Anuj K. Chandel

Bioconversion of sugarcane biomass into ethanol: an overview about composition, pretreatment methods, detoxification of hydrolysates, enzymatic saccharification, and ethanol fermentation.

Depleted supplies of fossil fuel, regular price hikes of gasoline, and environmental damage have necessitated the search for economic and eco-benign alternative of gasoline. Ethanol is produced from food/feed-based substrates (grains, sugars, and molasses), and its application as an energy source does not seem fit for long term due to the increasing fuel, food, feed, and other needs. These concerns have enforced to explore the alternative means of cost competitive and sustainable supply of biofuel. Sugarcane residues, sugarcane bagasse (SB), and straw (SS) could be the ideal feedstock for the second-generation (2G) ethanol production. These raw materials are rich in carbohydrates and renewable and do not compete with food/feed demands. However, the efficient bioconversion of SB/SS (efficient pretreatment technology, depolymerization of cellulose, and fermentation of released sugars) remains challenging to commercialize the cellulosic ethanol. Among the technological challenges, robust pretreatment and development of efficient bioconversion process (implicating suitable ethanol producing strains converting pentose and hexose sugars) have a key role to play. This paper aims to review the compositional profile of SB and SS, pretreatment methods of cane biomass, detoxification methods for the purification of hydrolysates, enzymatic hydrolysis, and the fermentation of released sugars for ethanol production.

Detoxification of Lignocellulose Hydrolysates: Biochemical and Metabolic Engineering Toward White Biotechnology

Chemical hydrolysis of lignocellulosic biomass (LB) produces a number of inhibitors in addition to sugars. These inhibitors include lignin-derived phenolics, carbohydrate-derived furans, and weak acids that have shown a marked effect on the productivities of various metabolites and the growth of biocatalysts in the fermentative reaction. In the past, a number of physicochemical and biological approaches have been proposed to overcome these fermentation inhibitors, including modified fermentative strategies. Additionally, the timely intervention of genetic engineering has provided an impetus to develop suitable technologies for the detoxification of lignocellulosics in biorefineries. However, the improvements in detoxification strategies for lignocellulose hydrolysates have resulted in significant loss of sugars after detoxification. Hydrolysis of myco-LB (LB after fungal pretreatment) has been recognized as a promising approach to avoid fermentation inhibitors and improve total sugar recovery. Biotechnological inventions have also made it possible to widen the range of suitable biocatalysts for biorefineries by microbial-routed induction of enzymatic expression for the elimination of inhibitors, eventually improving ethanol production from acid hydrolysates. This article aims to highlight the strategies that have been adopted to detoxify lignocellulosic hydrolysates and their effects on the chemical composition of the hydrolysates to improve the fermentability of lignocellulosics. In addition, genetic manipulation could widen the availability and variety of substrates and modify the metabolic routes to produce bioethanol or other value-added compounds in an efficient manner.

The realm of cellulases in biorefinery development

Geopolitical concerns (unstable supply of gasoline, environmental pollution, and regular price hikes), economic, and employment concerns have been prompting researchers, entrepreneurs, and policy makers to focus on harnessing the potential of lignocellulosic feedstock for fuel ethanol production and its commercialization. The carbohydrate skeleton of plant cell walls needs to be depolymerised into simpler sugars for their application in fermentation reactions as a chief carbon source of suitable ethnologic strains for ethanol production. The role of cellulolytic enzymes in the degradation of structural carbodydrates of the plant cell wall into ready-to-fermentable sugar stream is inevitable. Cellulase synergistically acts upon plant cell wall polysaccharides to release glucose into the liquid media. Cellulase predominantly dominates all the plant cell wall degrading enzymes due to their vast and diverse range of applications. Apart from the major applications of cellulases such as in detergent formulations, textile desizing, and development of monogastric feed for ruminants, their role in biorefinery is truly remarkable. This is a major area where new research tools based upon fermentation based formulations, biochemistry, and system biology to expedite the structure–function relationships of cellulases including cellulosomes and new designer enzymatic cocktails are required. In the last two decades, a considerable amount of research work has been performed on cellulases and their application in biomass saccharification. However, there are still technical and economic impediments to the development of an inexpensive commercial cellulase production process. Advancements in biotechnology such as screening of microorganisms, manipulation of novel cellulase encoding traits, site-specific mutagenesis, and modifications to the fermentation process could enhance the production of cellulases. Commercially, cheaper sources of carbohydrates and modified fermentation conditions could lead to more cost-effective production of cellulases with the goal to reduce the cost of ethanol production from lignocellulosics. Implementation of integrated steps like cellulase production and cellulase mediated saccharification of biomass in conjunction with the fermentation of released sugars in ethanol in a single step so called consolidated bio-processing (CBP) is very important to reduce the cost of bioethanol. This paper aims to explore and review the important findings in cellulase biotechnology and the forward path for new cutting edge opportunities in the success of biorefineries.

