Scholarly article on topic 'Potential of Fermentable Sugar Production from Napier cv. Pakchong 1 Grass Residue as a Substrate to Produce Bioethanol'

Potential of Fermentable Sugar Production from Napier cv. Pakchong 1 Grass Residue as a Substrate to Produce Bioethanol Academic research paper on "Agriculture, forestry, and fisheries"

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Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — Bunthita Pensri, Pruk Aggarangsi, Thanongsak Chaiyaso, Nopakarn Chandet

Abstract Bioethanol is one of the most significant renewable fuels. The major sources of bioethanol production are food crops such as corn, sugarcane, rice, wheat and sugar beet. However, utilization of food crops to produce bioethanol could affect the food sources and disrupt the food to population ratio. To overcome these issues, the utilization of lignocellulosic materials such as wheat straw, grass and crop residues to produce bioethanol has been developed for second-generation fuel, since those resources are abundant, cheap and renewable. Napier Pakchong 1 grass (NPG) residue is a lignocellulosic waste obtained from the process of biogas production that can be used as an alternative material for bioethanol production. This research aims to study on the potential of fermentable sugar production from NPG residue. The materials were pretreated with different concentrations of sodium hydroxide (NaOH), followed by enzymatic hydrolysis for saccharification. The results suggested that pretreatment with 3.0% (w/v) NaOH solution at 121̊C for 60 minutes provided the highest lignin removal of 86.1% (w/w) and enriched cellulose fraction from 36.4 to 75.6% (w/w). The enzymatic hydrolysis was conducted by varying enzyme loading volume and total solid contents (TS) at pH 4.8, 50̊C for 72h. The hydrolysis with enzyme loading volume of 2.0 ml/g of substrate and 10% (w/v) of TS were optimal for saccharification giving the reducing sugar yield of 768 mg/g of pretreated biomass or equal to 64 g/L and glucose yield of 522 mg/g of pretreated biomass or equal to 43 g/L. The reducing sugar will be used as a starting material for yeast to produce bioethanol.

Academic research paper on topic "Potential of Fermentable Sugar Production from Napier cv. Pakchong 1 Grass Residue as a Substrate to Produce Bioethanol"

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Energy Procedia 89 (2016) 428 - 436

CoE on Sustainable Energy System (Thai-Japan), Faculty of Engineering, Rajamangala University

of Technology Thanyaburi (RMUTT), Thailand

Potential of Fermentable Sugar Production from Napier cv. Pakchong 1 Grass Residue as a Substrate to Produce Bioethanol

Bunthita Pensria,b,e, Pruk Aggarangsib, Thanongsak Chaiyasoc, Nopakarn Chandeta,dA*

aBiotechnology Program, Graduate School, Chiang Mai University, Suthep District, Ampher Muang, Chiang Mai 50200, Thailand. bEnergy Research and Development Institute, Nakornping Chiang Mai University, Suthep District, Ampher Muang, Chiang Mai 50200, Thailand. cDivision of Biotechnology, Faculty of Agro-Industry, Chiang Mai University, Suthep District, Ampher Muang, Chiang Mai 50200, Thailand.

dDepartment of Chemistry, Faculty of Science, Chiang Mai University, Suthep District, Ampher Muang, Chiang Mai 50200, Thailand. eMultidisciplinary Science Research Centre, Faculty of Science, Chiang Mai University, Suthep District, Ampher Muang, Chiang Mai 50200,

Thailand.

Abstract

Bioethanol is one of the most significant renewable fuels. The major sources of bioethanol production are food crops such as corn, sugarcane, rice, wheat and sugar beet. However, utilization of food crops to produce bioethanol could affect the food sources and disrupt the food to population ratio. To overcome these issues, the utilization of lignocellulosic materials such as wheat straw, grass and crop residues to produce bioethanol has been developed for second-generation fuel, since those resources are abundant, cheap and renewable. Napier Pakchong 1 grass (NPG) residue is a lignocellulosic waste obtained from the process of biogas production that can be used as an alternative material for bioethanol production. This research aims to study on the potential of fermentable sugar production from NPG residue. The materials were pretreated with different concentrations of sodium hydroxide (NaOH), followed by enzymatic hydrolysis for saccharification. The results suggested that pretreatment with 3.0% (w/v) NaOH solution at 121°C for 60 minutes provided the highest lignin removal of 86.1% (w/w) and enriched cellulose fraction from 36.4 to 75.6% (w/w). The enzymatic hydrolysis was conducted by varying enzyme loading volume and total solid contents (TS) at pH 4.8, 50°C for 72 h. The hydrolysis with enzyme loading volume of 2.0 ml/g of substrate and 10% (w/v) of TS were optimal for saccharification giving the reducing sugar yield of 768 mg/g of pretreated biomass or equal to 64 g/L and glucose yield of 522 mg/g of pretreated biomass or equal to 43 g/L. The reducing sugar will be used as a starting material for yeast to produce bioethanol.

