposes the application of consortia-based bioprocessing systems over the conventionally favorable single organism approach for efficient hydrolysis of cellulosic substrates to fermentable sugars.

Keywords Cellulolytic . Consortia NDMC-1 . Genome sequencing . CAZymes . Glycoside hydrolase . Bagasse . Biogas

Introduction of valuable bioproducts. However, the development of cost- effective industrial setup based on cellulosic biomass requires Cellulosic biomass offers a broad-spectrum renewable energy its efficient hydrolysis to simple fermentable sugars resource by serving as a potential substrate in the production (Shirkavand et al. 2016;Aggarwaletal.2017). This process is accomplished by the action of a multicomponent cellulolytic system comprising endoglucanase (EC 3.2.1.4), Electronic supplementary material The online version of this article exoglucanase (EC 3.2.1.91), and β-glucosidase (EC (https://doi.org/10.1007/s13213-019-01462-x) contains supplementary material, which is available to authorized users. 3.2.1.21) that plays an indispensable role in cellulose hydrolysis (Saini et al. 2015). Many cellulolytic microbes are dis- * Nishant A. Dafale covered till date; yet, substantial hydrolysis still remains a [email protected] major bottleneck in rapid and economic utilization of cellulosic biomass (Singhania et al. 2013). For that, several strate- 1 Environmental Biotechnology & Genomics Division, CSIR– gies including use of microbial consortium to produce a broad National Environmental Engineering Research Institute (NEERI), array of enzymes, implementation of unrefined enzyme ex- Nagpur, India tracts, heterologous expression of cellulolytic enzymes, 696 Ann Microbiol (2019) 69:695–711 formulation of enzyme cocktail, and enzyme recycling etc. SB used for the experiments was obtained locally. All other have been adopted to minimize production cost and maximize chemicals were of analytical grade and obtained from com- the hydrolytic efficiency (Zhu et al. 2009; Lee et al. 2010;Hu mercial sources. et al. 2011; Liu and Du 2012; Adsul et al. 2014; Leo et al. 2016; Poszytek et al. 2016; Orencio-Trejo et al. 2016). Of Selection of members for the designing of microbial these, application of microbial consortium is reported to be consortia simpler, economical, and equally productive for hydrolytic breakdown of cellulose (Leo et al. 2016;Poszyteketal. Isolation of diverse bacterial population from rumen habitat 2016). Application of microbial consortium or co-culture technique involves two or more microbes for better substrate Rumen samples from cow and buffalo were explored for iso- utilization resulting in increased productivity as compared to lation of cellulolytic bacteria as described by Bohra et al. the use of monocultures (Saini et al. 2016). Wongwilaiwalin (2018a). Initially obtained 847 morphologically distinct bac- et al. (2010) developed a stable microbial consortium desig- terial isolates were subjected to random amplified polymor- nated MC3F, capable of hydrolyzing bagasse, corn stover, and phic DNA (RAPD) profiling as described by Saxton et al. rice straw. Kato et al. (2005) developed a functionally stable (2016)withprimer60S(5′-CAGCAGCAGCAG-3′)todiffer- microbial consortium SF356 consisting of five microbial iso- entiate microbial populations at the genetic level. lates Pseudoxanthomonas sp. M1–3, Brevibacillus sp. M1–5, BioNumerics v 7.1 was used to identify distinct bacterial iso- Bordetella sp. M1–6, Clostridium straminisolvens CSK1, and lates based on UPGMA method. Clostridium sp. FG4. Another microbial consortium, WSD5 comprising both bacterial and fungal isolates, was developed Plate zymography for selection of efficient cellulolytic from plant litter and soil for degradation of wheat straw (Wang bacterial stain et al. 2011). This study evaluated the production of cellulolytic en- Based on RAPD profiling, 473 distinct isolates were obtained. zymes and biomass hydrolysis using monoculture and consor- These isolates were screened for cellulolytic efficiency tium NDMC-1 buildup of Microbacterium arborescens through plate zymography. The cellulose hydrolyzing ability ND21, Enterobacter cloacae ND22, Bacillus subtilis ND23, of the bacterial isolates was semi-quantitatively estimated by Paenibacillus polymyxa ND24, and Paenibacillus polymyxa calculating enzyme activity index (EAI): the ratio of hydroly- ND25. All these bacterial strains were isolated from cow and sis zone diameter to the colony diameter (Bohra et al. 2018a). buffalo rumen and displayed highest efficiency for cellulose Bacterial strains exhibiting EAI ≥ 3 were further assayed for decomposition. The hydrolyzed product was further utilized their ability to produce diverse cellulolytic and for biogas generation. Wet lab experiments were combined hemicellulolytic enzyme. with genome sequencing and annotation of selected strains to provide further insight into the enzymes responsible for Screening bacterial strains for diverse CAZyme using cellulose and hemicellulose hydrolysis. Results indicate the chromogenic substrates presence of highly proficient and indispensable enzymatic machinery efficient in deconstructing cellulose and hemicel- Forty-five isolates exhibiting EAI ≥ 3 were assayed for exo- lulose in the arsenal of bacterial consortium NDMC-1, for 1,4-β-glucanase; β-glucosidase; α-glucuronidase; endo- biotechnology applications. 1,4-β-xylanases; arabinosidase; and α-galactosidase producing efficiency. For this, all strains were individually grown in 5 ml of Berg minimal salt (BMS) media (Pawar et al. 2015) Materials and methods supplemented with 400 μg/ml chromogenic substrate; pNPC, pNPG, pNPGl, pNPX, pNPA, and pNPGa. After incubating Materials for 14 h at 37 °C, 150 μl aliquots were recovered, centrifuged at 8000 rpm for 5 min and 50 μl of the supernatant was trans- Carboxymethyl cellulose sodium salt (CMC), microcrystal- ferred to a 96 well plate. Then, 50 μlof0.1Msodiumcar- line cellulose avicel PH-101 (Avicel), p-nitrophenyl-β-D- bonate was added to each well and pNP concentration was glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside determined at 405 nm (Strahsburger et al. 2017). (pNPC), p-nitrophenyl-β-D-glucuronide (pNPGl), p- nitrophenyl-β- D -xylanopyranoside (pNPX), p- Molecular identification of cellulolytic strain using 16s rDNA nitrophenyl-α-L-arabinopyranoside (pNPA) and p- sequencing nitrophenyl-α-D-galactopranoside (pNPGa), p-nitrophenol (pNP), and 3,5-dinitrosalicylic acid were procured from Molecular identification of isolate exhibiting high cellulolytic Sigma–Aldrich. efficiency (EAI > 3) was achieved by amplifying 16S rDNA Ann Microbiol (2019) 69:695–711 697 sequence using bacteria universal primers 27F (5- Soluble chemical oxygen demand (sCOD) was determined AGAGTTTGATCCTGGCTCAG-3) and 1492R (5- using the standard method provided by APHA (2005). TACGGTTACCTTGTTACGACTT-3) (Bohra et al. 2018a; Dafale et al. 2010). PCR products were checked for size and Biomethane production using hydrolyzed substrates purity and sequenced using Sanger di-deoxy method. The similarity search for the sequence was carried out using the For biochemical methane potential (BMP) assay, hydrolysates BLAST program of the National Center of Biotechnology containing deconstructed SB were harvested after 48 h of Information (NCBI). incubation. Hydrolysate (20 ml) was mixed with equal volume of anaerobic sludge and made up to 100 ml using Nucleotide sequence accession number autoclaved distilled water. The reaction volume was trans- ferred to a 125 ml serum bottle subsequently sealed with an 16S rDNA gene sequences of cellulolytic cultures retrieved in aluminum crimp to maintain anaerobic conditions. BMP assay the study were deposited in GenBank and assigned accession was carried out for 4 days, and biogas volume and composi- nos. from KX404917 to KX404971. tion were monitored at regular interval of 24 h. Frictionless glass syringe was used for measuring biogas volume and gas Growth conditions and hydrolysis of different chromatography (Agilent 7890 A) for determining its compo- agro-waste material sition. The gas chromatograph was operated using thermal conductivity detector and packed column (Restek, 6 ft. × Based on ability to produce diverse cellulolytic and 2 mm ID) with hydrogen as carrier gas. The conditions used hemicellulolytic CAZymes, M. arborescens ND21, for analysis of biogas composition were as described by E. cloacae ND22, B. subtilis ND23, P. polymyxa ND24, and Gulhane et al. (2016). P. polymyxa ND25 were selected for designing the consortia NDMC-1. The ability of the consortia to hydrolyze different agro-waste (rice straw, rice husk, wheat straw, wheat husk, Analytical methods saw dust, corn stover, grass straw, sugarcane bagasse, filter paper, and newspaper) was estimated by inoculating one OD During hydrolysis, samples from the experimental flask were cell of each isolate into 50 ml of BMS media supplemented collected at regular time interval and centrifuged at 1000g for with one of the agro-waste material (2%). Optimal physio- 10 min. The supernatant was filtered through 0.2 μmsyringe chemical conditions for hydrolysis of SB by monoculture filter before performing the following analysis: and NDMC-1 were investigated after 48 h of incubation using different concentration of SB (1%, 2%, 3%, and 4%). Optimal Estimation of total reducing sugar Total reducing sugar gen- nitrogen source (peptone yeast extract, ammonium nitrate, erated was determined by 3,5-dinitrosalicylic acid solution ammonium chloride, and sodium nitrate) and its concentration (DNS) method (Miller 1959). An equal volume (0.5 ml) of (0.25%, 0.50%, 1%, 1.50%, 2%) were determined simulta- supernatant and DNS reagent were mixed and heated to 99 °C neously. All the experiments were performed at 37 °C and for 15 min in a boiling water bath. The reducing sugar gener- 120 rpm. ated was estimated at 540 nm using glucose as standard.

