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The current study encompasses the development of a combined pretreatment strategy using deep eutectic solvent (DES) i.e. choline chloride (ChCl)/glycerol and ultrasonic waves for the digestibility of sugarcane bagasse (SCB). Pretreated biomass was saccharified by an enzyme cocktail produced in house from a novel fungus Aspergillus assiutensis VS34. Combined ChCl/glycerol and ultrasound pretreatment yielded higher sugars (276.8 mg/g biomass) than either of the individual pretreatments i.e. ChCl/glycerol (235.3 mg/g biomass) or ultrasound (174.5 mg/g biomass). Optimization of pretreatment process variables (biomass loading, sonication amplitude and pretreatment time) by Design of Experiment enhanced sugar yield by 1.12-fold. Sugar hydrolysate obtained was fermented by dual yeast cultures i.e. Saccharomyces cerevisiae and Pichia stipitis to get an ethanol yield of 142.09 mg/g biomass. To understand the molecular mechanism of combined DES/ultrasound pretreatment which led to eased hydrolysis, and enhanced sugar yield, the pretreated biomass was studied by SEM, FT-IR, XRD and 1H NMR analysis for various structural aberrations that made the biomass more amenable to enzymatic hydrolysis. Insights of underlying functional mechanisms of pretreatment may help in developing new/novel designer pretreatment approaches for proficient biomass conversion.

Sugarcane tops (SCT) may be an imperative lignocellulosic feedstock for production of renewable energy in view of ever-rising global energy demands. The current study presents the first ever report of combinative pretreatment of sugarcane tops biomass using ionic liquid (1-ethyl-3-methylimidazolium chloride) and alkali (ammonium carbonate) for effective and enhanced saccharification. The enzymatic hydrolysis of combinatorially pretreated SCT biomass resulted in a significantly higher reducing sugar yield (172.34 mg/g biomass) as compared to that obtained after standalone IL (85.9 mg/g biomass) or alkali pretreatment (102.6 mg/g biomass). The saccharification enzymes (cellulase/xylanase) used for hydrolysis of pretreated SCT biomass were in-house produced from an IL-tolerant, newly isolated fungus Penicillium chrysogenum VS4. Optimization of process variables for combined pretreatment was accomplished based on design of experiments, and enhanced reducing sugar yield was obtained. The experimental design for ascertaining optimal level of process variables, i.e., biomass loading (5.33%, w/w), temperature (100 °C), and time (4.50 h) was validated. Optimization of the process parameters resulted in 25.27% increased reducing sugar yield (215.89 mg/g biomass) as compared to that under unoptimized process. Ultrastructural analysis of SCT biomass after combined pretreatment was investigated by scanning electron microscopy and Fourier transform infrared spectroscopy which indicated that the biomass had undergone drastic alterations and substantial disintegration due to pretreatment. The current study highlights the potential of combinative pretreatment strategy for efficient conversion of SCT biomass. Such combined pretreatment approaches may be extended to other biomass feedstocks as well for developing successful biorefineries.

A novel consolidated bioprocess was designed for processing of Parthenium hysterophorus biomass based on ultrasound-assisted deep eutectic solvent (DES) pretreatment followed by in situ enzymatic saccharification and fermentation of sugars to ethanol-biofuel. Among various DESs examined, choline chloride/sorbitol (CCS, molar ratio 1:5) pretreated P. hysterophorus biomass yielded maximum sugar (148.54 mg/g biomass) upon enzymatic saccharification. Furthermore, combined ultrasonic irradiation and CCS pretreatment resulted in substantially enhanced sugar yield as compared with either of the standalone pretreatment. A central composite design was employed for the optimization of combined ultrasound and choline chloride/sorbitol pretreatment that further aided in achieving increased reducing sugar yield (25.72%). The pretreated biomass was examined by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and proton nuclear magnetic resonance (1H-NMR) analysis to elucidate the functional mechanisms of combinative pretreatment. Understanding of pretreatment mechanisms may help custom designing of novel strategies for the efficient refining of biomass.