© 2016 The Authors.Publishedby Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of the organizing committee of the 12th EMSES 2015 Keywords: Fermentable sugar production; Napier cv. Pakchong 1 grass; Bioethanol.

* Corresponding author. Tel.: +66-53-942-007 ; fax: +66-53-903-763 E-mail address: carrey.tc@gmail.com

1876-6102 © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of the organizing committee of the 12th EMSES 2015 doi:10.1016/j.egypro.2016.06.287

1. Introduction

At present, the energy crisis has become an important issue. The rising cost and the rapid consumption of the fossil fuels have forced the world to seek and utilize the alternative energy. Energy crops are considered to be an interesting raw material to produce alternative energy. The energy crops are devided into 2 generation, Firstgeneration base on starch and sugar crops such as corn, cassava and sugar cane. However, the utilization of human-food might possibly lead to the ploblem of food crisis [1,2]. Therefore, the development of second-generation of energy crops that is derived from lignocellulosic material and agriculture waste such as grass, corn cob and wheat straw [3].

Napier Pakchong 1 grass (Pennisetum purpureum cv. denoted as NPG) is one of appropriate lignocellulosic materials to produce the alternative energy because of high productivity per hectare, low cost, fit for infertile land and utilization of whole plants [4]. Energy Research and Development Institute-Nakornping, Chiang Mai University was study the utilization of Napier Pakchong 1 grass to produced alternative energy by used press fluid grass for the biogas production. For the grass residue which is a waste obtained from the process of biogas production that can be used as an alternative material for lignocellulosic bioethanol production. It is less expensive than molasses and cassava starch. Moreover, it is abundant renewable resource and does not compete with food industry.

Bioethanol production from lignocellulosic materials requires three several steps as shown in Figure 1 [5]: pretreatment, hydrolysis (saccharification) and fermentation. The first step, pretreatment is the important step to free cellulose from its lignin seal and open up the crystalline structure of the cellulose to improve the enzyme accessibility [6-8]. Among pretreatment technologies, alkaline pretreatment has received much attention [9]. It is low energy demanding and relatively inexpensive technique which has studied with various lignocellulosic materials [10]. The second step is hydrolysis of cellulose and hemicellulose to releases fermentable sugar by acid or enzyme. Using acid is not appropriate because it requires the high temperature and there is a ploblem from acidic corrosion, whereas using enzyme is higher specificity and more neutral reaction without by product [4]. In enzymatic hydrolysis, cellulose is broken down by cellulases into cellobiose which in turn is cleaved by P-glucosidase into glucose [11]. For the final step, the fermentable sugar obtained from hydrolysis process is fermented to bioethanol by yeast. Bioethanol production technology can be classified into two major processes; separate hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF). In the SHF method, enzymatic hydrolysis and fermentation are carried out in separate vessels. This makes it possible to conduct optimal conditions of pH and temperature, but the activity of cellulase might be inhibited by the end-products (cellobiose and glucose). In the SSF method, the enzymatic hydrolysis and ethanol fermentation are carried out in the same vessel where glucose released by cellulase action is converted to ethanol immediately by yeast and this continuous removal of glucose from the hydrolysate to reduce end-product inhibition on enzyme activity. Moreover, the bioethanol presentation in the culture broth helps to avoid microbial contamination [12].

Therefore, the objective of this study is to investigate the potential of the fermantable sugar production from Napier Pakchong 1 grass residue which is a waste obtained from the process of biogas production. The effects of NaOH pretreatment on lignin removal and the optimum of enzyme loading volume and total solid contents (TS) on saccharification were investigated to evaluate the fermentable sugar yield, which is further utilized for bioethanol production.

lignocellulosic biomass

Pretreatment -

Cellulose, hemicellulose enriched biomass

Hydrolysis -

Extracellular

Glucose

Xylose, arabinose

Intracellular

Pyruvate

Acetate

Acetyl-CoA

Ethanol

Fig. 1. Simplified metabolic pathway from biomass to ethanol by solventogenic clostridia modified from [5].