Hydrolytic potential of isolates and microbial Estimation of mono- and oligosaccharide Glucose, cellobiose, consortia (NDMC-1) for SB and cellotriose concentration were determined using high- performance liquid chromatography (HPLC, make: Perkin Flask experiments were carried out to study the cellulolytic Elmer) with RI detector. Separation of the hydrolyzed prod- potential of individual microbes. For these, 250-ml flasks con- ucts was performed through zorbax NH2 column (4.6 × taining 50 ml of BMS media supplemented with 2% SB as 12.5 mm, 5 μm, Agilent) using acetonitrile and water in the sole carbon source and 0.5% peptone as nitrogen source were ratio 70:30 as the mobile phase at a flow rate of 0.5 ml/min. used. The flasks were inoculated with 10 OD cells/100 ml and The detector temperature was maintained at 35 °C. incubated for 72 h at 37 °C and180 rpm. Co-culture study involving all five bacterial isolates were performed in same Exoglucanase activity Reaction mixture for estimation of media with equal proportion (2.0OD/100 ml) of seed cultures. exoglucanase activity consisted of 0.5 ml enzyme along with For evaluating the hydrolytic ability of designed consortia 0.5 ml 2% (w/v) avicel substrate in phosphate buffer (100 mM, NDMC-1 and its individual member, supernatants were col- pH 7) at 37 °C and 60 min. lected to determine cellulase activity (endoglucanase, exoglucanase, and β-glucosidase) and concentration of glu- Endoglucanase activity Endoglucanase activity evaluation cose, cellobiose, and cellotriose at regular time intervals. was done similarly by incubating reaction mixture consisting 698 Ann Microbiol (2019) 69:695–711

0.5 ml of enzyme along with 0.5 ml of 2% (w/v) CMC solution hydrolase (GH) were functionally classified by in phosphate buffer (100 mM, pH 7), at 37 °C and 30 min. BLASTP algorithm using the public database KEGG In both the assay, reaction was inhibited using 1 ml and NR. Positional locations of the endoglucanase, dinitrosalicylic acid solution (DNS) and boiled for 5 min. exoglucanase, and β-glucosidase were mapped in the The released reducing sugars were estimated at 540 nm genome by using the NCBI server (Bohra et al. 2018b). (Nitisinprasert and Temmes 1991).