This work aimed to study the hydrolysis of ionic liquid (IL) pretreated sugarcane tops (SCT) biomass with in-house developed IL-stable enzyme preparation, from a fungal isolate Aspergillus flavus PN3. Maximum reducing sugar yield (181.18 mg/g biomass) was obtained from tris (2-hydroxyethyl) methylammonium-methylsulfate ([TMA]MeSO4) pretreated biomass. Pretreatment parameters were optimized to attain enhanced sugar yield (1.57-fold). Functional mechanism of IL mediated pretreatment of SCT biomass was elucidated by SEM, XRD, FTIR and 1H NMR studies. Furthermore, nanobiocatalysts prepared by immobilization of enzyme preparation by covalent coupling on magnetic nanoparticles functionalized with amino-propyl triethoxysilane, were assessed for their hydrolytic efficacy and reusability. Nanobiocatalysts were examined by SEM and FTIR analysis for substantiation of immobilization. This is the first ever report of application of magnetic nanobiocatalysts for saccharification of IL-pretreated sugarcane tops biomass.

Biorefining of lignocellulosic biomass to fuels/chemicals has recently gained immense research momentum. Current study reports sequential pretreatment of sunflower stalk (SFS) biomass in a combinatorial regime involving alkali (NaOH) and ionic liquid 1-butyl-3-methyl imidazolium chloride. The pretreatment enhanced the enzymatic digestibility, and resulted in increased sugar yield (163.42 mg/g biomass) as compared to standalone pretreatment using alkali (97.38 mg/g biomass) or ionic liquid (79.6 mg/g biomass). Ultrastructural and morphological analysis (FTIR and SEM) of pretreated biomass showed that the combined ionic liquid and alkali pretreatment causes more drastic alterations in the biomass ultrastructure as compared to alone ionic liquid or alkali pretreatment. Thus, combined pretreatment led to ease of enzymatic saccharification and consequent increased sugar yield, and this observation was corroborated by physicochemical analysis of the pretreated biomass. The pretreated SFS biomass was subjected to consolidated bioprocessing for its direct conversion to bioethanol in a single vessel.

Lignocellulosic biomass (LB) despite its huge potential as a renewable bioenergy resource faces bottlenecks due to its recalcitrance and lack of appropriate pretreatment approaches. The current study evaluates the combinatorial application of alkali and acid pretreatment of pine needle biomass (PNB), for achieving high sugar release upon enzymatic saccharification. Pine needle accumulation poses a big threat to the forest soil fertility and overall ecosystem and environment. However, pine needle waste can be valorized after appropriate pretreatment and enzymatic saccharification for production of renewable energy, i.e. biofuel–ethanol. In combinatorial pretreatment strategy, first PNB was subjected to ammonium carbonate pretreatment, and parameters like ammonium carbonate concentration, incubation time and pretreatment temperature were optimized using design of experiment (DoE) approach. The relative influence of parameters on efficacy of pretreatment was established individually and in interactive terms. Based on DoE, sugar yield of 7.56 mg/g of PNB was obtained. Furthermore, DoE-based pretreated PNB was subjected to sulphuric acid pretreatment, followed by enzymatic saccharification. The sugar released during various steps was pooled (8.19 g/100 g), concentrated and subjected to ethanol fermentation with dual yeast cultures using Saccharomyces cerevisiae and Pichia stipitis. An ethanol yield of 8.8%, v/v (6.94% w/v), was obtained. This represents the process efficiency of 19.34% for bioethanol production from PNB.

Ionic liquid (IL) pretreatment represents an effective strategy for effective fractionation of lignocellulosic biomass (LB) to fermentable sugars in a biorefinery. Optimization of combinatorial pretreatment of sugarcane bagasse (SCB) with IL (1-butyl-3-methylimidazolium chloride [Bmim]Cl) and surfactant (PEG-8000) resulted in enhanced sugar yield (16.5%) upon enzymatic saccharification. The saccharification enzymes (cellulase and xylanase) used in the current study were in-house produced from a novel IL-tolerant fungal strain Aspergillus assiutensis VS34, isolated from chemically polluted soil, which produced adequately IL-stable enzymes. This is the first ever report of IL-stable cellulase/xylanase enzyme from Aspergillus assiutensis. To get the mechanistic insights of combinatorial pretreatment physicochemical analysis of variously pretreated biomass was executed using SEM, FT-IR, XRD, and 1H NMR studies. The combined action of IL, surfactant and ultrasound had very severe and distinct effects on the ultrastructure of biomass that subsequently resulted in enhanced accessibility of saccharification enzymes to biomass, and increased sugar yield.