Multi-scale structural and chemical analysis of sugarcane bagasse in the process of sequential acid-base pretreatment and ethanol production by Scheffersomyces shehatae and Saccharomyces cerevisiae

BackgroundHeavy usage of gasoline, burgeoning fuel prices, and environmental issues have paved the way for the exploration of cellulosic ethanol. Cellulosic ethanol production technologies are emerging and require continued technological advancements. One of the most challenging issues is the pretreatment of lignocellulosic biomass for the desired sugars yields after enzymatic hydrolysis. We hypothesized that consecutive dilute sulfuric acid-dilute sodium hydroxide pretreatment would overcome the native recalcitrance of sugarcane bagasse (SB) by enhancing cellulase accessibility of the embedded cellulosic microfibrils.ResultsSB hemicellulosic hydrolysate after concentration by vacuum evaporation and detoxification showed 30.89 g/l xylose along with other products (0.32 g/l glucose, 2.31 g/l arabinose, and 1.26 g/l acetic acid). The recovered cellulignin was subsequently delignified by sodium hydroxide mediated pretreatment. The acid–base pretreated material released 48.50 g/l total reducing sugars (0.91 g sugars/g cellulose amount in SB) after enzymatic hydrolysis. Ultra-structural mapping of acid–base pretreated and enzyme hydrolyzed SB by microscopic analysis (scanning electron microcopy (SEM), transmitted light microscopy (TLM), and spectroscopic analysis (X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Fourier transform near-infrared (FT-NIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy) elucidated the molecular changes in hemicellulose, cellulose, and lignin components of bagasse. The detoxified hemicellulosic hydrolysate was fermented by Scheffersomyces shehatae (syn. Candida shehatae UFMG HM 52.2) and resulted in 9.11 g/l ethanol production (yield 0.38 g/g) after 48 hours of fermentation. Enzymatic hydrolysate when fermented by Saccharomyces cerevisiae 174 revealed 8.13 g/l ethanol (yield 0.22 g/g) after 72 hours of fermentation.ConclusionsMulti-scale structural studies of SB after sequential acid–base pretreatment and enzymatic hydrolysis showed marked changes in hemicellulose and lignin removal at molecular level. The cellulosic material showed high saccharification efficiency after enzymatic hydrolysis. Hemicellulosic and cellulosic hydrolysates revealed moderate ethanol production by S. shehatae and S. cerevisiae under batch fermentation conditions.