2. Materials and methods

2.1. Raw material and enzyme

Napier Parkchong 1 grass (NPG) residue used in this experiment was obtained from Energy Research and Development Institute-Nakornping, Chiang Mai University (ERDI-Nakornping CMU). It was dried at 60°C for 2 days, ground to small particles, later filtered through 100 mesh and stored in sealed plastic bags at room temperature until used for characterization and pretreatment.

The cellulose hydrolyzing enzyme used in this research was the commercial enzyme (iKnowzyme, Acid Cellulase, Reach Biotechnology, Thailand), the activity of enzyme was 50 FPU.

2.2. Lignocellulosic pretreatment

Dried NPG residue was pretreated with 1.0, 2.0 and 3.0% (w/w) sodium hydroxide (NaOH) solution with total solid content 10% (w/v), in sealed Elenmeyer flask. The slurry was pretreated in the autoclave at 121°C for 60 min. The alkaline pretreatment step used in this study was modified from the method which firstly described by Zhang et al. [3]. After the mixture was cooled to room temperature, the extract residue was washed with tab water to adjust pH to neutral. After filtration, the collected solid residue was dried in hot-air oven at 80°C to maintain the constant weight for at least 48 h and stored in sealed plastic bag at room temperature for the subsequent enzymatic hydrolysis. The aim of the pretreatment is to break down the lignin component and disrupt crystalline structure of the cellulose to increase accessibility of the hydrolyzing enzyme as shown in Figure 2.

2.3. Enzymatic hydrolysis

Enzymatic hydrolysis of pretreated biomass was done in 50 cm3-plastic polypropylene tubes. The pretreated biomass equivalent to 1.0, 1.5 and 2.0 g dried basis was immersed in 1.0 M sodium citrate buffer to maintain a pH of 4.8 with working volume of 10 ml, as total solid content (TS) 10, 15 and 20% (w/v) respectively. The enzyme was added to the mixture at loading volume of 0.5, 1.0, 2.0 and 3.0 ml/g substrate. The hydrolysis was carried out for 72 h at 50°C with shaking 150 rpm. All sample was withdrawn after 0, 12, 24, 36, 48, 60 and 72 h to monitor the progress of hydrolysis. The hydrolysate was centrifuged at 4°C, 4000 rpm for 15 min and the supernatant was analyzed for total reducing sugar and the monomeric sugars by DNS method and high performance liquid

chromatography (HPLC) technique. The effect of enzyme loading volume and TS on fermentable sugar production were studied. The enzymatic digestibility was calculated as follows:

Soluble glucose (g) x 0.9 x 100

Enzymaticdigestibility (%) =--(1)

Cellulose contained in substrate

2.4. Analytical methods

The NPG residue was characterized prior and later alkaline pretreatment to determine neutral detergent fiber (NDF), acid detergent fiber (ADF), and permanganate lignin (PML) according to the method of Animal Nutrition Laboratory, Faculty of Agriculture, Chiang Mai University. The content of hemicellulose was determined as (NDF-ADF), lignin content as (ADF-PML) and cellulose content as (PML-residue after ash). The total reducing sugar in the enzymatic hydrolysate was determined by the 3,5-dinitrosalicylic acid (DNS) method [13] and the monomeric sugar in the hydrolysate was determined using HPLC technique [14] (Model 1200, Agilent). The mobile phase consisted of 5 mM H2SO4 as an eluent at a flow rate of 0.45 ml/min, and the column thermostat was set at 40°C. Sugar was detected using an RI detector (refractive index detector RID-10A) in a linear gradient over 20 min, and glycerol was used as an internal standard.

Cellulose

Fig.2. Schematic representation of the pretreatment effect on lignocellulosic biomass [23].