Huge industrial application potential of xylanases is stalled due to lack of process suitable characteristics like thermostability, broad range pH stability, and high catalytic efficiency in the available enzymes. Current study presents the first ever report of a pH stable (pH 6–11) and thermostable (80-100 °C) xylanase from a novel strain of Aspergillus terreus S9. The xylanase was purified to homogeneity (6.67-fold) by ammonium sulphate precipitation, ion exchange chromatography, and molecular exclusion chromatography. SDS-PAGE analysis revealed an estimated molecular mass of ~33 kDa for the xylanase. Metal ions and surfactants such as K+, Ca2+, Mn2+, Mg2+, CTAB and Tween-80 enhanced the xylanase activity while Cu2+ and Hg2+ strongly inhibited the activity. Kinetic parameters i.e. Km, Vmax, Kcat and Kcat/Km of A. terreus S9 xylanase were 2.94 mg/ml, 285.71 μmol/min/mg, 1587.28 s−1and 539.89 ml/mg/s, respectively. The substrate specificity confirmed the true endoxylanolytic nature of xylanase. The conserved domain analysis, and Blastn and Blastx results showed that the xylanase belonged to GH10 family. A. terreus S9 xylanase may be used as model system for understanding the molecular basis of robust nature of enzymes, and the knowledge generated may help designing novel enzymes that are suitable for industrial applications.

The current study presents the first ever report of surfactant (Tween-20) assisted ionic liquid IL, (1-ethyl-3-methylimidazolium methane sulphonate [Emim][MeSO3]) pretreatment of Parthenium hysterophorus biomass, its saccharification by in-house developed enzyme cocktail from Aspergillus aculeatus PN14, and fermentation of sugars to bioethanol under consolidated bioprocess. Optimization of pretreatment process variables viz. biomass loading, temperature and time, resulted in enhanced sugar yield (40.1%) upon saccharification of pretreated biomass with IL-stable cellulase and xylanase enzymes from an IL-tolerant newly isolated fungus Aspergillus aculeatus PN14. Physicochemical analysis of surfactant assisted IL-pretreated biomass by SEM, FT-IR and XRD provided molecular insights into inter/intra molecular ultrastructural changes in the biomass that eased the saccharification. Thorough understanding of chemical/molecular structure of biomass may help developing customized pretreatment regimes of apt severity which might result in enhanced accessibility of enzymes to biomass, and hence more sugar content.

Pretreatment is the requisite step for the bioconversion of lignocellulosics. Since most of the pretreatment strategies are cost/energy intensive and environmentally hazardous, there is a need for the development of an environment-friendly pretreatment process. An ionic liquid (IL) based pretreatment approach has recently emerged as the most appropriate one as it can be accomplished under ambient process conditions. However, IL-pretreated biomass needs extensive washing prior to enzymatic saccharification as the enzymes may be inhibited by the residual IL. This necessitated the exploration of IL-stable saccharification enzymes (cellulases). Current study aims at optimizing the bioprocess variables viz. carbon/nitrogen sources, medium pH and fermentation time, by using a Design of Experiments approach for achieving enhanced production of ionic liquid tolerant cellulase from a bacterial isolate Bacillus subtilis SV1. The cellulase production was increased by 1.41-fold as compared to that under unoptimized conditions. IL-stable cellulase was employed for saccharification of IL (1-ethyl-3-methylimidazolium methanesulfonate) pretreated pine needle biomass in a newly designed bioprocess named as “one pot consolidated bioprocess” (OPCB), and a saccharification efficiency of 65.9% was obtained. Consolidated bioprocesses, i.e., OPCB, offer numerous techno-economic advantages over conventional multistep processes, and may potentially pave the way for successful biorefining of biomass to biofuel, and other commercial products.