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Natural Product Research: Formerly
Natural Product Letters
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Effect of alkaline pretreatment on
delignification of wheat straw




Umar Asghar , Muhammad Irfan , Mehvish Iram , Zile Huma ,



Rubina Nelofer , Muhammad Nadeem & Quratulain Syed

Food and Biotechnology Research Center (FBRC), PCSIR
Laboratories Complex, Ferozpur Road, Lahore 54600, Pakistan
Published online: 06 Oct 2014.

To cite this article: Umar Asghar, Muhammad Irfan, Mehvish Iram, Zile Huma, Rubina
Nelofer, Muhammad Nadeem & Quratulain Syed (2014): Effect of alkaline pretreatment on
delignification of wheat straw, Natural Product Research: Formerly Natural Product Letters, DOI:
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Among these methods. 2012). which is produced as a byproduct of wheat after cultivation. SEM. Lahore 54600.biotechnologist@gmail. Pretreated biomass after saccharification is employed in *Corresponding the production of bioethanol has attracted the researchers and proved that ethanol can be used as an alternate fuel. During the study. Email: mirfanashraf@yahoo. The pretreatment is a crucial step to breaking down lignocellulosic biomass by removing lignin to expose cellulose content for proper application of saccharifying enzyme. Ferozpur Road. The main characteristic of alkaline pretreatment is that it modified the structure of cellulose fibre. Mehvish Iram. 90 and 120 min). 2014 http://dx. Muhammad Nadeem and Quratulain Syed Food and Biotechnology Research Center (FBRC). This result indicates that the above-mentioned pretreatment conditions create accessible areas for enzymatic hydrolysis.5% NaOH for 1 h at room temperature and then autoclaved at 1218C for various steaming time (30.5% enzyme concentration using 2.5% substrate concentration for 8 h of incubation at 508C.200] at 09:54 10 October 2014 (Received 27 July 2014. Several method of pretreatment has been used for maximum degradation of lignin (Kim & Holtzapple 2006). This study investigated wheat straw. Zile Huma.5%. PCSIR Laboratories Complex. Keywords: alkaline pretreatment. Maximum saccharification yield of 52.93% was achieved with 0. Several countries have used bioethanol blend with gasoline (Hahn-Ha¨gerdal et al. wheat straw contains 35 – 45% cellulose. fermentation and distillation. Various types of lignocellulosic biomass are carbohydrates (cellulose and hemicelluloses) being used for the production of Pakistan Downloaded by [39. FTIR 1. In general. making it thermodynamically stable than the control fibre.Natural Product Research.doi.34. Results revealed that residence time of 90 min at 1218C has strong effect on substrate. The conversion of lignocellulosic biomass to ethanol is performed in four steps. Pure ethanol can also be exploited in vehicles after some modifications in their engines (Ibrahim 2012). irfan. hemicellulose and 8 –15% lignin.153.2014. pretreatment. The production of biofuel from lignocellulosic biomass is more feasible and economical than fossil fuel (Ibrahim 2012). Introduction Over the few decades. A small amount of wheat straw is used for livestock but large amounts are burned in fields causing global warming (Han et al. 2 mm size of substrate was soaked in 2. Further SEM and FTIR spectroscopy confirmed structural modification caused by alkaline pretreatment in substrate. delignification of 81% and hemicellulose content of 10. achieving a maximum cellulose content of 83%. final version received 31 August 2014) This study was conducted to analyse structural changes through scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) after alkaline pretreatment of wheat straw for optimum steaming period. 20– 30%. 2006). wheat straw. Muhammad Irfan*. hydrolysis of cellulose. 60.1080/14786419. Rubina q 2014 Taylor & Francis .964712 Effect of alkaline pretreatment on delignification of wheat straw Umar Asghar. alkali pretreatment especially by NaOH has advantages over acid pretreatment to expose maximum cellulosic contents.

