Chapter V. treated sugarcane bagasse

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1 Chapter V Production of xylooligosaccharides by an alkali treated sugarcane bagasse 167

2 5.1. Introduction Cellulose is the most abundant biomass in the world, and its biological degradation is a key step in the global carbon cycling as well as a promising approach for the production of bio-energy (Lynd et al. 2002). Cellulose is the principle component of lignocellulosic biomass and its concentration ranges from 40 to 50 % of dry weight. Cellulose is a homopolysaccharide composed of repeating β-d-glucopyranose units. The degree of polymerization and crystallinity of cellulose varies from species to species and this is shown to have a significant impact on hydrolytic process (acidic and enzymatic) (Zhang and Lynd, 2004). Plant biomass particularly the lignocellulosic materials are the major renewable carbon reservoir that offers sustainable generation of numerous valuable products having diverse industrial significance and non-food consumer products such as fuel, chemicals, polymeric materials (Peng et al. 2010). The exploitation of cellulolytic microorganisms is the most efficient process for cellulose transformation into useful products. The cellulolytic microorganisms, which possess very active and complex hydrolytic systems, are thus potential biocatalysts for the synthesis of highly added value products. To date, several glycosynthases have been developed from different families of glycosidases, including both exoglycosidases and endoglycosidases. The exoglycosynthases derived from exoglycosidases (Mackenzie et al. 1998; Trincone et al. 2000; Nashiru et al. 2001; Jakeman and Withers 2002; Drone et al. 2005; Faijes et al. 2006; Mullegger et al. 2006) have moderate substrate specificity and regioselectivity, based on that of the original wild-type enzymes, and can catalyze the formation of various glycosidic linkages of short-chain oligosaccharides (di-, tri-, and tetrasaccharides) 168

3 as their major products. The endoglycosynthases derived from endoglycosidases (Malet and Planas 1998; Fort et al. 2000; Hrmova et al. 2002; Jahn et al. 2003; Van Lieshout et al. 2004; Kim et al. 2006; Sugimura et al. 2006) have high regioselectivity and catalyze the synthesis of one specific glycosidic linkage. The lignin-cellulose-hemicellulose-pectin complex forms one of the most stringent seals around cellulose. The first step in the overall process of lignocellulosic fermentation is breaking this barrier (pre-treatment). This is the most important and rate limiting step in the overall process. Further steps involve isolation and hydrolysis of cellulose and hemicellulose to generate fermentable sugars (saccharification) followed by fermentation and distillation. The pre-treatment processes involve the use of acids, alkalis and/or organic solvents. The aim of this process is to separate lignin, cellulose, hemicellulose and pectin from lignocellulosic biomass. Post pre-treatment, the recalcitrant lignocellulosic biomass becomes susceptible to acid and/or enzymatic hydrolysis as the cellulosic microfibrils are exposed and/or accessible to hydrolyzing agents. In the pretreatment process, small amounts of xylose and most of hemicellulose is hydrolyzed to sugar monomers; mainly D-xylose and D-arabinose. The crux of problem lies in its complex character and linkages with lignin. In order to maximize the yield, fractionation process of hemicellulose is generally aimed to break down those linkages with other biomolecules of lignocellulosic materials. At the same time, precautions are taken that none of the step will be able to hydrolyze the other moieties such as hemicellulose or lignin as these will affect the quality of cellulose. 169

4 The lignocellulosic materials are the most abundantly available and renewable raw materials in the globe and its total quantity are estimated around MT (Sanchez and Cardonna, 2008). Keeping in mind the present trend of agricultural production, renewability and recyclability of lignocellulosic materials, researchers are considering generating wide spectrum of products ranging from inexpensive composite materials to high value prebiotic like xylo-oligosaccharides (XOS). Like all other lignocellulosic materials, sugarcane bagasse (SCB) is a substrate of high potential for biotechnological processes, which comprises % cellulose, % hemicelluloses, and % lignin. Conversion of hemicellulose into value added useful products by enzymatic routes holds strong promise for the use of a variety of unutilized and underutilized agricultural residues for practical purposes. However, development of efficient and cost effective conversion of any lignocellulosic biomass to prebiotics is a key issue. The present paper focused on the optimization of conditions for alkaline treatment of cellulose in order to maximize its recovery. Many oligosaccharides are found naturally in plant species including fruits, vegetables and some also in milk and honey. They include the disaccharides sucrose, lactose and trehalose, and various oligosaccharides are made up of sucrose known as the raffinose family oligosaccharides that are ubiquitous in the plant kingdom being present in many different varieties of seeds. Oligosaccharides are not digested by the human gastro-intestinal enzymes and are passed intact into the large intestine where they are fermented by colonic bacteria. Some disaccharides such as maltose and cellobiose can be prepared through hydrolysis 170