3. Results and discussion

3.1. Effect of NaOHpretreatment on NPG residue composition

NPG residue was pretreated with sodium hydroxide (NaOH) concentration varied from 1.0 to 3.0% (w/v) at 121°C for 60 min. The composition of NPG residue after NaOH pretreatment compared to non-pretreatment was shown in Table 1. After pretreatment, the cellulose composition was increased whereas the lignin and hemicellulose composition in pretreated NPG residue decreased. The highest lignin removal of 86.1% was observed in pretreated with a 3.0% (w/v) NaOH. After 3.0% (w/v) NaOH pretreatment, the cellulose composition was increased from 36.4 to 75.6% whereas the lignin and hemicellulose composition in pretreated NPG residue decreased from 6.0 to 2.3 and 21.4 to 11.0% (w/w), respectively. It could be due to the NaOH solution reacted with both aliphatic and aromatic structure of lignin leading to an enhancement of the internal surface area, a reduction in the degree of polymerization (DP) and disruption of cellulose crystallinity by seperation of structural linkages between cellulose, hemicellulose and lignin [15]. The recovery of NPG residue after pretreatment decreased as the NaOH concentration

increased which may be due to solubilization of the carbohydrate content during pretreatment. The cellulose, hemicellulose and lignin content were decreased to 76.3, 18.9 and 13.9 g per 100 g biomass, respectively.

After pretreatment, NaOH concentration of 3.0% (w/v) and residence time of 60 min at 121°C was found to be the best in term of lignin removal (86.1%) and cellulose to lignin ratio (5.5%). Lignin removal is an important process of the pretreatment because it can absorb cellulase enzymes decreasing the hydrolysis performance [16].

Table 1. Characterization and pretreatment of solid components of NPG residue.

NaOH concentration (% w/v) Recovery yield (% w/w) Component of the solid fraction (% w/w) Cellulose recovery Hemicellulose recovery Lignin removal Cellulose /Lignin

cellulose hemicellulose lignin (% w/w) (% w/w) (% w/w) ratio

Non pretreated 100.0 36.4 21.4 6.0 100.0 100.0 0.0 1.0

1.0 51.7 64.0 16.7 6.1 91.0 40.3 46.9 1.7

2.0 43.7 69.3 15.8 3.8 83.3 32.3 71.9 3.0

3.0 36.7 75.6 11.0 2.3 76.3 18.9 86.1 5.5

3.2. Effect of total solid contents on the fermentable sugar production

The hydrolysis of cellulose in pretreated biomass via enzymatic action is critical in releasing monomeric sugars for bioethanol fermentation. The reducing sugar and glucose in the hydrolysate were measured. The pretreated NPG residue was further used as a substrate for enzymatic hydrolysis with various total solid contents (TS 10, 15 and 20% w/v) and enzyme loading volumes (0.5, 1.0, 2.0 and 3.0 ml/g-substrate). The amount of reducing sugar and glucose yield were expected to increase at longer hydrolysis duration due to the extension of enzyme-cellulose interaction, producing more fermentable sugar (mainly glucose). The result of reducing sugar production with different total solid contents were shown in Figure 3. It was found that the reducing sugar yield increased rapidly within 24 h of saccharification and then slightly increased through the end of saccharification (72 h). The yield of reducing sugar obtained from 10, 15 and 20% (w/v) of total solid contents were 768, 716 and 612 mg/g-pretreated biomass, respectively. The increasing of total solid contents from TS 10 to 20% (w/v), the reducing sugar yield was also observed to gradually decrease. This observation was unexpected because a high total solid contents does not giving the high amount of reducing sugar. This optimum total solid content occurs because when the total solid content is increased beyond its optimum value, the fixed amount of enzyme used then becomes a limiting factor, because the end-product (cellobiose and glucose) in the hydrolysis step inhibits the activity of enzyme and mass transfer was limited within the reaction mixture due to the high viscosity of the slurry, which leads to a low product yield [17]. The pattern of increasing amount of glucose in various total solid contents showed similarly as observed in the amount of reducing sugar as shown in Figure 4. Moreover, the enzymatic digestibility of pretreated NPG residue at the high total solid contents was lower than the low total solid contents, which leads to a low of glucose yield (Table 2). Thus, the total solid contents of 10% (w/v) was selected to use as an optimum concentration of pretreated biomass for further experiment.

Table 2. Enzymatic digestibility of pretreated NPG residue after 72 h (loading enzyme 2.0 ml/g-substrate).

Total solid contents Glucose yield Enzymatic digestibility

(%) (mg/g-pretreated biomass) (%)

10 522 ± 8.5 62 ± 1.0

15 454 ± 6.0 54 ± 0.7

20 440 ± 7.8 52 ± 0.9

Fig. 3: Reducing sugar production in various total solid contents of pretreated NPG residue.

Fig. 4: Glucose production in various total solid contents of pretreated NPG residue.