β-Glucosidase activity The activity of β-glucosidase was de- Results and discussion termined using substrate p-nitrophenyl β-glucopyranoside (pNPG). The assay mixture consisted of 0.5 ml of enzyme Mining of rumen microbial population for design along with 0.5 ml of pNPG in 100 mM phosphate buffer. of effectual consortia The reaction mixture was incubated for 30 min at 37 °C and reaction was stopped with 2 ml of 1% (w/v) solution of sodium Selection of cellulolytic bacterial stain based on plate carbonate. p-Nitrophenol liberated during the reaction was zymography estimated at 410 nm (An et al. 2004; Svasti et al. 2003). One unit of the enzyme activity is defined as the amount of Plant biomass can be deconstructed by complex microbial com- enzyme units required to liberate 1 µmol of glucose or p- munities existing in natural environments, such as cow rumen nitrophenol from the respective substrates per minute. (Hess et al. 2011). Biomass hydrolysis in these environments is achieved by the synergistic action of hydrolytic enzymes exhib- DNA extraction and genome sequencing ited by different bacterial species (Hemsworth et al. 2015; López-Mondéjar et al. 2016). In this study, rumen diversity of Total genomic DNA (gDNA) was isolated from all the five cow and buffalo has been exploited for the isolation of bacterial bacterial isolates grown over night in Luria Bertani broth strains able to produce cellulolytic enzymes. In total, 847 mor- using Fast DNA SPIN Kit for soil from MP Biomedicals fol- phologically distinct bacterial colonies were obtained. RAPD lowing manufacturer’s instruction. Nanodrop 8000 and profiling and band pattern analysis of these isolates revealed Qubit® 2.0 Fluorometer were used for determination of 473 distinct isolates. Of this, 58.13% (275 isolates) displayed A260/280 ratio and concentration, respectively. Illumina cellulolytic efficiency on plate zymography with EAI ranging TruSeq Nano DNA HT Library Preparation Kit was used for from 1.2 to 5.8. Of all the cellulolytic isolates, 14% exhibiting generating paired-end sequencing library which was analyzed EAI ≥ 3 were selected for further study. The EAI following using High Sensitivity DNA chip in Bioanalyzer 2100. CMC hydrolysis could be helpful in the selection of strains Cluster generation and sequencing were performed using the having a high cellulose degradation activity (Florencio et al. Illumina MiSeq platform. The high-quality data thus generat- 2012). 16S rDNA identification of 65 (14%) cellulolytic mem- ed were Denovo assembled using Velvet on optimized k-mer bers exhibiting high cellulolytic efficiency (EAI ≥ 3) revealed (Bohra et al. 2018b). 45 distinct isolates. Majority of these isolates belong to genera Staphylococcus, Paenibacillus, Bacillus, Exiguobacterium,and Genome annotation Enterococcus of phyla Firmicutes and dominated the entire rumen microflora. Phyla Proteobacteria were represented by gen- The genome was annotated using the NCBI prokaryotic era Achromobacter, Stenotrophomonas, Pseudomonas, genome automatic annotation pipeline (PGAAP) for Acinetobacter, Enterobacter, Pantoea, Roultella,andErwinia finding functional protein. Genome sequence data of whereas Phyla Actinobacteria is represented by only two genera P. polymyxa ND24, P. polymyxa ND25, B. subtilis Microbacterium and Arthrobacter. Cellulolytic members of ND23, M. arborescens ND21, and E. cloacae ND22 Proteobacteria exhibited low cellulolytic activity (EAI range were submitted to NCBI under the accession nos. 3.0–3.42) as compared to members of Firmicutes (EAI range LZCE00000000, LZEL00000000, LZCF00000000, 3–5.3). In Firmicutes, most abundant genera Bacillus has less LZEM00000000, and LZEN00000000, respectively. enzyme activity (EAI range 3–3.8) than Paenibacillus (EAI Sequenced genomes were analyzed using database for range 3.2–5.3) which are less abundant as compared to automated carbohydrate-active enzyme annotation Bacillus (Table S1). (dbCAN) algorithm from dbCAN pipelines (http://csbl. bmb.uga.edu/dbCAN/index.php)(Huangetal.2017) Screening bacterial strains for diverse cellulolytic against the carbohydrate-active enzyme database (http:// and hemicellulolytic enzyme using chromogenic substrate www.cazy.org/)(Lombardetal.2013) for identification of genes responsible in lignocellulose degradation. Formerly selected 45 distinct isolates exhibiting EAI (ratio of Coding sequences annotated by dbCAN as glycoside hydrolysis zone to colony diameter) ≥ 3 were further Ann Microbiol (2019) 69:695–711 699 examined for production of diverse cellulolytic and wastes can be accredited to the presence of processed cellu- hemicellulolytic enzymes. Five isolates, M. arborescens lose, with low lignin, and hemicellulosic content in them. ND21, E. cloacae ND22, B. subtilis ND23, P. polymyxa Despite the variations observed in the hydrolytic potential of ND24, and P. polymyxa ND25 producing variety of these NDMC-1 towards various agro-wastes, the consortium dis- enzymes were selected for designing of the consortium plays potential for substantial hydrolysis of diverse biomass. NDMC-1. The consortium is comprised of both aerobic/ facultative anaerobic bacteria which further avoid the compli- Optimal physiochemical conditions for SB hydrolysis cation of establishing an anaerobic environment required for biomass hydrolysis by anaerobic species. Microbial consortia Based on high efficiency of consortia NDMC-1 to hydrolyze designed from complex lignocellulolytic microbial communi- SB, it was selected as sole carbon source for further experi- ties offers a promising alternative for efficient hydrolysis of ments. Optimal physiochemical conditions of monoculture cellulolytic biomass as compared to monoculture (Zuroff and and NDMC-1 to enhance cellulase production were deter- Curtis 2012;Pengetal.2016). Several studies have reported mined. As the percentage of SB in culture media increased the application of stable microbial consortium for hydrolyzing from 1 to 4%, maximal endoglucanase activity of 0.467 U/ml diverse agricultural waste (Wongwilaiwalin et al. 2010;Kato at 2% SB was observed using consortia NDMC-1; afterwards, et al. 2005;Wangetal.2011). All individual isolates were endoglucanase production remained nearly constant. Peptone found effectual in production of β-glucosidase; endo-1,4-β- was found to be the most suitable nitrogen source for enhanc- xylanases; and α-galactosidase. The exo-1,4-glucanase was ing enzyme production. The maximal endoglucanase activity produced by isolate P. polymyxa ND24, P. polymyxa ND25, 0.988 U/ml of NDMC-1 was observed in media supplemented and M. arborescens ND21, while α-glucuronidase and with 2% SB and 1% peptone as additional nitrogen source. arabinosidase were only produced by P. p o ly m yx a ND24 The comparison of endoglucanase activity achieved using and P. polymyxa ND25 (Table S1). Endoglucanase activity monoculture and consortia NDMC-1 under different physio- of these isolates was estimated after 48 h of growth on BMS chemical conditions is tabulated in Tables S2–S4. medium supplemented with 1% CMC. Highest endoglucanase activity of 0.149 U/ml was observed for Study of hydrolytic potential of isolates and consortia P. polymyxa ND25 and 0.143 U/ml for P. polymyxa ND24 (NDMC-1) for sugarcane bagasse followed by B. subtilis ND23, E. cloacae ND22, and M. arborescens ND21. Experiments on hydrolysis of SB were conducted using both single isolates and consortia (NDMC-1). The special preference Hydrolysis of different agricultural waste biomass was given to microbial consortia as it had a broad array of en- using consortium NDMC-1 zymes showcasing high activities to complement each other. The five bacterial isolates were mixed equally constituting a novel The consortium NDMC-1 was found proficient in consortium. Hydrolysis of SB (2%) was observed for the time deconstructing rice straw, rice husk, wheat straw, wheat husk, period of 72 h at 37 °C, and its efficiency was evaluated based saw dust, corn stover, grass straw, filter paper, and newspaper on endoglucanase, exoglucanase and β-glucosidase produced, to a variable extent. Maximum sCOD values of 2778 mg/l and changes in the concentration of sCODs, and production of mono-, 2674 mg/l were observed when filter paper and newspaper di-, and trisaccharides. Consortia exhibited highest extracellular were respectively used as a substrate. These results were CMCase activity (0.98 U/ml) after 48 h followed by monoculture found to be coherent with a high concentration of reducing of P. polymyxa ND24 (0.526 U/ml), P. polymyxa ND25 (0.49 U/ sugar 1634 mg/l and 1572 mg/l, produced respectively during ml), M. arborescens ND21 (0.195 U/ml), B. subtilis ND23 hydrolysis of filter paper and newspaper as a sole carbon (0.185 U/ml), and E. cloacae ND22 (0.174 U/ml) (Fig. 2a). source. NDMC-1 was equally efficient in hydrolyzing SB as Exoglucanase or avicelase activity was exhibited by only three reflected from the resulting sCOD of 2589 mg/l along with the isolates P. polymyxa ND24 (0.247 U/ml), P. polymyxa ND25 release of 1523 mg/l of reducing sugar. Although NDMC-1 (0.24 U/ml), and M. arborescens ND21 (0.108 U/ml). was capable of hydrolyzing diverse agro-waste, comparative- Exoglucanase activity of consortia was found to be 1.7 to 3.9 ly low elevation in sCOD was observed when rice straw, rice times higher than individual isolates (Fig. 2b). β-Glucosidase husk, wheat husk, and grass straw were used as the substrate, activity is shown by all the five isolates and ranges between with a concurrent lower concentration of reducing sugar 0.482–0.185 U/ml with maximum production by P. polymyxa (Fig. 1a, b). The resulting differences in hydrolytic efficiency ND25 and minimum production by B. subtilis ND23. Four to of consortium NDMC-1 towards various substrates are mainly 2.2-fold increase in β–glucosidase activity was achieved when due to variation in composition and structural complexity of consortia was employed for SB hydrolysis (Fig. 2c). Substantial agro-waste materials. High hydrolytic efficiency of NDMC-1 improvement in cellulase activities was observed for microbial towards filter paper and newspaper over other cellulosic consortia NDMC-1, indicating its significance in hydrolyzing 700 Ann Microbiol (2019) 69:695–711

