Chapter 7: Liquefaction of lignocellulosic biomass for the production of
7.2. Materials and methods
In the present study, a wild type sorghum biomass (Sorghum bicolor ssp.
Verticilliflorum) has been used as a model lignocellulosic substrate. Preparation of biomass for compositional analysis and determination of structural carbohydrates and lignin contents are performed according to NREL procedures and the results obtained are shown in Table 7.1.
Table 7.1: Compositional analysis of a wild type sorghum biomass before and after pretreatment
Composition
Raw (%)
Pre-treated (%)
Pre-treated 628 (mg)
Water extractives 10.34 - -
Ethanol extractives 1.97 - -
Cellulose 38.4 59.88 376.07
Hemicellulose 27.1a 8.2b 51.94b
Lignin 21.3 30.c 189c
aXylan- 22.2%, Arabinan- 2.1% and Acetic acid- 2.7%; b Xylan;c Acid insoluble residue.
7.2.2. Pretreatment
According to our earlier published papers [51,82,148], optimum condition for sorghum stalks pretreatment was found to be 121 ºC, 0.2 M sulfuric acid for 120 min with a solid loading of 5% (w/v). Therefore, in the present study, pretreatment was conducted at the solid loading of 15% (w/v) to acquire the fermentable sugars (glucose plus xylose) concentration of 40 g/L and above. After the pretreatment, solid and liquid fractions were separated through 0.2 µm nylon membrane under vacuum condition.
Solid fraction was washed with distilled water to attain the neutral pH and then dried at 45 ºC for 48 h. Chemical compositional analysis of pre-treated biomass was carried out according to the NREL Procedure [31] and listed in Table 7.1.
7.2.3. De-lignification of pre-treated biomass
Dilute sodium hydroxide was used as a de-lignifying agent for the pre-treated biomass. Different sodium hydroxide strengths (1–5%) were used to maximize the de- lignification process with minimal cellulose loss. This process was conducted at 121
ºC for 20 min with a solid loading of 10% (w/v). After the delignification process, solid and liquid fractions were separated through 0.2 µm nylon membrane under vacuum condition. The solid fraction was washed with distilled water under vacuum condition using 0.2 µm nylon membrane to attain neutral pH and then dried at 45 ºC for 48 h.
Weight of the biomass was noted for mass balance analysis. Chemical compositional analysis of de-lignified biomass was carried out according to the NREL procedure and listed in Table 7.2.
Table 7.2: Compositional analysis of samples after delignification process
Composition 1% NaOH 2% NaOH 3% NaOH 4% NaOH 5% NaOH
Measurement (%)
422
(mg) (%)
355
(mg) (%)
337
(mg) (%)
322
(mg) (%)
311 (mg)
Cellulose 84 354 91 324 92 311 92 297 92 288
Xyaln 6.7 28.5 5.9 21 58 19.6 5.7 18.4 5.4 16.8
AIR 8.6 36.3 2.3 8.1 1.9 6.4 1.4 4.5 1.2 3.7
Based on the above results, another set of de-lignification process was conducted at similar conditions (like temperature, time and NaOH strength and solid to liquid ratio) and the biomass was washed with distilled water and then directly subjected to enzymatic hydrolysis without drying. In general, drying of either acid or alkali treated biomass prior to the enzymatic hydrolysis shows negative impact on cellulose conversion that could be due to the hornification of biomass components.
7.2.4. Enzymatic hydrolysis
Residual materials that remained after delignification process were hydrolysed with cellulase (Celluclast 1.5L®) at 50 ºC for 24‒96 h at 140 rpm. Instead of using 50
enzymatic hydrolysis. Optimization of cellulose hydrolysis has been carried out by varying the enzyme concentrations (20‒60 mg cellulase protein/g of cellulose) with 1%
(w/v) substrate loading. After obtaining the optimum conditions for cellulose hydrolysis, effect of solid loading (2–10% w/v) were also performed for the evaluation of cellulose hydrolysis efficiency. An aliquot of 100 µL was collected from the reaction mixture at every 24 h interval, and after appropriate dilution was boiled for 10 min to inactivate the enzyme activity. Further samples were analysed by HPLC for the determination of glucose yield. After the enzymatic hydrolysis, solid and liquid fractions were separated through 0.2 µm nylon membrane under vacuum condition.