Ultra-structural mapping of sugarcane bagasse after oxalic acid fiber expansion (OAFEX) and ethanol production by Candida shehatae and Saccharomyces cerevisiae

BackgroundDiminishing supplies of fossil fuels and oil spills are rousing to explore the alternative sources of energy that can be produced from non-food/feed-based substrates. Due to its abundance, sugarcane bagasse (SB) could be a model substrate for the second-generation biofuel cellulosic ethanol. However, the efficient bioconversion of SB remains a challenge for the commercial production of cellulosic ethanol. We hypothesized that oxalic-acid-mediated thermochemical pretreatment (OAFEX) would overcome the native recalcitrance of SB by enhancing the cellulase amenability toward the embedded cellulosic microfibrils.ResultsOAFEX treatment revealed the solubilization of hemicellulose releasing sugars (12.56 g/l xylose and 1.85 g/l glucose), leaving cellulignin in an accessible form for enzymatic hydrolysis. The highest hydrolytic efficiency (66.51%) of cellulignin was achieved by enzymatic hydrolysis (Celluclast 1.5 L and Novozym 188). The ultrastructure characterization of SB using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform–near infrared spectroscopy (FT-NIR), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) revealed structural differences before and after OAFEX treatment with enzymatic hydrolysis. Furthermore, fermentation mediated by C. shehatae UFMG HM52.2 and S. cerevisiae 174 showed fuel ethanol production from detoxified acid (3.2 g/l, yield 0.353 g/g; 0.52 g/l, yield, 0.246 g/g) and enzymatic hydrolysates (4.83 g/l, yield, 0.28 g/g; 6.6 g/l, yield 0.46 g/g).ConclusionsOAFEX treatment revealed marked hemicellulose degradation, improving the cellulases’ ability to access the cellulignin and release fermentable sugars from the pretreated substrate. The ultrastructure of SB after OAFEX and enzymatic hydrolysis of cellulignin established thorough insights at the molecular level.

Dilute Acid Hydrolysis of Agro-Residues for the Depolymerization of Hemicellulose: State-of-the-Art

Geo-political, long-term economic and sustainable concerns are promoting researchers and entrepreneurs to harness the potential of lignocellulosic feedstock (LCF) into industrially significant products. Agro-residues (sugarcane bagasse, wheat straw, rice straw, corn stover, etc.) constitute the principal fraction of LCF and are available in large amounts globally. The judicious exploration of agro-residues into important products such as d-xylitol, an artificial sweetener, may provide a strong platform for its sustainable supply to the medical and non-medical applications-based sectors. Pretreatment of agro-residues by dilute acid hydrolysis is an inevitable process for the depolymerisation of hemicellulosic fraction into xylose and other sugars. Dilute acid catalyses hemicellulose fractionation at high temperature within short reaction times. Significant developments have been made in the past towards the chemical hydrolysis of agro-residues, particularly for the hemicellulose breakdown. Critical parameters such as acid load, temperature, residence time and solid-to-liquid ratio play pivotal roles in the kinetics of dilute acid hydrolysis of agro-residues. Furthermore, reactor configurations such as counter-current, plug-flow, percolation and shrinking-bed have been designed in order to maximize the sugars recovery with minimum inhibitors generation. This chapter reviews the process parameters, kinetics, methods and reactor engineering for the dilute acid catalysed processes employed for agro-residues.

Bioremediation: a genuine technology to remediate radionuclides from the environment

Radionuclides in the environment are a major human and environmental health concern. Like the Chernobyl disaster of 1986, the Fukushima Daiichi nuclear disaster in 2011 is once again causing damage to the environment: a large quantity of radioactive waste is being generated and dumped into the environment, and if the general population is exposed to it, may cause serious life‐threatening disorders. Bioremediation has been viewed as the ecologically responsible alternative to environmentally destructive physical remediation. Microorganisms carry endogenous genetic, biochemical and physiological properties that make them ideal agents for pollutant remediation in soil and groundwater. Attempts have been made to develop native or genetically engineered (GE) microbes for the remediation of environmental contaminants including radionuclides. Microorganism‐mediated bioremediation can affect the solubility, bioavailability and mobility of radionuclides. Therefore, we aim to unveil the microbial‐mediated mechanisms for biotransformation of radionuclides under various environmental conditions as developing strategies for waste management of radionuclides. A discussion follows of ‘‐omics’‐integrated genomics and proteomics technologies, which can be used to trace the genes and proteins of interest in a given microorganism towards a cell‐free bioremediation strategy.