a rice straw rice husk wheat straw wheat husk saw dust corn stover 3000 grass straw filter paper newspaper Sucarcane bagasse 2500 )l/gm(DOCelbuloS

2000

1500

1000

500

0 0 122436486072 Time (Hour)

b 1800 1600 1400 )l/gm(ragusgnicudeR 1200 1000 800 600 400 200 0

Agro waste material Fig. 1 Hydrolysis of diverse cellulosic waste materials using consortium straw, wheat husk, saw dust, corn stover, grass straw, filter paper, and NDMC-1. a Changes in the concentration of sCOD and b concentration newspaper. Hydrolysis was performed at 37 °C and 120 rpm for 72 h of reducing sugar generated on hydrolyzing rice straw, rice husk, wheat lignocellulosic biomass. These results are supporting the earlier variant. The observed rise in sCOD concentration reflects the reports on enhanced cellulase production by mixed/co-cultures hydrolysis of insoluble organic components of SB into soluble (Zuroff et al. 2013; Saini et al. 2016). fermentable sugars. After 48 h, the subsequent decrease in The treatment of SB using consortia caused significant rise sCOD of hydrolysate was observed. Hydrolysis of SB of sCOD in the hydrolysate from 609 to 2589 mg/l and in pure expressed as a function of change in sCODs and cellulase isolate (from 478 to 665 to 1376–2054 mg/l) after 48 h of production followed similar pattern for both, pure cultures pretreatment with no significant change in sCOD of control and the consortia as depicted in Fig. 3a. Several researchers Ann Microbiol (2019) 69:695–711 701 a )lm/U(ytivitcaemyznE 1.2 12 hour 24 hour 36 hour 48 hour 1 0.8 0.6 0.4 0.2 0

b 0.5 0.45 12 hour 24 hour 36 hour 48 hour )lm/U(ytivitcaemyznE 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

)lm/U(ytivitc 12 hour 24 hour 36 hour 48 hour 1.2 1 0.8

aemyznE 0.6 0.4 0.2 0

Fig. 2 Hydrolysis of SB (2% w/v) by individual strains P. polymyxa endoglucanase. b Avicelase/exoglucanase. c β-Glucosidase activity after ND24, P. polymyxa ND25, B. subtilis ND23, M. arborescens ND21 48 h of incubation at at 37 °C and pH 7 and E. cloacae ND22 and consortia NDMC-1. a CMCase/ 702 Ann Microbiol (2019) 69:695–711

Fig. 3 a Changes in the a 3000 concentration of sCOD during P. polymyxa ND24 hydrolysis of SB (2% w/v)by P. polymyxa ND25 individual strains P. p oly my xa B. subtilis ND 23 2500 ND24, P. polymyxa ND25, E. cloacae ND 22

B. subtilis ND23, M. arborescens )l/gm(DOCelbuloS M. arborescence ND 21 Consortia ND21 and E. cloacae ND22, and 2000 consortiaNDMC-1atdifferent Blank time intervals for 72 h at 37 °C and pH 7. b Biogas production 1500 using hydrolyzate derived after treating SB by individual strains P. polymyxa ND24, P. polymyxa 1000 ND25, B. subtilis ND23, M. arborescens ND21, E. cloacae ND22, and consortia NDMC-1 500 for 96 h 0 0 122436486072 Time (hour) b P. polymyxa ND24 P. polymyxa ND25 B. subtilis ND23 60 M. arborescence ND 21 E. cloacae ND 22 Consortia ) lm(emulovsagyliaD 50 Unhydrolyzed SB control Blank (water+ sludge) 40

