Silvio Silvério da Silva

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.

A study on the pretreatment of a sugarcane bagasse sample with dilute sulfuric acid.

Experiments based on a 23 central composite full factorial design were carried out in 200-ml stainless-steel containers to study the pretreatment, with dilute sulfuric acid, of a sugarcane bagasse sample obtained from a local sugar–alcohol mill. The independent variables selected for study were temperature, varied from 112.5°C to 157.5°C, residence time, varied from 5.0 to 35.0 min, and sulfuric acid concentration, varied from 0.0% to 3.0% (w/v). Bagasse loading of 15% (w/w) was used in all experiments. Statistical analysis of the experimental results showed that all three independent variables significantly influenced the response variables, namely the bagasse solubilization, efficiency of xylose recovery in the hemicellulosic hydrolysate, efficiency of cellulose enzymatic saccharification, and percentages of cellulose, hemicellulose, and lignin in the pretreated solids. Temperature was the factor that influenced the response variables the most, followed by acid concentration and residence time, in that order. Although harsher pretreatment conditions promoted almost complete removal of the hemicellulosic fraction, the amount of xylose recovered in the hemicellulosic hydrolysate did not exceed 61.8% of the maximum theoretical value. Cellulose enzymatic saccharification was favored by more efficient removal of hemicellulose during the pretreatment. However, detoxification of the hemicellulosic hydrolysate was necessary for better bioconversion of the sugars to ethanol.

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.

Utilization of sugar cane bagasse hemicellulosic hydrolyzate by Candida guilliermondii for xylitol production

Abstract Sugar cane bagasse hemicellulosic hydrolyzate from steam explosion was treated by seven different methods in which the pH was altered by bases (including Ca(OH) 2 , CaO and KOH) and H 2 SO 4 . The effectiveness of the treatment was judged by measuring the conversion of the hydrolyzate to xylitol by Candida guilliermondii . The best treatment was found to be the alteration of pH to 10 with Ca(OH) 2 and its subsequent decrease to 6·5 with H 2 SO 4 , since 95% of the original 70 g/liter xylose contained in the hydrolyzate was converted to xylitol with a yield of 0·48 g/g (53% of the theoretical maximum).

Pretreatment of Sugarcane Bagasse Hemicellulose Hydrolysate for Xylitol Production by Candida guilliermondii

In order to remove or reduce the concentrations of toxic substances present in the sugarcane bagasse hemicellulose hydrolysate for xylose-to-xylitol bioconversion, the hydrolysate was pretreated by changing the initial pH level through the combination of different bases and acids with or without the subsequent addition of activated charcoal. Attention was given to the influence of the fermentation time as well.

Xylitol production by Candida guillermondii as an approach for the utilization of agroindustrial residues

Abstract Different substrates based on hydrolyzed hemicellulosic fractions of agroindustrial residues were used for xylitol production by Candida guilliermondii FTI 20037 under semi-aerobic conditions. Batch fermentation performances were characterized and compared with those attained in a synthetic medium using d -xylose as a major carbon source. For all media tested, simultaneous utilization of hemicellulosic sugars (glucose and xylose) was observed and the highest substrate uptake rate was attained in sugar cane bagasse medium. Increased xylitol concentrations (40 g/litre) were achieved in synthetic and rice straw-media, although the highest xylitol production rate was obtained in sugar cane bagasse hydrolysate. These results show that both hydrolysates can be converted into xylitol with satisfactory yields and productivities.

Detoxification of Lignocellulosic Hydrolysates for Improved Bioethanol Production

Lignocelluloses are the most abundant raw materials on Earth comprised of cellulose, hemicelluloses and lignin. After cellulose, hemicellulose is the principal fraction of the plant cell wall that could serve as a potential substrate for the production of value-added products under optimized conditions [Chandel & Singh, 2011; Chandel et al., 2010a; Hahn-Hagerdal et al., 2007; Saha, 2003]. Largely, the secondary cell wall of plants contains cellulose (40– 80%), hemicellulose (10–40%), and lignin (5–25%). The carbohydrate fraction of the plant cell wall can be converted into fermentable monomeric sugars through acidic and/or enzymatic (hemicellulase/cellulase) reactions, which have been exploited to produce ethanol, xylitol, n-butanol and 2, 3-butanediol via microbial fermentation processes [Sun, 2009.; Chandel et al., 2010a; Carvalheiro et al., 2005; Saha, 2003]. Until now the pretreatment is unavoidable necessity, which has been examined and employed extensively in the past [Moiser et al., 2005, Taherzadeh & Karimi 2007; Kumar et al., 2009; Chandel et al., 2010b]. The acidic pretreatment of lignocellulosics hydrolyzes the hemicellulose fraction, enabling subsequent enzymatic digestion of the cellulose in fermentation reaction [Kumar et al., 2009; Chandel et al., 2007a; Chandel et al., 2007b; Chandel et al., 2007c]. However, the non-specificity of acidic treatment led to the formation of complex sugars and compounds inhibitory to the microorganisms for ethanol production [Parawira & Tekere, 2011]. The depolymerization of hemicellulose by chemical process yields xylose as the major fraction and arabinose, mannose, galactose, and glucose in smaller fractions in addition to potential microbial inhibitors [Chandel et al., 2010a; Girio et al., 2010; Chandel et al., 2009; Chandel et al., 2007a]. These inhibitors can be divided into three major groups (Fig. 1), i.e. organic acids (acetic, formic and levulinic acids), ii. furan derivatives [furfural and 5hydroxymethylfurfural (5-HMF)], iii. phenolic compounds [Chandel et al., 2010a; Chandel et al, 2007b; Mussatto and Roberto, 2004; Palmqvist and Hahn-Hagerdal, 2000a], affecting overall cell physiology and often result in decreased viability, ethanol yield, and productivity [Chandel et al, 2007a; Palmqvist & Hahn-Hagerdal, 2000a]. The ethanologenic microorganisms have ability to degrade some of the inhibitors; however, the toxicity of hydrolysate was determined by the aggregate effect of compounds [Mussatto and Roberto, 2004; Zaldivar et al., 2001]. Progress has been made to achieve higher levels of sugars by diminishing the overall impact of fermentative inhibitors which in-turn improves the fermentability of lignocellulosic hydrolysates [Alriksson et al., 2011; Sun & Liu, 2011;

Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian Forest.

Background This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. Methodology/Principal Findings A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L·h to 0.75 g/L·h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L·h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. Conclusions/Significance This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

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.