we report on the changes in physical properties and cellulose microstructure of the pretreated wheat straw. whereas the hemicelluloses decreased further to 10. In the same way. 2010.30%) and lignin (19%) were determined. In this study. fermented material used in distillation for removing excess water from ethanol. wheat straw was subjected to compositional analysis on dry basis weight (w/w) and the contents of cellulose (45%).5%) increased but reduction was observed in hemicelluloses (14. the substrate was pretreated for 60 min the cellulose (77. the wheat straw was kept at 1218C steam under pressure for 30 min. 90 and 120 min) on structural changes that take place in wheat straw.51%) and delignification (72.34. the steaming time was further increased where the substrate was treated for 90 min. In continuation of our research work reported earlier (Irfan et al. delignificaion (%) Downloaded by [39. hemicelluloses (21. Moreover. 2011) regarding the delignification of pretreated plant biomass for ethanol production. As reported earlier. 17. 2013).5% sodium hydroxide (Nadeem et al. 2010). Figure 1 represents the effect of 2. this work is conducted to enhance the delignification of wheat straw by various steaming time.5% sodium hydroxide (alkaline solution) at various steaming times (30. Initially. The reduction in hemicelluloses and highest value of cellulose indicate the removal of hemicelluloses from wheat straw. resulting in the maximum cellulose content of 83% and delignification of 81%. The main objective of this study was maximum exposure of cellulose and maximum removal of lignin content from wheat straw after alkaline pretreatment. The result of earlier study showed that maximum cellulose and delignification was achieved at 2. Result and discussion Before pretreatment.153.6%) as compared with 30 min steam under pressure. 60. the major problem in bioethanol production from lignocellulosic biomass is the high cost of fermentation process based on various steps (Gupta 2008).5% sodium hydroxide and also the observed effect of steaming time on substrate. The previous study also reported that reduction of Delignification Hemicellulose 25 80 20 60 15 40 10 20 5 0 0 Control 30 60 Steam Time (min) Figure 1.2 U.5% delignification were achieved. . 2. Moreover. Recently. the alkaline pretreatment as well as under steam pressure for various times removes maximum lignin and also solubilises hemicelluloses (Irfan et al.50%. However. Comparison of control and pretreated wheat straw. a cheaper and abundantly available substrate having high cellulosic content play an important role to overcome such issues involved in commercial production of bioethanol.200] at 09:54 10 October 2014 fermentation process to produce bioethanol (Zhang et al. 90 120 Hemicellulose (%) Cellulose 100 Cellulose. where 67% cellulose. Asghar et al. Alkaline pretreatment has stronger effects on cleavage of ester bonds which are present in lignin and hemicelluloses (Modenbach 2013). 2010).30% hemicelluloses and 63. This study provides the information about the effect of 2.

The peak at 1030 . CZCZO band stretching present in control as well as pretreated which indicates the band of cellulose. Han et al (2012) reported maximum enzymatic hydrolysis at 30 h of incubation period at 508C. CvO. FTIR analysis revealed changes in pretreated and untreated wheat straw (Figure 3).153. (2013) reported that the band at 1032 cm21 was associated with polysaccharide. Further structural modification was studied by Fourier transform infrared (FTIR) spectroscopy. Scanning electron microscopy (SEM) was done to study the morphological changes in untreated and treated biomass. The earlier work reported that the peak 3334 cm21 represents the absorption of ZOH group of alcoholic hydroxyl (Irfan et al. a reduction in cellulose contents (76%) was observed. The peaks at 3336 cm21 expressed to hydroxyl group (—OH) which is present in control wheat straw and after treatment. Saliu and Sani (2012) obtained maximum sugars (7. Zhang et al.200] at 09:54 10 October 2014 Natural Product Research 3 hemicelluloses and lignin content indicates the effect of sodium hydroxide and steam explosion (Rahnama et al. These results proved that alkali pretreatment effectively removes the lignin content.26 cm21.34. when the substrate was treated for 120 min. 2011). These results expressed that treatment at 1218C for 120 min has some harsh effect on lignocellulosic biomass as compared with 90 min which may degrade the lignin. Khokhar et al. The intensity of hydroxyl group (ZOH) decreased which indicated the effect of 2. Figure 4(A) describes the effect of incubation time on saccharification process. . Moreover. The red circles (in B) represent the morphological changes of pretreated wheat straw (visible as holes) which indicates the degradation of lignin and hemicelluloses. pH 5. enzyme concentration and substrate concentration. this band was stretched and appeared as 3315. SEM of wheat straw: (A) control and (B) pretreated substrate. 2013). The previous work showed that alkaline 2% NaOH degrades the linkage between lignin and hemicelluloses.5% NaOH on wheat straw. 2011. The previous work reported that the peak at 1315 cm21 indicated hemicelluloses in control sample. 2014). Results revealed that (Figure 2) alkaline pretreatment caused pores in substrate.63 mg/mL) from alkali-treated corncobs hydrolysed with Figure 2. Enzymatic hydrolysis was optimised at 508C.0 by changing the incubation time. hemicelluloses and also solubilised cellulose contents. The pretreated substrate (wheat straw) was evaluated for enzymatic hydrolysis by commercial cellulases. (2012). Further increase or decrease in incubation period beyond this limit resulted in the decline in saccharification. These pores were created by dissolution of lignin and hemicellulose content. This peak formation was similar to the work of Han et al. increasing the surface area and making cellulose more accessible for enzymatic hydrolysis. Results revealed that maximum saccharification was obtained at 8 h of incubation period. Our findings were consistent with previous reports (Irfan et al.Downloaded by [39. These morphological changes in pretreated substrates revealed that these pores provide surface area for the penetration of cellulase enzyme to enhance the saccharification process. the second peak at 1316 cm21 corresponds to lignin which was observed in untreated wheat straw while this peak disappeared in treated (2. 1034 cm21 linked with CZO. Furthermore.5% NaOH) wheat straw.