0 0 24487296 Time (hour) have formerly reported the increase degradation of natural HPLC after 24 and 48 h of incubation (Table 1). Analyses re- cellulosic material using mixtures of different microorgan- vealed the presence of glucose, cellobiose, and cellotriose in the isms. Efficient cellulose hydrolyzing microbial consortia hydrolysate. The highest amount of reducing sugar 1295 mg/l, XDC-2 was developed from soil samples ameliorated with comprising 59.8%, 8.23%, and 6.16% of glucose, cellobiose, and composted agricultural waste by Guo et al. (2010). Yuan cellotriose was obtained using consortia NDMC-1. For the indi- et al. (2014) reported a rapid increase in the concentration of vidual member, reducing sugars yields varied from 240 to sCOD during 4 days of hydrolysis of corn stalk while using 578 mg/l with glucose accounting for more than 37% of total the consortium, MC1. In this study, a similar trend in rise of reducing sugar. Among individual member, maximum glucose sCOD was observed during the first 48 h of the culture. The production 578.34 and 533.23 mg/l were observed in increased sCOD concentration suggests the high cellulolytic P. polym yxa ND24 and P. polym yxa ND25, respectively, which activity of the consortia. Results indicate that synergy and were nearly half the amount of glucose produced by the consor- cooperation among selected isolates enhance their degradation tium NDMC-1. The increasing content of glucose and almost abilities and is relatively more efficient than pure cultures. constant concentration of cellobiose and cellotriose in the hydrolysate confirms the simultaneous action of cellobiohydrolase and Estimation of reducing sugars through HPLC β -glucosidase. However, In case of consortium NDMC-1, the low increase in glucose concentration at 48 h and high cellobiose Sugars released during SB hydrolysis with consortia NDMC-1 and cellotriose content is probably due to the inhibitory action of and its individual isolates were identified and quantified by glucose on β-glucosidase. Similar considerably reduction of Ann Microbiol (2019) 69:695–711 703

fermentable sugars yields due to inhibition of β-glucosidases (EC 3.2.1.21) by glucose, endoglucanases (EC 3.2.1.4), and cellobiohydrolases (EC 3.2.1.91) by cellobiose and glucose was also reported earlier (Miao et al. 2012).

Enhanced biogas production using consortium Total reducing sugar 24 h 48 h NDMC-1 pretreated SB as a substrate

Hydrolysis of SB using consortia and pure culture was reflected through elevated levels of cellulase activity and bax NH2 column operated at 35 °C with sCOD. However, high sCOD concentration does not neces-

48 h sarily signify efficient generation of biomethane, as certain by-products may inhibit methanogenic archaea. Therefore, it is essential to determine the effect of isolates and consortia on

iose SB hydrolyzate which has a direct impact on biogas production. Thus, serum bottle experiments were carried out in an- Cellotr 24 h aerobic condition for 96 h. The SB hydrolyzate derived after 48 h was the sole source of organic compounds to carry out anaerobic digestion. The biogas production for 96 h is shown in Fig. 3b. The biogas production was enhanced using previ- (3.96) 16.04 (2.18) 24.04 (2.46) 732.75 977.5 (2.98) 9.69 (1.75) 20.41 (2.42) 554.25 843.5

nd SB. HPLC was performed using zor ously hydrolyzed SB as a substrate for anaerobic digestion, in comparison to controls containing inoculum and substrate 48 h with 2% bagasse. Maximum biogas production was detected within 48 h and reached 86 ml, 81 ml, 65 ml, 69 ml, 42 ml, and 108 ml for P. polymyxa ND24, P. polymyxa ND25, B. subtilis ND23, M. arborescence ND21, E. cloacae ND22, and con- Cellobiose 24 h sortia NDMC-1, respectively. The decrease was observed in biogas production for all the hydrolysates after 48 h. Pretreatment of SB with consortia NDMC-1 resulted in highest volume (186 ml) of biogas production. Similarly, 145 ml, 139 ml, 110 ml, 105 ml, and 76 ml biogas production 41 (59.8) 19.34 (1.96) 106.58 (8.23) 15.02 (1.53) 79.772 (6.16) 984.5 1295

1 were observed when SB hydrolysate derived from −

48 h P. polymyxa ND24, P. polymyxa ND25, M. arborescence ND21, B. subtilis ND23, and E. cloacae ND22, respectively, were used as the substrate. Results indicate 5.5-fold increase in biogas production using consortium-treated SB which was higher than 2.2 to 4.0-fold increase while employing 378 (51.6) 533.23 (54.55) 17.44 (2.38) 38.71 123 (25.58) 240.13 (37.23) 8.06 (1.67) 11.22 (1.74) 27.83 (5.8) 50.70 (7.86) 480.75 645 170 (30.15) 301.16 (41.03) 7.72 (1.3) 10.64 (1.45)monoisolate. 35.67 (6.32) 62.023 (8.45) 563.75 The average 734 methane percentage was 24 h Glucose Concentration (mg/l) found to be 40–55% of the total biogas produced by the individual isolates and consortia. The methane con- d cellotriose concentration generated during enzymatic hydrolysis of CMC a tent was found to be 45 to 54% when consortia

ND21 219 (41.32) 392.64 (46.55)pretreated 14.8 (2.67) 25.14 SB was used as a substrate and 43 to 52%

ND24ND25 411.5 (54.50) 578.34 (57.32) 16.32 (2.16) 34.51 (3.42)when 19.84 (2.63) SBpretreated 31.78 (3.15) 755.5 using 1009 monoisolates were used as a ND22 ND23 substrate. These resulted in corresponding methane yield of100ml,75ml,73ml,54ml,52ml,and38mlfor consortia NDMC-1, P. polymyxa ND24, P. polymyxa P. polymyxa P. polymyxa M. arborescence E. cloacae B. subtilis Consortium NDMC-1 554.25 (56.3) 774. Organisms ND25, M. arborescence ND21, B. subtilis ND23, and E. cloacae ND22, respectively (Table 2), while insignif- icant (2–5%) increase in biogas production was noted for the control. Increased biogas production and meth- Estimation of glucose, cellobiose, an ane yield using consortia treated SB indicates the potential of consortia NDMC-1 for efficient hydrolysis of Sugarcane bagasse Table 1 Substrate acetonitrile and water (70:30) as the mobile phase at a flow rate of 0.5 ml min cellulosic biomass. 704 Ann Microbiol (2019) 69:695–711

Table 2 Biogas and methane production by anaerobic digestion Inoculum for SB hydrolysis Cumulative biogas Methane (%) Corresponding methane of hydrolyzate from P. polymyxa volume (ml) yield (ml) ND24, P. polymyxa ND25, B. subtilis ND23, P. polymyxa ND24 145 44–52 75 M. arborescence ND21 and P. polymyxa ND25 139 44–52 73 E. cloacae ND22, and consortia B. subtilis ND23 105 43–48 52 NDMC-1 M. arborescence ND21 110 44–47 54 E. cloacae ND22 76 44–49 38 Consortia NDMC-1 186 45–54 110