Thereafter, weight of the solid fraction after drying was noted for mass balance analysis. Liquid fraction derived from enzymatic hydrolysis was concentrated under the reduced pressure using rotary evaporator to reach desired sugar concentration for industrial titer of ethanol production.
7.2.5. Conditioning of pre-hydrolysates
Pretreatment derived acid hydrolysate (pre-hydrolysate-PH) was divided into 2 equivalent fractions (F) and are further referred as PHF-1 and PHF-2. These two fractions, PHF-1 and PHF-2, were heated up to 50 ºC and then neutralized with calcium hydroxide [Ca(OH)2] and magnesium hydroxide [Mg(OH)2], respectively under continuous stirring condition. The resultant CaSO4 of PHF-1 was separated through centrifugation at 8000 rpm for 10 min. The neutralized fractions (PHF-1 and PHF-2) were further filter sterilized under vacuum condition, and pH of both the fractions (PHF-1 and PHF-2) were readjusted to cultivation pH (6) of Pichia stipitis aseptically with 10 N H2SO4 and was further used in fermentation studies.
7.2.6. Fermentation 7.2.6.1. Microorganisms
The actively growing cultures, Pichia stipitis NCIM 3498 and Saccharomyces cerevisiae NCIM 3090 were procured from NCIM Pune, India, which are supplied on MGYP agar slants. The composition of MGYP agar medium contains (g/L) 3, Malt extract; 10, Glucose; 3, Yeast extract; 5, peptone; 20, agar; Temperature and pH of the MGYP agar medium maintained at 30° C and 6.4–6.8, respectively.
7.2.6.2. Sub-culturing and seed culture preparation
Pichia stipitis 3498 and Saccharomyces cerevisiae NCIM 3090 have been used in the present study. Sub-culturing and seed culture preparation was performed according to the method described in section 6.2.4.1.
7.2.6.3. Xylulosic ethanol production from pretreatment derived hydrolysate
To evaluate the ethanol conversion efficiency from the filter sterilized hydrolysates of PHF-1 and PHF-2, fermentation experiments were conducted in batch mode for the production of xylulosic ethanol. Each fermentation medium contains, 2%
(v/v) of sterilized 50X concentrated nutrient solution (1.7 g of yeast nitrogen base, 1 g of urea and 6.56 g of peptone in 20 mL of distilled water), and 5% (v/v) of inoculums (which gives an initial cell concentration of 2 g/L). Initial pH of the media was adjusted to 5.5 with 10N H2SO4 and incubated at 30 °C and 140 rpm. All fermentation samples were taken periodically for HPLC analysis.
7.2.6.4. Cellulosic ethanol production from enzymatic hydrolysis derived hydrolysate The fermentation experiments were also conducted in batch mode with varying enzymatic hydrolysate loading with respect to glucose concentration i.e., 96 g/L and
170 g/L. Along with this, both the fermentation media contain, 4% (v/v) of 25X YP nutrient solution (10 g of yeast extract and 20 g of peptone in 40 mL of distilled water), and 6% (v/v) of seed culture (which provides an initial concentration of 1.6 g/L on cell dry weight basis). Initial pH of the fermentation broth was maintained at 5.5 and incubated at 30 °C and 150 rpm for 48 h.
7.2.7. X-Ray Diffractometer (XRD) Analysis
The X-Ray diffractometer (Bruker, D8- Advanced XRD measurement systems, Japan) analysis of untreated, pretreated and delignified biomass samples was performed to evalute the crystalline nature of cellulose present in the biomass samples. The XRD was equipped with Cu Kα (λ=1.541 Å) radiation settled at 40 KW voltage and 40 mA current. The diffraction angle (2θ) was 8 to 40° at a step size of 0.05° and scan speed 1°/min. The following equation (Eq. 7.1) was used for the determination of crystallinity index (CI) which was based on the diffraction intensities of crystalline and amorphous regions [185].
002 amp
002
I - I
Crl (%) = ×100
I (7.1) Where, I002 = the peak corresponding to 002 lattice plane of a cellulose at 2θ = 22.4°, and Iamp = amorphous region peak intensity at 2θ = 15.6° [186].
7.3. Results and Discussion