Elsevier

Renewable Energy

Volume 152, June 2020, Pages 653-663
Renewable Energy

Sugarcane bagasse saccharification using Aspergillus tubingensis enzymatic cocktail for 2G bio-ethanol production

https://doi.org/10.1016/j.renene.2020.01.063Get rights and content

Highlights

  • Zymography of Aspergillus tubingensis enzymatic cocktail displayed multiplicity of cellulases and hemicellulases.

  • Optimized saccharification of sugarcane bagasse using enzyme cocktail lead to release of 16.5% of fermentable sugars.

  • Hydrolysate containing glucose-xylose was fermented to bioethanol (0.415 g/g) by Candida shehatae.

Abstract

In order to meet the increasing demand of 2G ethanol, sugarcane bagasse (SCB), a low cost by-product of sugarcane industry, can be utilized. However, cost-effective degradation of SCB into constituent fermentable sugars and subsequent ethanolic fermentation remains to be a far-fetched goal. In the present study, cellulase and hemicellulase cocktail produced by Aspergillus tubingensis NKBP-55 was applied for SCB hydrolysis under parametrically optimized conditions. Enzyme treatment (SCB: 7%, enzyme load: 1U = 1 mg) resulted in the liberation of fermentable mono-sugars 20 mg/mL (glucose, xylose and arabinose). Powder X-ray diffraction (PXRD) analysis revealed increase in the crystallinity index from 58.4 to 63.9% as a result of degradation of hemicellulose and amorphous cellulose. SCB hydrolysate was fermented to ethanol (0.415 g/g) by glucose and xylose-fermenting yeast, Candida shehatae NCIM 3501. Maximum ethanol concentration (15.54 ± 0.3 g/L) with 77.9% fermentation efficiency and 0.161 g/L/h productivity was achieved. The present study revealed that A. tubingensis enzyme cocktail can be used for efficient SCB hydrolysis and conversion of resulting hydrolysate into bioethanol using C. shehatae.

Introduction

In recent times, bioconversion of plant biomass into 2G bioethanol, a substitute to fossil fuel, has emerged to the fore [1]. By-products of agro-industry such as sugarcane bagasse (SCB) and rice straw (RS) are potential candidates for generation of alternative renewable biofuel such as 2G bioethanol as they are the source of large quantities of rapidly utilizable sugars [2]. A critical step in the bioconversion of lignocellulosic substrate to bioethanol is the use of expensive enzymes in the liberation of simple sugars from these complex polymers [3]. At present, the effective cost of 2G bioethanol from lignocellulosic biomass is not competitive with the price of petroleum-derived liquids because of cost-intensive bioprocesses and inefficient conversion technology [1]. Cellulase is the major enzyme that plays a pivotal role in biomass degradation. Efficient strategies for production and sugar generation from biomass are required to make the process economically viable [3,4]. A comprehensive approach to address above issues would require (1) a potential cellulase-hemicellulase cocktail producing strain (2) cheap and easy method of enzyme production (3) parametrically optimized process of SCB bioconversion and (4) bioethanol generation using hexose and pentose fermenting yeast. SCB is one of the largest agro-industrial wastes produced worldwide with India producing more than 350 million metric tons annually [5]. It is a continuously available by-product of sugarcane industry. SCB is composed of 50% cellulosic and 30% hemicellulosic content with better porosity for enzyme action. It has low ash and lignin content as compared to other agricultural residues such as wheat straw and rice straw [6]. Alkali pretreatment of SCB decreases lignin content thereby increasing porosity that renders the complex cellulose and hemicellulose more amenable to enzymatic hydrolysis [7]. Alkali pretreatment also cleaves glycosidic and ester side chains of various polymeric complexes leading to hemicellulose salvation and decrystallization and swelling of cellulose [8]. Owing to their capability to secrete massive spectra of hydrolases, Trichoderma spp. have been extensively used as a source of cellulolytic enzymes. Potential hyper-cellulase producer Trichoderma reesei and its mutants have been used predominantly in industrial production of cellulases [9]. More recently, Aspergillus spp. such as A. niger [10,11], A. fumigatus [12], A. flavus [13] and A. nidulans [14] are reported as efficient producers of cellulases. Aspergillus tubingensis NKBP-55 has been shown to produce cellulases and hemicellulases on copra meal [15]. Response surface methodology (RSM) is a useful statistical modeling method used for obtaining optimal interplay among process variables. Reports on statistical optimization strategy for enhanced cellulase production [16,17] and enzymatic saccharification of lignocelluloses [18,19] indicate its relevance.

In the present report, cellulolytic and hemicellulolytic enzyme profile produced by A. tubingensis NKBP-55 on various lignocellulosic substrates was studied in terms of titres and types. RSM was applied to optimize process variables for maximizing cellulase production in solid-state fermentation (SSF) using copra meal as substrate. Sugar generation by SCB hydrolysis using enzymatic cocktail (cellulase and hemicellulase) was also optimized. Generation of bioethanol from fermentable sugars obtained from SCB was demonstrated using a glucose-xylose fermenting yeast, Candida shehatae NCIM 3501.

Section snippets

Chemicals

4-nitrophenyl β-d-glucopyranoside (pNPG), 4-nitrophenyl α-d-galactopyranoside (pNPGa),4-nitrophenyl β-d-xylopyranoside (pNPX), 4-nitrophenyl β-d-mannopyranoside (pNPM),4-methylumbelliferyl β-d-glucopyranoside (4-MUG), 4-methylumbelliferyl β-d- galactopyranoside (4-MUGa), birchwood xylan, locust bean gum (LBG), carboxymethyl cellulose (CMC), Sigmacell, Avicel, Solka-Floc, guar gum, 7-amino-1, 3-naphthalene disulfonic acid monopotassium salt monohydrate (ANDS), sodium cyanoborohydride, 3,5

Chemical composition of sugarcane bagasse

Analysis of chemical composition of raw SCB based on NREL lab method [20] showed presence of cellulose (36.4%), xylan (4.37%), arabinan (4.42%), total lignin 26.35 (24.86% insoluble, 1.49% acid soluble) and ash (1.55%) excluding extractives. The alkali pretreated SCB showed relatively high amounts of cellulose (57.7%), xylan (5.66%) and arabinan (5.07%) while significant decrease in the total lignin content (15.98%) and ash (1.53%) was noticed. In case of enzyme treated SCB, decrement in

Conclusion

The cellulolytic and hemicellulolytic spectrum of A. tubingensis produced on CM is effective in the generation of fermentable sugars (glucose and xylose) from SCB. The enzyme cocktail hydrolyzed different agro-wastes efficiently. Central composite design was found promising for achieving maximum cellulase production and SCB saccharification. Cooperative action of enzymes resulted in the increased saccharification efficiency without any extraneous supplementation of commercial cellulases or

Ethics approval and consent to participate

Not applicable.

CRediT authorship contribution statement

Bhanu Pratap Prajapati: Formal analysis, Writing - original draft, Writing - review & editing. Uttam Kumar Jana: Formal analysis, Writing - original draft, Writing - review & editing. Rahul Kumar Suryawanshi: Formal analysis, Writing - original draft, Writing - review & editing. Naveen Kango: Writing - original draft, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Authors thank sophisticated instrumentation center (SIC), Dr. Harisingh Gour V.V., Sagar for SEM and other sophisticated instrumentation facilities and financial support by DST-PURSE (II). Author BPP and RKS are grateful to the University Grants Commission (UGC), New Delhi for financial support as NFOBC fellowship. Author UKJ is grateful to ICMR, New Delhi for providing financial assistance as Senior Research Fellow (SRF).

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