Genome analysis of members comprising consortia particular relationship are detailed in Table 3. GH family which NDMC-1 for CAZymes was absent in one isolate is complemented by other isolate in consortium NDMC-1, thus forming a complete system that acts Draft genomic sequencing and analysis of M. arborescens in synergy for efficient biomass hydrolysis. Based on this, ND21, E. cloacae ND22, B. subtilis ND23, P. polymyxa P. polymyxa can be anticipated as driver organism while ND24, and P. polymyxa ND25 present in the consortium was M. arborescence ND21, E. cloacae ND22, and B. subtilis performed for further characterization of complex lignocellulose- ND23 play auxiliary role in this consortium for lignocellulose hydrolyzing machinery. Genome-centric approach emphasizes decomposition. Surface layer homology domains were annotated on the analysis of complete or near-complete genomes to provide only in Paenibacillus sp. and absent in all other bacterial ge- more extensive data for the presence and abundance of entire nomes. The richness of various CAZymes in M. arborescens gene repertories in an organism (Jovanovic et al. 2009), thus ND21, E. cloacae ND22, B. subtilis ND23, P. polymyxa allowing thorough understanding of microbial physiologies. ND24, and P. polymyxa ND25 reflects its potential to utilize or Outcomes of NCBI prokaryotic genome annotation pipeline degrade diverse types of organic compound such as cellulose, are summarized in Table S5. Results from dbCAN algorithm hemicellulose, pectin, and lignin. Paenibacillus polymyxa ND24 are summarized in Fig. 4a. Total 1258 different CAZyme genes, (115 of total CAZyme) and P. polym yxa ND25 (140 of total covering all six CAZy families, were annotated in the genomes, CAZyme) also stands out for being the richest in diversity and comprising the following: 37.44% GHs, 21.06% GTs, 13.91% abundance of GHs compared to the other genomes. The other CEs, 2.94% PLs, 5.80% AAs, and 16.21% CBM. dbCAN sys- three species contribute collectively with 20 GH families not tem annotated around 185, 157, 200, 327, and 389 carbohydrate encoded in the P. polymyxa genome (GH77, GH38, GH20, utilizing enzyme in M. arborescens ND21, E. cloacae ND22, GH73, GH114, GH78, GH9, GH125, GH92, GH15, GH71, B. subtilis ND23, P. polymyxa ND24, and P. polym yxa ND25 GH103, GH19, GH80, GH8, GH37, GH39, GH102, GH126, genome, respectively, which is 3.8–8% of the total CDS encoded GH45). Overall, 16 of the GH families (GH8, GH9, GH15, by the genome. Distribution of various classes of CAZy seems to GH19, GH37, GH39, GH45, GH71, GH78, GH80, GH92, be in proportionate number, i.e., glycosides hydrolases (GH), GH102, GH103, GH114, GH125, GH126) are specific, occur- 30.5 to 45.4%; carbohydrate esterases (CE),11.5 to 19%; poly- ring only in one of the three genomes other than P. polym yxa. saccharides lyases (PL), 2.0 to 3.5%; glycosyltransferases (GT), Among these specific GH families: GH9 and GH78 (in 18.5 to 24.5%; and carbohydrate-binding modules (CBM), 12.1 M. arborescence ND21), GH8 and GH39 (E. cloacae ND22), to 19%, in annotated genomes. P. polymyxa ND24 (327 CDSs, andGH45(B. subtilis ND23) are worth noting, because it in- 26%) and P. polymyxa ND25 (389 CDSs, 30.92%) genome cludes enzymes involved in cellulose and hemicellulose decom- shows the highest number of CDSs annotated as CAZymes. position (Lombard et al. 2013). Venn diagram (Fig. 4b) was constructed for the comparison of Based on the functional role of the enzymes, such as CAZyme within P. polymyxa ND24, P. polymyxa ND25, hydrolysis of oligosaccharide, cellulose, and hemicellu- B. subtilis ND23, M. arborescens ND21, and E. cloacae loses, GH family can be further classified. A total of 45 ND22. It shows the number of proteins shared or unique within different GH families comprising cellulase and a particular relationship. As evident, 23 CAZyme (GH23, GH3, hemicellulase enzymes were found, among which major- GH5,CBM50,CE1,CE9,CE3,GH43,GT35,GT51,GT2, ity included those playing role as oligosaccharide CBM48, GT4, GH109, GT28, GH13, CE4, GH65, GT5, degrading enzymes (21 families) followed by CE10, GH1, GT26, GH18) are common in all the five isolates, debranching enzymes (9 families), hemicellulases (6 whereas 1, 5, 9, 15, and 14 CAZyme were exclusively present in families), and cellulases (6families) (Fig. 5). Out of P. polymyxa ND24, P. polymyxa ND25, B. subtilis ND23, these, oligosaccharide degrading GH13, GH1, GH43, M. arborescens ND21, and E. cloacae ND22, respectively. The GH3, and GH18 and debranching enzyme family GH number and kind of CAZyme shared or unique within a 23 and cellulase family GH5 were most highly Ann Microbiol (2019) 69:695–711 705

Fig. 4 a Annotation of carbohydrate active enzyme (CAZyme) using dbCAN algorithm from dbCAN pipeline. b CAZyme comparison of P. polymyxa ND24, P. polymyxa ND25, B. subtilis ND23, M. arborescens ND21, and E. cloacae ND22. The Venn diagram shows the number of proteins shared or unique within a particular relationship

represented. The abundance of the GH families GHs family mainly comprised of endo-1,4-β- encoding gene varied among the indiviadual memebrs glucanase; exo-1,4-β-glucanase; and β-glucosidase that of consortia. The cellulases, GH6 and GH9, were exclu- plays crucial role in the decomposition of cellulosic sively present in M. arborescens ND21, and GH45 component of lignocellulose. Collectively, 33 cellulase remained restricted to B. subtilis ND23 although GH8 genes were identified in the genome of five isolates, family hemicellulase was only detected in E. cloacae comprising of 16 β-glucosidases (8 from GH1 and 8 ND22 (Fig. 5). Likewise, various GH families absent from GH3 family), 12 endoglucanases (GH5), and three in one isolate are complemented by other isolate in exoglucanases (3 from GH6 and two from GH48 fami- consortium NDMC-1, thus forming a complete system ly). Maximum numbers of cellulase were annotated in that acts in synergy for efficient biomass hydrolysis. P. polymyxa species. Paenibacillus polymyxa ND25 706 Ann Microbiol (2019) 69:695–711