5 (chemical or enzymatic hydrolysis) from starch or cellulose respectively (Hirayama, 2002; Collins and Rastall, 2008), while higher oligosaccharides e.g. hexa-, hepta- and nona- saccharides have been obtained through hydrolysis by the action of specific enzymes (hydrolases), or via organic synthesis mediated by specific enzymes such as glycosyltransferases (Zhang and Kong, 2003; Yang and Kong, 2005). Oligosaccharides consist of a class of biomolecules that function in biological processes of recognition, like viral or bacterial infections, cell adhesion, cellular signal transduction and intercellular communications (Davies et al. 2001). Potential applications of these sugars in the areas of food, animal feeds, pharmaceuticals, cosmetics as well as prebiotics and immunomodulator agents has promoted new research to foster their production and to elucidate their biological and functional properties (Remaud-Simeon et al. 2000). A rich source of xylan decorated with branches of L-arabinose and 4-O-methyl- Dglucuronic acid is sugarcane bagasse (Brienzo et al. 2009). Since xylan bound in bagasse is directly not susceptible to enzymatic attack, it has to be extracted before the enzyme hydrolysis. Hence the present investigation aimed to produce XOS from the cellulose of SCB through cellulase by Exiguobacterium sp. VSG-1 with the variables such as ph, temperature, enzyme dose and incubation time Materials and Method All chemicals were of analytical grade. Standards of glucose, xylose, arabinose and oat spelt xylan (OSX) were purchased from Sigma- Aldrich (USA). Xylobiose, xylotriose and xylotetraose were purchased from Megazyme, An alkaliphilic bacterium Exiguobacterium sp. VSG-1 was used for the secretion of cellulase. Sugarcane bagasse 171

6 (SCB) was obtained from a local maize field in Gulbarga, Karnataka, India and was dried, powdered (60-80 mesh) and stored in polycarbonate containers Milling of Sugarcane bagasse A two stage grinding of SCB has been carried out to get a powder of mesh size. Firstly, the corncob chips were ground in a Multimill (Gansons Ltd, Bombay-55, India) using a 10 mm sieve followed by 6 mm sieve, Secondly, the powder obtained was ground again in a Plate Mill (Madras Standard Engineering Works, Madras, India) to mesh size. This powder was used as the inducer for cellulase production and also for various pre-treatment studies and XOS production Pre-treatments of SCB Previously reported protocols were adopted for alkali pre-treatment of SCB powder. After the pre-treatment excess alkali/acid was washed with de-ionized water till the ph of the washings was 9.0 and the treated SCB powder was dried at 40 C and stored till further use. In another set of experiments, the acid treated corncob obtained as mentioned above was further cooked under pressure at 121 C for 30 min and was dried at 40 C and stored until use Microorganism Exiguobacterium sp. VSG-1 is an isolate from our laboratory used for the production of cellulase Xylanase production The strain VSG-1 was grown in 250 ml Erlenmeyer flask containing 50 ml of mineral salt medium at 40 C of ph 9.0 containing 1 % OSX and 0.5% peptone as carbon and nitrogen 172

7 sources. Xylanase production was carried out by inoculating with 1 ml of 24 h old culture and incubated at 37 o C in a rotary shaker at 200 rpm for 48 h. The contents of the flasks were removed at regular intervals and centrifuged at 8,000 rpm for 10 min at 4ºC (Remi cooling centrifuge, Mumbai, India). The supernatant was used as an enzyme source for the assay of xylanase activity. The growth was monitored by measuring optical density at 660 nm in a spectrophotometer (UV-6450; Jenway, UK) Xylanase assay Xylanase activity of supernatant was assayed using standard oat spelt xylan (OSX) as the substrate by DNS method (Miller, 1959) as explained earlier in the chapter Production of XOS from untreated and pre-treated sugarcane bagasse For evaluating the efficiency of various pre-treatment methods, XOS production was carried out from untreated/pre-treated sugarcane bagasse (SCB) powder xylanase from Exiguobacterium sp. VSG-1. The effects of various reaction parameters on the XOS production from alkali pre-treated (SCB) powder was carried out using extracellular endoxylanase obtained by submerged fermentation of Exiguobacterium sp. VSG HPLC analysis XOS was estimated by HPLC method and sample preparations and conditions were based on previous reports (Aachary and Prapulla, 2009). The products of hydrolysis were analyzed by high performance liquid chromatography (HPLC) on a micro Bond pack Amino Carbohydrate column (4.1 mm 300 mm). Samples (20 µl) were injected and eluted with acetonitrile-water (70:30 ratio) at a flow rate of 1 ml/min. The hydrolyzed products were detected using a refractive index detector. The XOS formed was 173

