With concerns over issues relating to global warming and depletion of fossil fuel reserves, the search for sustainable and renewable energy sources has become a matter of global interest [1]. To reduce the net contribution of green house gases (GHGs) to the atmosphere, bioethanol has been recognized as one of the alternatives to petroleum-derived transportation fuels [2]. Ethanol production from biomass (lignocellulosics) has received major research attention due to their abundance and potential for biochemical conversion to fuels and chemicals. Various lignocellulosic feedstocks e.g. rice straw, corn cobs, wheat straw, sugarcane bagasse etc. have been investigated in this context. Sweet sorghum bagasse, an agro-industrial residue left after juice extraction from its stalk, is yet another potential source. Moreover, with an expected increase in the number of sweet sorghum-based ethanol plants in countries like China, sweet sorghum bagasse is expected to become widely available [3].

Lignocellulosics consist of cellulose (a linear polymer of glucose, a C 6 sugar), which is associated with hemicellulose (a heteropolymer consisting mainly of xylose, a C 5 sugar) and surrounded by lignin seal which makes the structure resistant to enzymatic attack. Pretreatment is required to alter the structure of biomass so that cellulose becomes more accessible to the enzymes that break it into its component monosaccharide (glucose). Pretreatment effectiveness is usually evaluated in terms of hemicellulose solubilization, extent of delignification, enzymatic convertibility of cellulose fraction and recovery of fermentable sugars from both cellulose and hemicellulose fractions. Pretreatment not only modifies the chemical composition of lignocellulose, but also the physical characteristics e.g. crystallinity of cellulose, disintegration of the compact structure etc. This, in turn, may affect the rate of enzymatic saccharification. One of the important goals of pretreatment is to increase the accessible surface area of cellulose for enzymes to enhance the conversion of cellulose to glucose [4, 5].

Various lignocellulosic pretreatment methods such as acid, alkaline and steam pretreatment have been investigated. Acid treatment solubilizes the hemicellulose while alkaline treatment solubilizes mainly lignin. Steam pretreatment/explosion also solubilizes the hemicelluloses by ‘exploding’ and rupturing the biomass structure to make the cellulose better accessible for enzymatic saccharification [6].

The objective of this work was to compare two pretreatment methods viz., chemical (acid followed by alkaline peroxide) pretreatment and steam pretreatment for sweet sorghum bagasse fractionation. The performance was evaluated on the basis of the total amount of fermentable sugars released in the hydrolyzate and after enzymatic saccharification.

Sweet sorghum bagasse (Sorghum bicolor (L) Moench) was received from Directorate of Sorghum Rasearch (DSR), Hyderabad (Figure 1). Chemical treatment was conducted with bagasse samples of > 180 micron particle size obtained after grinding, sieving and overnight drying at 105°C. Steam pretreatment was carried with as-received bagasse.

Lignin (acid soluble and insoluble), extractives (water and ethanol) and ash content of the untreated bagasse and the solid residue remaining after pretreatment were analyzed by Laboratory Analytical Procedures (LAP) from the National Renewable Energy Laboratory [7]. Hemicellulose was estimated spectrophotometrically using para-bromo aniline. The method is free of interference from most of the non-pentose components of lignocellulosic substrates [8]. Cellulose estimation was standardized based on the method developed by Updegraff [9].

Two-stage chemical pretreatment was conducted with 1% (w/w) H₂SO₄ at a substrate loading of 10% (w/w) in a 1L flask. The temperature was 121°C with 1 h residence time. This was followed by washing the residue with distilled water till pH of the wash water became neutral. The residue was delignified with 1.5% (w/w) NaOH and 0.5% (w/w)H₂O₂ at 121° C for 1 h. The reaction mixture was filtered through a 50 mm with 50 micron nylon sieve, washed till pH became neutral and the solid residue so obtained was stored at 4°C prior to enzymatic saccharification. Steam pretreatment was performed at 10% (w/w) substrate loading in a locally fabricated pressure vessel connected to a 10 kg/cm² boiler (Kaytherm, Delhi, India). The study was performed at varying steam pressures (6 and 10 kg/cm²) and residence time (5 and 10 minutes). Part of the pretreated solid residue was washed repeatedly with distilled water. Both washed and unwashed fractions were stored at 4°C prior to enzymatic saccharification.

Enzymatic saccharification for 10% (w/v) substrate was carried at 50°C using cellulase and cellobiase at a loading of 0.1 g cellulase/g dry substrate and 0.01 g cellobiase/g dry substrate respectively. Both enzymes were procured locally (Aum Enzymes, Gujarat, India). The reducing sugars released were analyzed by 3, 5-dinitro-salicylic acid (DNS) method [10].

The composition of the as-received sweet sorghum bagasse was 42.6% (w/w) cellulose, 26.2% (w/w) hemicellulose, 13% (w/w) lignin, 3% (w/w) ash, and 11.3% (w/w) extractives.

Figure 2 presents a comparison of the reducing sugars released by the two pretreatment methods. In the two stage chemical pretreatment, dilute acid hydrolysis resulted in reducing sugars concentration of 27.5 g/L. Since hemicellulose is mainly solubilized in this step, the concentration of C 5 sugars was higher than that of C 6 sugars (data not shown). The left-over solid residue is rich in cellulose and acid-insoluble lignin. This was subjected to alkali-peroxide treatment to remove residual lignin before enzymatic saccharification of the cellulosic fraction. Compared to dilute acid hydrolysis, steam pretreatment (10 kg/cm² and 10 minutes residence time) led to a marginally lower reducing sugars concentration (25.5 g/L). Further, the total reducing sugars content was significantly lower (15.6 g/L) at lower steam pressure (6 kg/cm²).

Figure 3 presents the results of enzymatic saccharification of the residues obtained after different pretreatments. The highest reducing sugars concentration (26.6 g/L) was obtained with the unwashed residue obtained after steam treatment at 10 kg/cm² for 10 minutes. For a given steam pressure and residence time, the unwashed fraction resulted in higher reducing sugars concentration than the washed fraction. At 10 kg/cm² steam pressure, the values were 26.6 g/L (unwashed) and 19.3 g/L (washed). Reducing the steam pressure to 6 kg/cm² lowered the reducing sugars content in the saccharified fraction as well viz. 22.7 g/L (unwashed) and 17.4 g/L (washed). The sugars released from the two stage pretreatment (18 g/L) was comparable to sugars released from the washed, steam pretreated fraction at lower pressure.

The combined steam treatment-enzymatic saccharification process resulted in overall release of 75.7% of the total reducing sugars originally present in the biomass. The value was lower (66.1%) for the two-stage chemical pretreatment-enzymatic saccharification process.

Cost-effective production of fermentable sugars from biomass remains the largest obstacle to emerging lignocellulosic ethanol plants. The unwashed solid residue obtained after steam pretreatment resulted in higher reducing sugar concentration as compared to the residues which were washed before the enzymatic saccharification. This was attributed to the presence of residual or free sugars in the biomass. Physical pretreatment like size reduction as done prior to two –stage chemical pretreatment has a major advantage in increasing the substrate accessibility to the enzymes; however, it comes at the cost of increased energy consumption required for milling. In contrast, size reduction can be omitted for the steam pretreatment process. The steam pretreatment process opens the complex lignocellulosic matrix by physical rupture to release the hemicellulose and lignin from the matrix and exposes the cellulose for enzymatic attack. Thus steam pretreatment followed by enzymatic saccharification is an effective process for converting sweet sorghum bagasse into fermentable monosaccharides for bioethanol production.

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