Table 3 CAZyme comparison of P. polymyxa ND24, P. polymyxa Names Total Elements ND25, B. subtilis ND23, M. arborescens ND21, and B. subtilis ND23 23 GH23 GH3 GH5 CBM50 CE1 CE9 CE3 GH43 GT35 GT51 GT2 CBM48 E. cloacae ND22.The table shows E. cloacae ND22 GT4 GH109 GT28 GH13 CE4 GH65 GT5 CE10 GH1 GT26 GH18 the CAZyme family shared or M. arborescence ND21 unique within a particular P. polymyxa ND24 P. polymyxa ND25 relationship B. subtilis ND23 1 AA6 E. cloacae ND22 M. arborescence ND21 P. polymyxa ND24 E. cloacae ND22 3 GH36 GH2 GH88 M. arborescence ND21 P. polymyxa ND24 P. polymyxa ND25 B. subtilis ND23 8 GH51 PL11 GH42 GH30 GH53 CE12 CE7 PL9 M. arborescence ND21 P. polymyxa ND24 P. polymyxa ND25 B. subtilis ND23 4 CBM34 GH105 GH4 GH32 E. cloacae ND22 P. polymyxa ND24 P. polymyxa ND25 B. subtilis ND23 2 GH76 AA7 M. arborescence ND21 P. polymyxa ND25 M. arborescence ND21 12 CBM4 GH127 CBM40 CBM13 GH6 CBM56 GH95 CBM35 GH10 CBM32 PL12 GH27 P. polymyxa ND24 P. polymyxa ND25 E. cloacae ND22 4 GH31 CE8 GH28 CBM44 P. polymyxa ND24 P. polymyxa ND25 B. subtilis ND23 15 CBM80 GH16 GT83 CBM6 CBM66 GH68 GH11 CBM63 GT1 GH26 PL1 CBM37 P. polymyxa ND24 CE14 CBM3 CBM26 P. polymyxa ND25 E. cloacae ND22 7 AA2 AA3 GH77 GH38 GH20 GT20 GT30 M. arborescence ND21 M. arborescence ND21 3 CBM61 GH35 CBM22 P. polymyxa ND25 B. subtilis ND23 3 GT19 GH73 PL22 E. cloacae ND22 E. cloacae ND22 1 GH24 P. polymyxa ND25 B. subtilis ND23 1 GH46 P. polymyxa ND24 B. subtilis ND23 1 PL3 P. polymyxa ND25 P. polymyxa ND24 22 CBM46 CBM36 GH14 PL10 CBM16 GH112 GH48 GH52 CBM54 CBM41 GT81 P. polymyxa ND25 CBM59 GH74 GH44 CE2 GT17 GH94 GH130 GH25 GH84 GH67 SLH M. arborescence ND21 15 GH114 GH78 GT27 CBM9 GH9 GH125 GH92 GT87 GH15 GH71 PL5 GT94 GT39 GT76 CBM67 E. cloacae ND22 14 GH103 GT25 CBM5 GT56 GH19 CE11 CBM73 GH80 GH8 GT9 AA10 GH37 GH39 GH102 B. subtilis ND23 9 CE6 CBM68 GH126 GH45 GT46 AA4 GT44 GT62 GT8 P. polymyxa ND24 1 CBM51 P. polymyxa ND25 5 GH99 AA12 GT41 GH115 GT57 contains one extra endoglucanase and β-glucosidases hemicellulose hydrolysis were also annotated in the ge- when compared to P. polymyxa ND24. Because of the nome comprising 9 1,4-β-xylanase, 2 α-glucuronidase, structural complexity of lignocellulose, sheer number of 7 β-xylosidase, 10 α-N-arabinofuranosidase, 4 α- enzymes is required in addition to cellulases to hydro- arabinosidase, 15 α-galactosidase, 2 β-mannanase, 4 lyze plant biomass into fermentable oligosaccharide. β-mannosidase, 3 1,3-β-glucanase, 2 xyloglucanase, Hemicellulase along with other polysaccharides and 1 β-glycosidase were also annotated. Moreover, degrading enzymes will be of great commercial impor- 48 esterase genes, including 4 acetyl esterase, 12 acyl- tance(Bohraetal.2018a). Additionally, 59 genes for CoA thioesterase, 9 carboxylesterase, 23 esterases, and Ann Microbiol (2019) 69:695–711 707

M. GHs E. Cloacae B. subtilis P. polymyxa P. Polymyxa Enzyme arborescence family ND22 ND23 ND24 ND25 ND21 Cellulase GH5 2.38 2.82 1.64 3.48 4.29 GH6 2.38 0.00 0.00 0.87 0.71 GH9 1.19 0.00 0.00 0.00 0.00 GH44 0.00 0.00 0.00 0.87 0.71 GH45 0.00 0.00 1.64 0.00 0.00 GH48 0.00 0.00 0.00 0.87 0.71 Endohemicellulase GH8 0.00 1.41 0.00 0.00 0.00 GH10 2.38 0.00 0.00 1.74 1.43 GH11 0.00 0.00 3.28 0.87 0.71 GH26 0.00 0.00 1.64 4.35 4.29 GH28 0.00 2.82 0.00 0.87 0.71 GH53 3.57 0.00 1.64 0.87 0.71 Xyloglucanases GH16 0.00 0.00 1.64 1.74 0.71 GH74 0.00 0.00 0.00 6.09 7.14 GH94 0.00 0.00 0.00 0.87 1.43 Debranching GH23 1.19 8.45 6.56 1.74 1.43 enzymes GH36 1.19 1.41 0.00 3.48 2.86 GH51 4.76 0.00 3.28 2.61 2.14 GH67 0.00 0.00 0.00 1.74 0.71 GH77 1.19 1.41 0.00 0.00 0.00 GH78 3.57 0.00 0.00 0.00 0.00 GH84 0.00 0.00 0.00 0.87 0.71 GH103 0.00 1.41 0.00 0.00 0.00 GH127 2.38 0.00 0.00 0.87 1.43 Oligosaccharide GH1 4.76 8.45 8.20 6.09 7.14 degrading GH2 4.76 1.41 0.00 1.74 2.14 enzymes GH3 4.76 4.23 1.64 1.74 2.86 GH4 0.00 4.23 6.56 1.74 2.14 GH13 15.48 15.49 13.11 7.83 6.43 GH18 2.38 2.82 4.92 2.61 2.14 GH20 1.19 1.41 0.00 0.00 0.00 GH27 2.38 0.00 0.00 1.74 1.43 GH30 3.57 0.00 1.64 0.87 1.43 GH31 0.00 5.63 0.00 0.87 0.71 GH32 0.00 1.41 6.56 6.09 5.00 GH35 2.38 0.00 0.00 0.00 0.71 GH36 1.19 1.41 0.00 3.48 2.86 GH38 2.38 1.41 0.00 0.00 0.00 GH39 0.00 1.41 0.00 0.00 0.00 GH42 2.38 0.00 3.28 2.61 2.14 GH43 3.57 2.82 6.56 6.96 6.43 GH52 0.00 0.00 0.00 0.87 0.71 GH92 1.19 0.00 0.00 0.00 0.00 GH95 1.19 0.00 0.00 0.87 0.71 GH130 0.00 0.00 0.00 1.74 1.43

Fig. 5 Distribution and abundance of predicted glycoside hydrolase enzymes in P. polymyxa ND24, P. polymyxa ND25, B. subtilis ND23, M. arborescens ND21, and E. cloacae ND22 that target cellulose and hemicellulose in plant biomass 708 Table 4 Diversity and abundance of potential lignocellulolytic enzymes predicted in the M. arborescens ND21, E. cloacae ND22, B. subtilis ND23, P. polymyxa ND24, and P. polymyxa ND2 genome using Blast NCBI annotation and Pfam analysis

