CHAPTER 6 STUDY ON THE EFFECT OF PRETREATMENT AND ENZYMATIC HYDROLYSIS IN BIOETHANOL PRODUCTION

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1 CHAPTER 6 STUDY ON THE EFFECT OF PRETREATMENT AND ENZYMATIC HYDROLYSIS IN BIOETHANOL PRODUCTION 6.1 INTRODUCTION The growing industrialization and population has increased the energy requirement due to the limited fossil fuel quantities in reserve. Renewable energy fuel have gained importance in emerging worldwide acceleration of energy demand. The production of bioethanol from renewable biomass have created great interest towards the long term energy supply and environmental friendly technologies. Bioethanol is mostly produced by sugar and starch containing food crops which pertains the world food security. But recently well carbon balanced lignocellulosic biomass like agricultural wastes, woody crops and weeds have been represented as a second generation renewable biomass energy. To one side other than crop biomass the availability of weedy lignocellulosic biomass have been explored as potential biomass for lignocellulosic ethanol production. In recent decades many studies have been focused on common weedy biomass for their proficiency for bioethanol production and identified Eichhornia crassipes, Lantana camara, Prosopis juliflora, Chromolaena odorata and Saccharum spontaneum as an effective feedstocks (Huber and Dale 2009, Chandel and Singh 2011). Water hyacinth will be an efficient biomass from an environmental and economic elucidation if it is used for wastewater treatment and as raw materials for the production of bioenergy. In this way the cost and labor for the cultivation of biomass can be minimized. Many studies have investigated the potential use of water hyacinth as a raw material for the production of bioenergy, animal feed and fertilizer (Tantimongcolwat et al. 2007, Mishima et al. 2008). Since, water hyacinth is mainly 89

2 composed of lignocelluloses which typically contain 55 57% of dry weight (DW) of carbohydrates are polymers containing sugar units of five and six carbon atoms and it contains cellulose, 34.19% hemicelluloses, 17.66% and lignin, 12.22% (Vásquez et al. 2007). Unlike other starch or sugar substrate pretreatment is also necessary to use water hyacinth in bioconversion for the production of ethanol which represents a promising alternative fuel to reduce environmental problems. Conversion of lignocellulosic materials to fermentable sugars and finally to ethanol must be performed at low cost. Enzymatic hydrolysis is one of the effective and economically applicable methods to generate monomeric sugars for the conversion into ethanol as shown in equation 6.1 (Farrell et al. 2006, Lee et al. 2009). (C6H10O5)n + n H2O nc6h12o6 2n C2H5OH + 2n H2O Equation 6.1 In this chapter water hyacinth substrate have been subjected to dilute sulphuric acid pretreatment followed by enzymatic hydrolysis using fungi Trichoderma reesei and ethanol fermentation by yeasts Saccharomyces cerevisiae, Pachysolen tannophilus, Candida intermedia and Pichia stipitis. 90

3 6.2 MATERIALS AND METHODS Substrate preparation chapter The preparation of the substrate was carried out as described earlier in the Microbial strains and culture media Four yeasts namely Saccharomyces cerevisiae NRRL Y , Pachysolen tannophilus NRRLY-2460, Candida intermedia NRRL Y , Pichia stipitis NRRL Y 7124 and one fungal strain Trichoderma reesei NRRL were acquired from Agricultural Research Service - New York. Stock of the fungal and yeast cultures were maintained on Potato Dextrose Agar (PDA) and Yeast Malt Agar (YMA) slants at 4 C Acid pretreatment Water hyacinth was treated with various concentrations of H2SO4 viz., 1%, 2%, 3% and 4% (v/v) to find out the optimum concentration of H2SO4 that yielded maximum reducing sugars Estimation of reducing sugars The total reducing sugar was estimated using DNSA method by the procedure given in the chapter

4 6.2.5 Enzymatic hydrolysis The solid biomass obtained after pretreatment was added in a medium 2 g/l of xylose and 2 g/l mixture of yeast extract and peptone, autoclaved at 121 C for 30 minutes. Inoculation was done with T. reesei mycelium agar plugs taken from PDA plate and incubated at ph 5 at 32 C for 120 hours (shaking at 150 rpm). Enzyme production was carried out in the table top fermenter (2.5 L Lark, India). Three replicates were used in all the experiments and autoclaved but uninoculated substrates were taken as controls Enzyme activity The cellulase and xylanase activity was assayed using the methods given in the previous chapter Structural change in the biopretreated biomass Alteration in structure of pretreated substrate and controls were characterized by analytical techniques using SEM, FTIR and XRD Scanning electron microscopy analysis The surface morphological changes in native and enzymatic hydrolyzed water hyacinth were analyzed by Scanning Electron Microscope (SEM) (JOEL JSM 6390, Japan). Specimen to be coated were mounted on a conductive tape and coated with gold palladium using a JOEL JFC 1600 auto fine coater and observed using a voltage 10 to 15 KV. 92

