Genomic Annotation and Validation of Bacterial Consortium NDMC-1 for Enhanced Degradation of Sugarcane Bagasse
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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 hydro- lytic 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 produc- tion. 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 cellulolyt- ic 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 hydro- lysis (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 cellu- losic 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 produc- ing 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