Enzymes Specific activity Locus tag

P. polymyxa ND 24 P. polymyxa ND 25 B. subtilis ND23 E. cloacae ND22 M. arborescence ND21

Cellulose Endoglucanases A9P44_RS14040 A9Z39_RS03585 A9D36_RS07230 A9Z41_RS05110 A9Z40_RS10670 hydrolyzing enzyme A9P44_RS05395 A9Z39_RS15650 –– – A9P44_RS11815 A9Z39_RS21070 –– – A9P44_RS15720 A9Z39_RS25850 –– – – A9Z39_RS23700 –– – Exoglucanases A9P44_RS14680 A9Z39_RS08400 –– A9Z40_RS04120 A9P44_RS02675 A9Z39_RS20820 –– – β-Glucosidases A9P44_RS16545 A9Z39_RS00395 A9D36_RS15400 A9Z41_RS10470 A9Z40_RS10545 A9P44_RS18720 A9Z39_RS02380 A9D36_RS15790 A9Z41_RS15710 A9Z40_RS09965 A9P44_RS02365 A9Z39_RS08065 A9D36_RS16195 – A9Z40_RS02060 A9P44_RS03290 A9Z39_RS17230 –– – – A9Z39_RS19335 –– – Hemicellulose 1,4-β-Xylanase A9P44_RS17000 A9Z39_RS10975 A9D36_RS06615 A9Z41_RS07850 A9Z40_RS11675 hydrolyzing enzyme A9P44_RS19465 A9Z39_RS11750 A9D36_RS12750 –– – A9Z39_RS19790 –– – α-Glucuronidase A9P44_RS20000 A9Z39_RS11100 –– – β-Xylosidase A9P44_RS00040 A9Z39_RS11095 –– – A9P44_RS19895 A9Z39_RS11205 –– A9Z40_RS03110 A9P44_RS20005 A9Z39_RS21905 –– – α-N-arabinofuranosidase A9P44_RS03320 A9Z39_RS02435 A9D36_RS09320 A9Z41_RS06385 A9Z40_RS01405 A9P44_RS07405 A9Z39_RS12365 A9D36_RS20370 –– A9P44_RS09130 A9Z39_RS15295 –– – α-Arabinosidase A9P44_RS04795 A9Z39_RS09345 –– – A9P44_RS07410 A9Z39_RS15300 –– – α-Galactosidase A9P44_RS02680 A9Z39_RS01995 A9D36_RS00670 A9Z41_RS20135 A9Z40_RS01435 A9P44_RS05320 A9Z39_RS02585 A9D36_RS21180 –– A9P44_RS11275 A9Z39_RS08405 –– – A9P44_RS11990 A9Z39_RS08545 –– – A9P44_RS15590 A9Z39_RS23940 –– – A9Z39_RS25470 –– – β-Mannanase A9P44_RS11960 A9Z39_RS25500 –– – β-Mannosidase A9P44_RS21615 A9Z39_RS01360 A9D36_RS12040 –– A9Z39_RS05635 –– – 1,3-β-Glucanase A9P44_RS12905 A9Z39_RS02725 –– – 69:695 (2019) Microbiol Ann A9P44_RS21805 –– – Xyloglucanase A9P44_RS15215 A9Z39_RS24390 –– – Acetyl esterase A9P44_RS12890 A9Z39_RS00060 –– – A9P44_RS20250 A9Z39_RS02710 –– – β-Glycosidase –– A9Z40_RS10310 Acyl-CoA thioesterase A9P44_RS00045 A9Z39_RS21460 A9D36_RS01885 A9Z41_RS18115 A9Z40_RS13475 A9P44_RS08305 A9Z39_RS21910 A9D36_RS09275 A9Z41_RS19145 A9Z40_RS13470 –––A9Z41_RS19375 A9Z40_RS04005

Carboxylesterase A9P44_RS01665 A9Z39_RS07380 A9D36_RS18400 A9Z41_RS13930 A9Z40_RS10470 – A9P44_RS04765 A9Z39_RS09375 A9D36_RS18810 – A9Z40_RS00375 711 Ann Microbiol (2019) 69:695–711 709

2 acetyl xylan esterases responsible for complex carbo- ND21 hydrate degradation have also been annotated. The distribution and abundance of all the enzymes annotated for lignocellulose decomposition are presented in Table 4. Presence of both types of plant cell wall-deconstructing enzyme is crucial for in hydrolyzing lignocellulosic biomass. A9Z40_RS04530 A9Z40_RS03640 A9Z40_RS06730 – – A9Z40_RS01730 M. arborescence Previous studies have reported that the diverse enzymes from various organisms work in synergy to improve lignocellulose hydrolysis and generation of simple sugars (Takasuka et al. 2013; Hiras et al. 2016). The presence of many other GH ND22 enzymes, combined with these findings in the genome, de- picts the great potential consortium NDMC-1 for hydrolysis of diverse plant biomass. A9Z41_RS09340 A9Z41_RS08490 A9Z41_RS02290 – A9Z41_RS18300 A9Z41_RS14575 E. cloacae

Conclusion

ND23 An efficient lignocellulolytic microbial consortium NDMC-1 comprising taxonomically diverse members has been designed in this study. The consortium NDMC-1 efficiently hy- – A9D36_RS15885 – A9D36_RS02945 B. subtilis A9D36_RS02550 drolyzed the SB cellulosic biomass generating 1295 mg/l reducing sugar, comprising 59.8%, 8.23%, and 6.16% of glucose, cellobiose, and cellotriose. The hydrolysis increased sCOD from 609 to 2589 mg l−1and leads to 5.5-fold enhance-

ND 25 ments in biogas production. Genome annotation reveals the presence of diverse CAZymes in NDMC-1 whose synergistic action supplements the capabilities of each individual isolates. The consortium was observed to be efficient decomposer of A9Z39_RS21910 A9Z39_RS20290 A9Z39_RS06675 A9Z39_RS05525 A9Z39_RS01310 P. polymyxa rice straw, rice husk, wheat straw, wheat husk, saw dust, corn stover, grass straw, filter paper, and newspaper. We believe that our designed consortium NDMC-1 with 471 GH family enzymes proposes effective utilization of cellulose-rich ba-

ND 24 gasse and is an important contribution to the research on hydrolysis of lignocellulosic biomass for further applications, especially the production of value added biobased products. –– A9D36_RS13915 A9P44_RS21565 A9P44_RS17830 A9P44_RS14865 A9P44_RS10805 P. polymyxa A9P44_RS07030 Acknowledgements Miss Varsha Bohra acknowledges Department of Science and Technology (DST) of India for awarding Senior Research Fellowship (SRF) for carrying out the work.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest. Acetylxylan esterase Esterase

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