5 Fourier transmission infrared analysis Structural and functional group changes in water hyacinth constituents during pretreatment were analyzed by FTIR (Shimadzu Spectrometer, Japan). Raw and pretreated biomass (3-4% w/w) were thoroughly mixed with dry powdered spectroscopic grade KBr and the mixture was pressed with 10,000 psi into a transparent pellet. The spectra were obtained at 4 cm -1 resolution accumulating 25 scans per spectrum over the wave number range cm X-ray diffraction Crystallographic structure was analyzed by X-ray diffraction peak height method of raw and pretreated samples using XRD 6000 (Shimadzu diffractometer, Japan). Diffraction patterns were recorded by using Cu-Kα radiation at 40 kv and 30 ma and grade range between 10 to 30 with a step size of Cellulose crystallinity was calculated using the pragmatic equation (Segal et al. 1959). where I020 is the intensity for the crystalline portion of cellulose whereas Iam is the amorphous portion and I020 is intensity diffraction at Fermentation of water hyacinth hydrolysates Hydrolysates obtained after acid and enzymatic pretreatments were subjected to fermentation by S. cerevisiae and xylose-fermenting yeasts Pachysolen tannophilus, Candida intermedia and Pichia stipitis. Log phase cultures (10% v/v) were used at ph 6.0 and incubated at 30 C with 125 rpm up to 72 hours. Fermentation was carried out in the table top fermenter (2.5 L Lark, India). The ph of the hydrolysates was adjusted with 1N HCL and 1N NaOH. Samples were taken for every 12 hours and the pentose 93

6 sugar (xylose) concentration was measured using phloroglucinol assay and the reducing sugars released were monitored by DNSA method. Samples were distilled at C using separate distillation unit and the distillate was used for ethanol quantification Estimation of xylose (Eberts et al. 1979; Johnson et al. 1984) At first 0.5 g of Phloroglucinol was mixed with 100 ml of glacial acetic acid and 10 ml of concentrated HCL to form a coloured reagent. 200 µl of sample was mixed with 5 ml of coloured reagent and heated at 100 C for 4 minutes and cooled down to room temperature and absorbance was measured at 540 nm (Hitachi U-2910 Spectrophotometer, Japan) Estimation of ethanol (Bennett 1971; Pilone 1985) Potassium dichromate assay method was followed to estimate the ethanol concentration in fermentation media. Add 34 g of K2Cr2O7 in 500 ml distilled water and 325 ml concentrated H2SO4 slowly added to the flask in an ice bucket. Added 7.5 ml of distillate with 12.5 ml of K2Cr2O7 solution and made up the volume to 25 ml with distilled water then kept at 60 C for 30 minutes and absorbance was measured at 600 nm (Hitachi U-2910 Spectrophotometer, Japan) Ethanol quantification At first the ethanol concentrations were determined by dichromate assay and the final distillate after incubation time was analyzed using gas chromatography (GC- 2010, Shimadzu, Japan) equipped with a flame ionization detector and a column of Porapak Q using N2 as carrier gas at a flow rate of 30 ml min -1 at an oven temperature of 130 C. The injector and detector temperature were kept at 200 and 230 C respectively. 94

7 6.3 RESULTS Acid pretreatment The maximum sugar yield of mg per gram of water hyacinth was obtained when the substrate was treated with 4% (v/v) of H2SO4 as shown in Fig.6.1. Figure 6.1 Sugar yield by using different concentrations of sulphuric acid Enzymatic hydrolysis of water hyacinth The cellulase and xylanase enzyme production started at the 3 rd day and showed high enzyme activity of cellulase IU/mL and xylanase IU/mL at 5 th day respectively as shown in Fig The amount of glucose, xylose and total reducing sugars produced per 1 g of water hyacinth biomass ranged from 0.07 to 0.41 g Fig

8 Figure 6.2 Production of cellulase and xylanase from WH hydrolysates by T. reesei Figure 6.3 Total reducing sugars concentration of the enzymatic hydrolysates of water hyacinth 96

9 6.3.3 Analysis of structural changes during pretreatment SEM analysis of pretreated biomass SEM observation revealed the physical change in cell wall structure of native and biopretreated biomass Fig 6.4. A B Figure 6.4 Scanning electron micrographs of Native water hyacinth (A) and Acid enzyme treated (B) water hyacinth 97