3.3. Effect of enzyme loading volume on the fermentable sugar production

The effect of enzyme loading volume on the fermentable sugars production was shown in Figure 5 and 6. It was found that the amount of reducing sugar increased rapidly within 24 h of incubation time in all loading volume of hydrolyzing enzyme. The amount of releasing sugar increased when the higher concentration of hydrolyzing enzyme was added into the mixture. The amount of reducing sugar at 72 h of saccharification were 644, 693, 768 and 789 mg/g-pretreated biomass in enzyme loading volume of 0.5, 1.0, 2.0 and 3.0 ml/g-substrate, respectively.

The amount of glucose at 72 h of saccharification was detected as almost the same amount, from 468 to 519 mg/g-pretreated biomass in enzyme loading volume from 0.5 to 3.0 ml/g-substrate (Figure 6). However, 2.0 ml/g-substrate of enzyme loading volume was selected as the optimum concentration for saccharification due to the lower cost for bioethanol production and the amount of reducing sugar obtained.

The HPLC chromatogram showed the fermentable sugars obtained from enzymatic hydrolysis with total solid contents of 10% (w/v) and enzyme loading volume of 2.0 ml/g-substrate. It was indicated that the fermentable sugars after hydrolysis step contained glucose as a major product including of xylose and arabinose as shown in Figure 7.

The reducing sugar yield (768 mg/g-pretreated biomass) and glucose yield (522 mg/g-pretreated biomass) obtained from enzymatic hydrolysis were considered mostly higher than other research working on grass pretreatment (Table 3). This could be due to the effect of using higher temperature with a long period of pretreatment step which leads to high sugar yield. This study indicated that NPG residue which is a waste obtained from the process of biogas production seems to be an alternative feedstock for the cost effective bioethanol production.

Fig. 5: Reducing sugar production with different enzyme loading volume

Fig. 6: Glucose production with different enzyme loading volume

Fig. 7: HPLC analysis result of fermentable sugars in enzyme saccharified hydrolysates

Table 3. Comparison of fermentable sugar yield with other research.

Substrates Pretreatment Enzyme source for saccharification or acid Product yield (mg/g-pretreated biomass) Refs.

Reducing sugar Glucose

Napier grass NaOH (90°C, 60 min) Commercial enzyme 347 - [18]

Napier grass NaOH (90°C, 60 min) Dilute H2SO4 259 - [18]

Napier grass NaOH (120°C, 60 min) Commercial enzyme 711 - [19]

Napier grass H2O2 (35°C, 24 h) Commercial enzyme 528 - [4]

Thai Mission Grass NaOH (120°C, 10 min) Dilute H2SO4 + crude enzyme from T. reesei - 681 [20]

Switchgrass NaOH (121°C, 10 min) Commercial enzyme 365 - [21]

Switchgrass NaOH (121°C, 60 min) Dilute H2SO4 510 - [22]

Napier grass residue NaOH (121°C, 60 min) Commercial enzyme 768 522 Present study

4. Conclusion

The alkaline pretreatment is a conventional strategy for delignification of biomass, at 3% (w/v) NaOH pretreatment, more than 86.1% (w/w) of lignin was removed and enriched cellulose fraction from 36.4 to 75.6% (w/w). Hence, the pretreated biomass contained the more surface area available for enzyme action to disrupted the cellulose and hemicellulose into fermentable sugar. The reducing sugar and glucose production from NPG residue were 768 mg/g of pretreated biomass or equal to 64 g/L and 522 mg/g of pretreated biomass or equal to 43 g/L, respectively with enzyme loading volume of 2.0 ml/g of substrate and 10% (w/v) of TS. The results showed that NPG residue which is a waste obtained from the process of biogas production has potential for saccharification before fermentation process. Therefore, the NPG residue can be used as an alternative material for lignocellulosic bioethanol production with a good cost-effective raw material.

Acknowledgements

This research was supported by Multidisciplinary Science Research Centre Chiang Mai University (MSRC), Center of Excellence for Renewable Energy, Chiang Mai University. Special thanks to Energy Research and Development Institute-Nakornping Chiang Mai University (ERDI-Nakornping) and Division of Biotechnology, Faculty of Agro-Industry, Chiang Mai University for providing the laboratory facilities of this research.

References

[1] Luo L, Van der Voet E, Huppes G. An energy analysis of ethanol from cellulosic feedstock-corn stover. Renewable and Sustainable Energy Reviews 2009; 13:2003-11.