Biodelignification of lignocellulose substrates: An intrinsic and sustainable pretreatment strategy for clean energy production.

Abstract Lignocellulosic biomass (LB) is a promising sugar feedstock for biofuels and other high-value chemical commodities. The recalcitrance of LB, however, impedes carbohydrate accessibility and its conversion into commercially significant products. Two important factors for the overall economization of biofuel production is LB pretreatment to liberate fermentable sugars followed by conversion into ethanol. Sustainable biofuel production must overcome issues such as minimizing water and energy usage, reducing chemical usage and process intensification. Amongst available pretreatment methods, microorganism-mediated pretreatments are the safest, green, and sustainable. Native biodelignifying agents such as Phanerochaete chrysosporium, Pycnoporous cinnabarinus, Ceriporiopsis subvermispora and Cyathus stercoreus can remove lignin, making the remaining substrates amenable for saccharification. The development of a robust, integrated bioprocessing (IBP) approach for economic ethanol production would incorporate all essential steps including pretreatment, cellulase production, enzyme hydrolysis and fermentation of the released sugars into ethanol. IBP represents an inexpensive, environmentally friendly, low energy and low capital approach for second-generation ethanol production. This paper reviews the advancements in microbial-assisted pretreatment for the delignification of lignocellulosic substrates, system metabolic engineering for biorefineries and highlights the possibilities of process integration for sustainable and economic ethanol production.

Bioethanol Production from Sugarcane Bagasse by a Novel Brazilian Pentose Fermenting Yeast Scheffersomyces shehatae UFMG-HM 52.2: Evaluation of Fermentation Medium

Bioconversion of hemicellulosic sugars into second generation (2G) ethanol plays a pivotal role in the overall success of biorefineries. In this study, ethanol production performance of a novel xylose-fermenting yeast, Scheffersomyces shehatae UFMG-HM 52.2, was evaluated under batch fermentation conditions using sugarcane bagasse (SB) hemicellulosic hydrolysate as carbon source. Dilute acid hydrolysis of SB was performed to obtain sugarcane bagasse hemicellulosic hydrolysate (SBHH). It was concentrated, detoxified, and supplemented with nutrients in different formulations to prepare the fermentation medium to the yeast evaluation performance. S. shehatae UFMG-HM 52.2 (isolated from Brazilian Atlantic rain forest ecosystem) was used in fermentations carried out in Erlenmeyer flasks maintained in a rotator shaker at 30°C and 200 rpm for 72 h. The use of a fermentation medium composed of SBHH supplemented with 5 g/L ammonium sulfate, 3 g/L yeast extract, and 3 g/L malt extract resulted in 0.38 g/g of ethanol yield and 0.19 g L.h of volumetric productivity after 48 h of incubation time.

Hemicellulosic ethanol production by immobilized cells of Scheffersomyces stipitis: Effect of cell concentration and stirring

Bioconversion of hemicellulosic hydrolysate into ethanol plays a pivotal role in the overall success of biorefineries. For the efficient fermentative conversion of hemicellulosic hydrolysates into ethanol, the use of immobilized cells system could provide the enhanced ethanol productivities with significant time savings. Here, we investigated the effect of 2 important factors (e.g., cell concentration and stirring) on ethanol production from sugarcane bagasse hydrolysate using the yeast Scheffersomyces stipitis immobilized in calcium alginate matrix. A 22 full factorial design of experiment was performed considering the process variables- immobilized cell concentration (3.0, 6.5 and 10.0 g/L) and stirring (100, 200 and 300 rpm). Statistical analysis showed that stirring has the major influence on ethanol production. Maximum ethanol production (8.90 g/l) with ethanol yield (Yp/s) of 0.33 g/g and ethanol productivity (Qp) of 0.185 g/l/h was obtained under the optimized process conditions (10.0 g/L of cells and 100 rpm).