10 FTIR analysis Chemical changes in the lignin skeleton of water hyacinth biomass constituents during T. reesei mediated biopretreatment were analyzed by FTIR shown in Fig.6.5. The typical lignin syringyl-guaiacyl-hydroxyphenyl (SGH) were observed by detection of peaks at 1127 cm -1 and 843 cm -1. The distribution of lignin associated bands especially at 1649 cm -1 suggested the C-H deformation within the methoxy groups of lignin. Figure 6.5 FTIR analysis of chemical changes in T.reesei pretreated water hyacinth. 98

11 Cellulose crystallinity measurement The XRD data illustrates further significant differences in the patterns in T. reesei biopretreated water hyacinth biomass as shown in Fig.6.6. Initially cellulose I characteristic diffraction pattern at two theta equal to 14.9, 17.1 and 22.8 were observed in raw WH. The crystallinity index of raw WH was 43.17%, which decreased to 41.03% after T.reesei biopretreatment. Figure 6.6 X-ray diffraction analysis of native and T.reesei pretreated water hyacinth for cellulose crystallinity. 99

12 6.3.4 Ethanol production by different strains of yeast The results of water hyacinth hydrolysates fermented by the yeast strain S.cerevisiae, P.tannophilus, C.intermedia and P.stipitis which yielded 0.13 to 0.37 g/g of ethanol concentration and 3.8 to 0.4 g/l xylose concentration as shown in Figures 6.7 and 6.8 Figure 6.7 Ethanol production from water hyacinth hydrolysates by yeast strains 100

13 Figure 6.8 Xylose sugar concentrations from water hyacinth hydrolysates by yeast strains Gas chromatography The gas chromatogram confirms the ethanol production with a retention time of 2.65 minutes by P. tannophilus strain from the water hyacinth biomass and 99.8% standard ethanol are shown in Figures 6.9 and

14 Figure 6.9 Gas chromatogram from distilled ethanol fermented by Pachysolen tannophilus Figure 6.10 Gas chromatogram of standard ethanol 102

15 6.4 DISCUSSION A number of reports exist on the pretreatment of water hyacinth using various methods such as dilute NaOH (Mishima et al. 2008), steam pretreatment (Harun et al. 2011). Shear stress and tensile forces during the pretreatment process will result in the breakdown of the substrate and consequently proliferate the surface area for ease cellulose accessibility (Chen et al. 2011). Pretreatment using sulphuric acid showed high sugar yield of mg per gram of water hyacinth sample treated with 4% (v/v) of H2SO4 compared to other acids and alkali. Though this pretreatment may not be appropriate for different feed stocks due to their lignocellulosic content and process requirements (Rogalinksi et al. 2008). Persisting boiling and steaming pretreatments resulted in reduced amount of sugar yield. The increased acid concentration due to the formation of organic acids in the course of extended retention time which ultimately leads to the degradation of sugars into aldehydes like furfural and 5-HMF (Balat et al. 2008). The cost of cellulase and xylanase plays a significant role in the cost of lignocellulosic ethanol production by enzymatic hydrolysis. Crude enzyme preparations attained after fermentation of acid pretreated WH were used in this study is to reduce the cost effect of ethanol production. The enzymatic hydrolysis and fermentation of the pretreated WH substrate can be done separately (SHF) or simultaneously (SSF). The ability of SHF is to carry out enzymatic hydrolysis and fermentation under optimized conditions. Whereas in SSF the immediate consumption of reducing sugars by microorganisms is an advantage avoiding enzyme inhibition (Ohgren et al. 2006). The final glucose concentration was high when both cellulase and xylanase enzymes are used together but decreases when either enzyme used alone. The amount of glucose, xylose and total reducing sugars ranged from 0.07 to 0.41 g and this values were similar to the values reported by Takagi et al. (2012). When 25 g/l of 103

16 WH was used the glucose and xylose conversion was 0.35 g and 0.25 g per gram biomass and it was 4.25 times higher than the conversion (0.18) reported by Kumar et al. (2009a). Structural changes in the SEM analysis showed the alteration in intact structure through formation of holes and crevices in biomass surface as a result of colonization by fungal hyphae. According to the earlier studies of Rollin et al. (2011) the intrinsic structural changes can shoot up the biomass reactivity by enhancing the cellulose accessibility towards better enzymatic hydrolysis. Significant lignin reduction was found in all these wave numbers. The absence or undetectable bands in FTIR analysis also suggested that the release of lignin in biopretreated biomass has occurred. Furthermore the ester linkage C=O with a peak at cm -1 which generally characterizes the acetyl group in the linkage between hemicelluloses and lignin was reduced in this band reflecting the breakage of ester group. Significant reduction of band intensity was also observed at cm -1 (C-O stretching in lignin and hemicellulose) and cm -1 (C-H deformation in hemicelluloses) which indicates the removal of hemicelluloses after biopretreatment (Kumar et al. 2009b, Li et al. 2010d). The spectrum also showed that the band intensity at cm -1 (OH vibration) cm -1 and cm -1 (C-H methyl and methylene groups) decreased after enzymatic hydrolysis (Wu et al. 2011). The crystallinity index of native water hyacinth is 20.49% and that of biopretreated water hyacinth is 54.37%. Similar observation has been reported by Sathyanagalakshmi et al. (2011). A contrary result by Li et al. (2010b) reported decrease in crystallinity index for ionic liquid pretreatment of switch grass indicating that the product is highly amorphous and therefore has an increase in cellulose surface accessibility and would theoretically enable more cellulose hydrolysis. The XRD data 104