8 quantified by comparing the peak area of XOS with that of standard xylose and is expressed as mg/ml of hydrolyzate Effect of ph, temperature, dose of xylanase and periods of incubation on reducing sugars in enzymatic hydrolysate of SCB The effect of ph ( ), temperature (20-70 C), dose of xylanase (0-20U/ml) and periods of incubation (0-30 h) were studied on reducing sugars in enzymatic hydrolysate of SCB Results Composition of sugarcane bagasse Proximate composition and analysis of sugarcane bagasse (SCB) has been reported by numerous researchers to evaluate its nutritive value. Because the production of XOS from the above SCB is based on hydrolysis of xylan, the compositional analysis of SCB was performed to ascertain its potentiality as raw material for XOS production. The SCB was oven dried and ground to obtain the uniform particle size of 1 mm and subjected to compositional analysis. It constituted of 89.17±0.08 % organic matter, 10.83±0.08 % ash and 3.35±0.15 % crude protein. 174

9 Table 5.1 Effect of alkali and extraction conditions on the true recovery of hemicellulose from sugarcane bagasse (SCB). Extractio n conditions NaOH followed by overnight incubation NaOH followed by steam explosion KOH followed by overnight incubation KOH followed by steam explosion Concentration of alkali (%) 2 % 4 % 6 % 8 % 10 % 12 % 7.26± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 Extraction of hemicellulose The xylan is an important structural component of plant biomass and its yield can vary depending on the method of isolation and the nature of raw materials. Extraction of hemicellulose from lignocellulosic biomass involves two stage process; alkaline hydrolysis of lignocellulosic matrix followed by extracting them into alkaline medium. Therefore, the present investigation attempted to extract the hemicellulose from SCB with incremental levels (2 %, 4 %, 8 % and 12 %) of both sodium hydroxide and potassium hydroxide. As higher temperature softens the lignin protective layer present in plant biomass, the present experiment further investigated the effect of different alkali on the recovery of hemicellulose from particular SCB under overnight incubation at room temperature (10 h, 40 C) or autoclaving (121 C, 15 lbs, 45 min). Evidently, during overnight incubation at room temperature, the incremental levels of either potassium hydroxide or sodium hydroxide resulted in increase in true recovery of hemicellulose from 2.47 % to % and 3.75 % to % of original biomass respectively (Table 5.1). These values corresponded to increase in the relative recovery from 8.79 % to % in case of extraction with potassium hydroxide and from % to %, while extracting with sodium hydroxide followed by overnight incubation. In the presence of steam, both the alkali was effective to further inching up the true and relative recovery of cellulose from SCB. As can be seen, the maximum true recovery of cellulose reached up to % of dried grass with potassium hydroxide coupled with steam application (corresponded to relative recovery % of original hemicellulose), while the values reached up to % of dried grass with sodium hydroxide plus steam application; 176

11 corresponding to the relative hemicellulose recovery as high as % of original hemicellulose Sugar composition of hemicellulose Though hemicellulose from different sources such as sugarcane bagasse, cereals, hardwood, softwood, differ in composition, it is mainly consisted of homopolymeric backbone chains of D-xylose units connected by b-1,4-xylosidic linkages (Saha et al. 2003). It is often substituted by arabinose, acetic acids, mannose (Yang et al. 2007). Reducing sugars detected in the alkaline extraction of plant biomass might be originated either from insoluble hemicellulose or from minute levels of XOS that generated during the processing of raw materials. The HPLC analysis of the alkali extracted hemicellulose of the SCB revealed xylose 59.3 %, arabinose 8.89 %, xylobiose 5.78 %, xylotriose and xylotetraose 6.25 % respectively. In fact, the sugar composition of cellulose could vary with the method of isolation as well as the raw materials from where xylan is extracted (Peng et al. 2009) Production and detection of xylo-oligosaccharides In the present investigation, the alkali extracted cellulase of SCB was hydrolyzed by cellulase enzyme secreted by strain VSG-1 to a number of oligosaccharides having the DP ranging from 2 to 3. The effect of ph, temperature, enzyme dose and reaction time on the production of XOS was evaluated through the estimation of reducing sugars and HPLC. The analysis of HPLC revealed the conversion of SCB cellulose into XOS namely xylobiose and xylotriose in addition to xylose under the influence of xylanase. 177