Results (Figure 4(B)) showed that maximum saccharification was achieved at 0. Substrate concentration is also another important factor in enzymatic reactions due to the limited availability of enzyme active sites. Downloaded by [39. Furthermore. . enzyme concentration (CMCase activity of 2900 IU/mL and filter paper activity of 1500 FPU/mL) in 50 mL of citrate buffer was optimised by varying the concentration from 0.U. From this experiment.93%) as shown in Figure 4(C).5% substrate concentration gave better saccharification rate (52. Han et al.200] at 09:54 10 October 2014 4 Figure 3. High concentration (3%) of enzyme did not increase the saccharification yield. it was observed that 2. (2012) reported that 3% concentration of substrate was optimum for maximum saccharification.34.5% to 3%.5% enzyme concentration.153. cellulases of Aspergillus niger after 48 h of hydrolysis time period. Asghar et al.5% NaOH) wheat straw.5% to 3%. FTIR spectrum of untreated and treated (2. Concentration of substrate (wheat straw) was also optimised by changing from 0.

3. 3. The biomass (sample ID WS001/2014) was washed and dried to remove dust particles and then chopped to powder form (2 mm) with hammer mill.5% of NaOH solution at the ratio of 1:10 (solid/liquid) for 1 h at room temperature. the samples were filtered and solid residues were washed up to neutrality (Irfan et al.34. (B) enzyme concentration. Lignocellulosic biomass Wheat straw was used as lignocellulosic biomass.153.A Saccharification (%) Natural Product Research 5 60 e 50 d 40 30 c b 20 10 a 0 4 6 8 Incubation time (h) 10 80 60 d c 40 c b a a 20 0 0.5 2 2.5 2 2.2. 90 and 120 min). . 508C. the samples were autoclaved at 1218C for various time periods (30. Later. Optimisation of saccharification process at pH 5. 3.5 1] at 09:54 10 October 2014 B Saccharification (%) 2 60 50 40 30 20 10 0 e f d c b a 0. 2010). Lignin content was estimated by using the method described by Milagres (1994). (A) Incubation time. (%) C Saccharification (%) Downloaded by [39. Materials and methods 3. which was procured from the local market of Lahore city. Then. (C) substrate concentration.5 3 Enzyme Conc.5 1 1. 60. Pakistan. Hemicellulose was estimated by using the method of Goering and Van Soest (1975). Compositional analysis The cellulose content was estimated by using the method of Gopal and Ranjhan (1980). Pretreatment of substrate The chopped sample of wheat straw was soaked in 2.5 3 Substrate Conc. (%) Figure 4.0.

4. Saccharification (%) was calculated using the following formula (Alam et al. and thick layers were supported in the sample holder fixed on a carbon ribbon. No. After termination of enzymatic hydrolysis. Tokyo.6. FTIR spectroscopy Downloaded by [39. This assembly was maintained in a vacuum-desiccator until the analysis. 81% cellulose.5. The spectra were recorded with 32 scans in the frequency range of 4000– 400 cm21 with a resolution of 4 cm21 (Irfan et al. . Funding The authors would like to thank the Ministry of Science and Technology (MoST). 3. SEM of wheat straw Samples of untreated and treated wheat straw were oven-dried at 508C for 1 h. 50 mL citrate buffer pH 5 and 1 mL of commercial cellulase enzyme (CMCase activity of 2900 IU/mL and filter paper activity of 1500 FPU/mL) with 2% pretreated wheat straw was incubated in a shaking water bath at 508C with agitation speed of 140 rpm for 8 h. Isolation. Pakistan for providing the financial support to carry out this research work under PSDP project entitled ‘Production of Bioenergy from Plant Biomass’. Van Soest PJ.50% hemicelluloses were achieved. Agricultural Research Service.153.05) values. 2011). References Alam MZ. Forage. Washington. Statistical analysis Statistical analysis was done by using computer-based software SPSS.200] at 09:54 10 October 2014 FTIR was used to check the chemical changes in treated and untreated samples. Agricultural Handbook. characterization of cellulolytic enzymes produced by the isolate Streptomyces omiyaensis. reduction of lignin and hemicelluloses indicates the effect of physiochemical pretreatment and the enhanced surface area for enzymatic hydrolysis. The difference in values was indicated in the form of probability ( p . Manchur MA. 3. During the study.7. DC: USDA. the material was centrifuged at 10. 379. The pretreated substrate showed maximum saccharification with 0. 3. Conclusion At this concentration.4. Biol Sci.5% substrate concentration for 8 h of incubation at 508C. 2004. Asghar et al.000 rpm for 10 min. 1975. purification. Japan) was used for observing the bagasse fibres in both treated and untreated samples (Irfan et al. Anwar MN. p. 20. Enzymatic hydrolysis of substrate In 500 mL flask.5% enzyme concentration using 2. 2011). Goering H. reagents. The SEM type S-3700 microscope (Hitachi. procedures and some applications). 2004): Saccharification ð%Þ ¼ Reducing sugars ðmg=mLÞ £ 100: Substrate used ðmg=mLÞ 3.6 U. Islamabad.34. 7:1647–1653. The supernatant was removed for sugar content analysis. fiber analysis (apparatus. Mixture of sample and KBr (5% sample: 95% KBr) was passed into a disk for FTIR spectroscopy measurement. 0. 83% delignification and 10.

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