17 also confirmed the improvement of amorphous nature of cellulose which provides many free β-glucosidic sites for endoglucanase activity (Zhang et al. 2012a). The xylose fermenting yeast P. tannophilus showed the maximum ethanol productivity, in 1 g of water hyacinth biomass yielded g ethanol followed by other two fermenting yeasts C. intermedia and P. stipitis yielding to g ethanol respectively, which was high when compared to the previous reports on ethanol production from WH hydrolysates using yeasts (Nigam 2002, Takagi et al. 2012, Ahn et al. 2012) whereas S. cerevisiae yielded very low amount (0.015 g) as this yeast cannot assimilate the pentose sugars. The toxic compounds (furfural and 5- dihydroxymethyl furfural) produced during the acid pretreatment of lignocellulosic biomass may have some inhibitory effect on the fermentation process (Adrados et al. 2005) the initial sugar loss during the hydrolysis may also resulted in the low ethanol production. The theoretical yield of ethanol was lower (6%) in hydrolysates obtained through enzymatic pretreatments when compared with synthetic sugars. This might be due to the low concentration of easily metabolizable sugars in the hydrolysates for yeast metabolism (Zhang et al. 2010). The GC results of the standard ethanol and Pachysolen tannophilus distilled ethanol seems to have the same retention time of 2.65 minutes and matrices in the previous studies the retention time for ethanol was 2.70 minutes (Helena et al. 2009). This confirms the ethanol production by chromatogram and also showed the presence of other compounds such as ethyl acetate, 3-methyl 1- butanol and propanol which are generally produced during the alcoholic fermentation (Dragone et a l. 2009). Hyum-Beom et al. (2009) has recommended using potassium dichromate for the determination of distilled ethanol obtained from yeasts fermentation and obtained similar results for gas chromatography. 105

18 Results reveal that using sulfuric acid hydrolysis followed by bioconversion of P. tannophilus yielded maximum ethanol (1.14 g/l) with 176 maximum yield coefficient (0.24 g g -1 ) and productivity (0.015 g L -1 h -1 ). These values are well comparable to those obtained from phenol-tolerant strain of xylose fermenting bacterium (Asli et al. 2002). This coefficient is greater than the results reported elsewhere using acid hydrolysis (0.14 g g - 1 ) and cellulase catalysis reaction (0.18 g g -1 ) (Dagino et al. 2013). Corresponding to the ethanol concentration of g/l, the ethanol yield was calculated as 0.42 g ethanol/g reducing sugar accounting for as high as 74% of the stoichiometric value. Many authors have reported on ethanol production by Saccharomyces cerevisiae (Benerji et al. 2010, Yah et al. 2010, Ahn et al. 2012, Takagi et al. 2012), Pichia stipitis (Nigam 2002) and Candida intermedia (Manivannan et al. 2012). Very few studies have been carried out on bioethanol production from lignocellulosic feed stocks using P.tannophilus (Sathesh Prabhu and Murugesan 2011). The present study is the first report on production of ethanol from water hyacinth using P.tannophilus (Manivannan and Narendhirakannan 2014). Feedstock availability and sustainability are the main limitations of bioethanol commercialization. In this present study, the weedy lignocellulosic biomass water hyacinth was explored as next generation biofuel resource due to its growth sustainability. The purpose of the present work is to investigate the possibility of using crude cellulases and xylanases produced by T. reesei using water hyacinth biomass to be more cost effective. Therefore, this report emphasizes that both hexose and pentose produced in the enzymatic hydrolysis of water hyacinth biomass is easily fermented by P. tannophilus yeast. In conclusion, the data obtained for fermentation in lignocellulosic residues revealed that the dilute acid pretreatment followed by enzymatic hydrolysis indicates that the water hyacinth biomass stands as an alternative feed stock and is economically favorable for fuel ethanol production. 106