12 Reducing sugars (mg/ml) ph Reducing sugar (mg/ml) Fig Effect of ph on the production of reducing sugars by enzymatic hydrolysis of sugarcane bagasse (SCB). 178

13 Reducing sugars (mg/ml) Temp ( C) Reducing sugars (mg/ml) Fig Effect of temperature on the production of reducing sugars by enzymatic hydrolysis of sugarcane bagasse (SCB). 179

14 Reducing sugars (mg/ml) Enzyme doze (U/ml) Reducing sugars (mg/ml) Fig Effect of enzyme dose on the production of reducing sugars by enzymatic hydrolysis of sugarcane bagasse (SCB). 180

15 Reducing sugars (mg/ml) Incubation time (h) Reducing sugars (mg/ml) Fig Effect of incubation time on the production of reducing sugars by enzymatic hydrolysis of sugarcane bagasse (SCB). 181

16 Effect of ph, temperature, dose of xylanase and periods of incubation on reducing sugars in enzymatic hydrolysate of SCB The reaction was carried out in 250 ml conical flask containing 50 ml reaction mixture, consisting of 1.0g untreated/ pre-treated SCB powder and 5 U/ml of xylanase and the volume was made up to 50 ml using phosphate buffer (0.05 M, ph 9.0). The enzymatic reaction was carried out in a shaking water bath maintained at 40 C for h. At the end of specified incubation time, the reaction was stopped by keeping the reaction mixture in a boiling water bath for 15 min. The effect of ph of 9.0 (Fig. 5.1), temperature 40 C (Fig. 5.2), enzyme concentration (5 U/m1) (Fig. 5.3) and incubation time of 10 h (Fig. 5.4) were used for the maximum production of xylo-oligosaccharides (XOS) Discussion Lignocellulosic (LCM) material is composed of three polymers namely, lignin of phenolic nature (Grima-Pettenati and Goffner, 1999), cellulose composed of glucose units linked through β1-4 linkage to form a linear polymer and hemicelluloses, a variety of monomers including xylose, glucose, arabinose, rhamnose and mannose branched result in a heteropolysaccharide (Puls and Schuseil, 1993). Source of substrate determines the nature of substantial part of hemicelluloses that can be a polymer of xylose (xylan), arabinose (arabinan) or mannose (mannan) (Huisman et al. 2000). Hemicellulose, the most abundant polysaccharide from plant source is a base material for preparation of several value added products. Hence, the use of agricultural by-products/wastes for xylooligosaccharides production is based on the philosophy called biomass refinery 182

17 which states fractionation of hemicellulosic component of these by-products is of interest to obtain separate streams useable for different product applications (Myerly et al. 1981). Since the SCB was rich in celluloses, the selection of raw material for extraction of hemicellulose was inappropriate. The SCB was also low in acid detergent lignin (4.80 ± 0.30 %) as reported earlier (Reddy and Reddy, 1992); which might facilitate maximization of hemicellulose recovery as lignin is one of the major hurdles in fractionation of hemicellulose from lignocellulosic biomass. Xylans are the second most plentiful hemicellulosic polymers, comprised of xylose units (Joseleau et al. 1992). Xylans correspond to an enormous resource of biopolymers for practical applications, accounting for % of the dry biomass of woody tissues of dicots and lignified tissues of monocots, and occur up to 50 % in some tissues of cereal grains (Vazquez et al. 2006). Xylans are heteropolysaccharide composed of a chain of xylose units joined through β-(1 4) glycosidic linkages (Bakir et al. 2001), with branching of short chain hexoses/pentoses (D-glucuronic acid or its 4-O-methylether, L- arabinose and/or various oligosaccharides, composed of D-xylose, L-arabinose, D- or L- galactose and D-glucose) (Subramaniyan and Prema, 2002). Polymers like xylans can be classified into homoxylans and heteroxylans, that include complex heteroxylans. In the presence of steam, both the alkali was effective to further inching up the true and relative recovery of cellulose from SCB. As can be seen, the maximum true recovery of cellulose reached up to % of dried grass with potassium hydroxide coupled with steam application (corresponded to relative recovery % of original hemicellulose), while the values reached up to % of dried grass with sodium hydroxide plus steam 183