[2] Brethauer S, Wyman CE. Rewiew: continuous hydrolysis and fermentation for cellulosic ethanol production. Bioresource Technology 2013;

101:4862-74.

[3] Zhang W, Lin Y, Zhang Q, Wang X, Wu D, Kong H. Optimisation of simultaneous saccharification and fermentation of wheat straw for ethanol production. Fuel 2013; 112:331-7.

[4] Wongwatanapaiboon J, Kangvansaichol K, Burapatana V, Inochanon R, Winayanuwattikun P, Yongvanich T, Chulalaksananukul W. The potential of cellulosic ethanol production from grasses in Thailand. BioMedical 2012; 303748.

[5] Ezeji TC, Qureshi N, Blaschek HP. Bioproduction of butanol from biomass: from genes to bioreactor. Biotechnology 2007b; 18:220-7.

[6] Mosier N, Wyman C, Dale B, Elander R, Lee YYY, Holtzapple M. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology 2005; 96:673-86.

[7] Laxman RS, Lachke AH. Bioethanol from lignocellulosic biomass, part 1: pretreatment of the substrates. In: Pandey A, editor. Handbook of plant-based biofuels. CRC Press; 2008. p. 121-39.

[8] Gupta R, Lee YY. Mechanism of cellulose reaction on pure cellulosic substrates. Biotechnology Bioengineering 2008; 102:1570-8.

[9] Xu J, Wang Z, Cheng JJ. Bermuda grass as feedstock for biofuel production: a review. Bioresource Technology 2011a; 102:7613-20.

[10] Kataria R, Ruhal R, Babu R, Ghosh S. Saccharification of alkali treated biomass of Kans grass contributes higher sugar in contrast to acid treated biomass. Chemical Engineering 2013; 230:36-47.

[11] Arshadi M, Sellatedt A. Production of energy from biomass. In: James H. Clark, Fabien Deswarte, editors. Handbook on introduction to Chemicals from Biomass. 2008. p. 143-78.

[12] Wyman CE. Ethanol production from lignocellulosic biomass. In: Charles E Wyman, Philip H Abelson, editors. Handbook on bioethanol production and utilization. 1996. p. 1-18.

[13] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 1959; 31:426-8.

[14] Akpinar O, Erdogan K, Bostanci S. Production of xylooligosaccharides by controlled acid hydrolysis of lignocellulosic materials. Carbohydrate Research 2009; 344:660-6.

[15] Broder G, Yau E, Badal k, Collier J, Ramachandran KB, Ramakrishnan S. Chemical and physiochemical pretreatment of lignocellulosic biomass: A review. Enzyme Research 2011; 787532.

[16] Mussato S, Fernandes M, Milagres A, Roberto I. Effect of hemicellulose and lignin on enzymatic hydrolysis cellulose from brewer's spent grain. Microbial Technology 2008; 43:124-9.

[17] Ingerson H, Zacchi G, Yang B, Esteghlalian AR, Saddler JN. The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose. Biotechnology 2001; 88:177-82.

[18] Pirasao T. Potential of bioethanol production from Pennisetum purpureum cv. Pakchong1 grass. Faculty of engineering, Chiang Mai University. 2013.

[19] Eliana C, Jorge R, Juan P, Luis R. Effects of the pretreatment method on enzymatic hydrolysis and ethanol fermentability of the cellulosic fraction from elephant grass. Fuel 2014; 118:41-7.

[20] Prasertwasu S, Khumsupan D, Komolwanich T, Chaisuwan T, Luengnaruemitchai A, Wongkasemjit S. Efficient process for ethanol production from Thai Mission grass (Pennisetum polystachion). Bioresource Technology 2014; 163:152-9.

[21] Gao K, Boiano S, Marzocchella A, Rehmann L. Cellulosic butanol production from alkali-pretreated switchgrass (Panicum virgatum) and phragmites (Phragmites australis). Bioresource Technology 2014; 174:176-81.

[22] Nlewem KC, Thrash JR ME. Comparison of different pretreatment methods based on residual lignin effect on the enzymatic hydrolysis of switchgrass. Bioresource Technology 2010; 101:5426-30.

[23] Haregewine T, Rafael L. Advances on biomass pretreatment using ionic liquids: An overview. Energy and Environmental Science 2011; 4:3913-29.