18 application; corresponding to the relative hemicellulose recovery as high as % of original hemicellulose. Though hemicellulose from different sources such as sugarcane bagasse, cereals, hardwood, softwood, differ in composition, it is mainly consisted of homopolymeric backbone chains of D-xylose units connected by b-1,4-xylosidic linkages (Saha et al. 2003). It is often substituted by arabinose, acetic acids, mannose (Yang et al. 2007). Reducing sugars detected in the alkaline extraction of plant biomass might be originated either from insoluble hemicellulose or from minute levels of XOS that generated during the processing of raw materials. Among the several process of XOS generation from xylan, enzymatic one is preferred over others as it neither generates toxic compounds nor requires special equipment (Akpinar et al. 2007; Kumar and Satyanarayana, 2011). As it can be seen that with an increase in the enzyme dose there was break down of both hemicellulose and XOS further which result into diminishing cellulose concentration and increasing glucose accumulation. Xylose and xylobiose were the predominant end products of hydrolysis. Arabinose liberation was also observed in the present experiment, especially from oat spelt xylan, and its level reached maximally 1 % of the hemicellulose. This indicates the presence of α-l-rabinofuranosidase in the used xylanolytic system, which was confirmed on 4-nitrophenyl α-l-arabinofuranoside. Akpinar et al. (2007) also noticed similar enzymatic hydrolysis pattern of xylan through TLC profile while producing XOS from cotton stalk xylan and suggested that with increasing enzyme dose there would increase in hydrolysis yield and rate. Yang et al. (2011) reported an increase in the rate of 184

19 hydrolysis of xylan from Populas tomentosa without any change in the yield of XOS, despite increasing the enzyme (15-35 U/g substrate) load. Increase in enzyme concentration during hydrolysis of xylan fractionated from oil palm fronds reflected increase in the production of reducing sugars upto certain level of enzymes, following which the increasing concentration of xylanase enzyme did not exhibit significant effect on final hydrolysis products (Sabiha-Hanim et al. 2011). As the reducing sugars contained xylose monomers along with XOS (xylobiose and xylotriose), enzymatic hydrolysate were further analyzed by HPLC. Several attempts were being made to produce XOS enzymatically from the xylan of diverse lignocellulosic biomass (Yang et al. 2007; Brienzo et al. 2010) there are few reports on XOS production from SCB. The yield of XOS from xylan varies according to the source of xylan, enzyme activity as well as incubation conditions. Upon exposure to same xylanase enzyme on xylan, originated from corn cob, wheat bran, peanut shell, oat spelt, the yield of XOS i.e. xylobiose ( mg/ml), xylotriose ( mg/ml) and xylotetrose (nil to 1.99 mg/ml) varied greatly (Yang et al. 2007). While generating prebiotic from alkali pretreated sugarcane bagasse, Brienzo et al. (2010) noticed XOS yield in the range of mg/ml while applying enzyme dose from 5 to 30 U/g of substrate. Use of higher enzyme loading (30 IU/ml) in the hydrolysis of wheat bran xylan resulted in the decrease of oligoxylosides yield along with reduction of degree of polymerization (Ochs et al. 2011). In the present investigation, a maximum yield of xylobiose (11 g/100 g xylan) could be possible at ph of 5.03, incubation temperature of C, enzyme dose of U for a reaction time of h. In order to obtain higher 185

20 xylotriose (7.06 g/100 g xylan) from the enzymatic hydrolysis of SCB xylan, the ideal conditions were ph 9.0, temperature 40 C, enzyme dose 5.0 U and reaction time 10.0 h. A lower yield of XOS in the present investigation might be ascribed to the nature of xylan that was extracted from the natural grass. The XOS behave as dietary fibre and since the dietary fibre has the properties mandatory for its consideration as an important ingredient in the preparation of functional foods, due to its beneficial effects such as increasing the volume of faecal bulk, decreasing the time of intestinal transit, cholesterol and glycaemia levels, trapping substances that can be dangerous for the human organism, stimulating the growth of the intestinal flora, etc. Furthermore, the XOS have been the latest and most exhilarating development, because even as they are already fermentable substances, their effects on bowel habit are minor and their contribution to the health relies on their noteworthy effects on the large intestinal flora. For this reason they are called prebiotics (Manning and Gibson, 2004). Conclusion The present study investigated to identify SCB as a raw material for the production of xylo-oligosaccharides. Sugarcane bagasse hemicellulose and birchwood xylan, which have similar composition, were hydrolyzed to about the same extent, similar as the results reported by others. The ability of fermentation of xylo-oligosaccharides by the bacteria varied with respect to the nature of xylooligosaccharides. It was possible to fractionate almost 97 % of original hemicellulose present in SCB with 12 % NaOH and steam application. Application of commercial xylanase over SCB cellulose enabled XOS 186

21 production. The various methods employed to define the variables (ph, temperature, enzyme dose, incubation time) wherein maximum production of XOS could be achieved keeping cellulose at minimum levels. Future perspective of XOS from SCB lies on its economic production on an industrial scale and their validation as prebiotics either through animal model or human clinical trials. 187

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