Production of Biofuel from Agricultural Plant Wastes: Corn Stover and Sugarcane Bagasse

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Corn stover was collected from farmer’s field after cob harvesting. The stalks were defoliated and cut in to small pieces. The pieces were then oven dried at 60 °C for 72 h (6 – 8 % moisture). It was treated at 60 °C because if the temperature was higher, it will affect the enzymes in the corn stover. Once dried, the materials were grinded using the grinding machine. The grounded sample was sealed in the seal bag or poly bag and stored in room conditions. The sugarcane bagasses were collected from the North Bengal Sugar Mill. The bagasse were also oven dried at 60°C for 72 h like corn stover, milled, packed in concealed poly bags and stored in room condition.

Lignocellulose is a complex carbohydrate polymer of cellulose, hemicellulose and lignin. Cellulose is linear and crystalline. It is a homopolymer of repeating sugar units of glucose linked by b-1,4 glycosidic bonds. Hemicellulose is a short and highly branched polymer. It is a heteropolymer of D-xylose, D-arabinose, D-glucose, D-galactose, and D-mannose. Lignin is hydrophobic in nature and is tightly bound to these two carbohydrate polymers. It thus protects these polymers from microbial attack. It is a three-dimensional aromatic polymer of p, hydroxyphenylpropanoid units connected by C-C and C-O-C links. Sugar compositions of various agro wastes (rice straw, wheat straw, corn straw, bagasse) are given in Table 2 ^[16]. Lignocellulosics are processed for bioethanol production through three major operations: pretreatment for delignification is necessary to liberate cellulose and hemicellulose before hydrolysis; hydrolysis of cellulose and hemicellulose to produce fermentable sugars including glucose, xylose, arabinose, galactose, mannose and fermentation of reducing sugars.

Table 2. Carbohydrate content of corn stover and sugarcane bagasse (%)

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2.3. Hydrogen Peroxide Pretreatment for Lignin Removal

The removal of lignin is necessary for cellulose to become readily available for the enzymes for asccharification, which permit the yeast to convert the glucose into ethanol ^[18]. In the present study the delignification was done with the pretreatment of feedstock with H₂O₂ in alkaline condition according to method developed by Dawson and Boopathy ^[19]. Milled corn stover and sugarcane baggase were slurried in water (12.5%,w/v unless otherwise stated) containing H₂O₂ (0-5%, v/v) were placed in 400ml Boro-silicate bottles (Boro 3.3, Isolab,Gemany) and adjusted to pH of the solution was 11.5 using NaOH and shaken in an incubator at 150 rpm at 25°C and 35°C for 6-24 h. Each H₂O₂treatment combination was repeated four times. After required time of soaking, the residue was removed from the solution by filtering through cheesecloth. The residues were washed three times with deionized water. The residues were then dried in an oven at 60°C for 12 h. Finally, the residues were re-weighed. The weight difference was equated to the amount of lignin removed. The both types of delignified feed stock materials were adjusted to P^H 4.5 using concentrated HCL before enzymatic saccharification. Although lignocellulose is the most abundant plant material resource, its susceptibility has been curtailed by its rigid structure. As the result, an effective pretreatment is needed to liberate the cellulose from the lignin seal and its crystalline structure so as to render it accessible for a subsequent hydrolysis step ^[20]. By far, most pretreatments are done through physical or chemical means. In order to achieve higher efficiency, both physical and chemical pretreatment are required. Physical pretreatment is often called size reduction to reduce biomass physical size. Chemical pretreatment is to remove chemical barriers so that the enzymes can access to cellulose for microbial destruction.

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Figure 1. Effect of H₂O₂ concentration (0-5%, v/v) on lignin removal of corn stover and sugarcane baggase

The effect of H₂O₂ pretreatment of corn stover and sugarcane bagasse in alkaline solution on lignin removal is shown in Figure 1. It shows that the pretreatment of both types’ raw materials with 2% H₂O₂at pH 11.5 and soaking for 48 h remove the most lignin content effectively. This treatment was chosen for the rest of the fermentation experiment. The amount of weight loss after H₂O₂pretreatment was due to lignin removal ^{[18, 19]}. The % of weight loss indicate that the removal of lignin from total amount of lignin (15.1% for corn stover and 18.4% for sugarcane baggase).

2.4. Enzymatic Saccharification of Delignified Sugarcane Bagasse and Corn Stover

The enzymatic saccharification of the alkaline peroxide pretreated corn stover and sugarcane baggase were performed by shaking gently (150 rpm) at 37°C in an incubator after adjusting the pH to 4.5 with HCl. An enzyme mix consist of two types of cellulytic enzymes viz., alpha-amylase and glucoamylase (Cabiochem, USA) were used for enzymatic saccharification of both types delignified feed stock materials. Saccharification was done in two steps. In first step 1µl of enzyme alpha- amylase diluted with phosphate buffer was added to the slury and incubated at 37°C in an incubated shaker at 150 rpm for 24 h. Then, 1 µL of secondary enzyme, glucoamylase diluted with acetic acid with sodium acetate buffer was added to the mixture. The mixture was maintained at 37°C for 24-120 h as the glucoamylase hydrolyzed the dextrin to fermentable glucose. Samples (1 mL) were withdrawn and kept at -20°C before analysis.

2.5. Preparation of Buffer

Buffer was used to dilute the enzyme alpha-amylase and glucoamylase. Enzymatic reaction will be more effective if dilute with buffer compared to distilled water. There were two types of buffers prepared which were phosphate buffer for α-amylase and acetic acid with sodium acetate buffer for glucoamylase. The prepared buffer was covered with aluminum foil and kept at room temperature for further use.

2.6. Microbial Source

Baker’s yeast (Saccharomyces cerevisiea) produced by S.I. Lesaffre, France, was prepared on agar plate prior to cultivation. S. cerevisiea culture was maintained in tryptic soy broth (TSB) and a fresh one week old culture grown on TSB was used in the fermentation experiments.

2.7. Fermentation Experiments

The hydrolysates from corn stover and sugarcane bagasse were filtered and 800 ml of filtered hydrolysate were transferred to one liter anaerobic fermentator. The pH of the hydrolysate was to 3.0, 3.5, 4.0, 4.5 and 5.0 (Table 4). The the volume of the hydrolysate was adjusted to one liter and autoclaved at 121°C (1.5 psi) for 20 min. After autoclaving the mixture was cooled down to 32°C and 10 ml of activated Saccharomyces cerevisiea (baker yeast) was added to the hydrolysate. The openings of each of the fermenator bottle were concealed with air lock system for ensuring anaerobic condition in side fermentator bottle. Then the fermentators were incubated at range of temperature from 25 to 50°C (Table 5) for 120 h.

2.8. Distillation of Ethanol

After 120 hours, the sample was filtered using Whatman Filter Paper to separate the ethanol from the residue. The bioethanol was distilled using rotary evaporator. The sample was heated at 80°C to get the bioethanol.

2.9. Analytical Procedure

Sugars, furfural, HMF, acetic acid, ethanol, and succinic acid were analyzed by high-pressure liquid chromatography (HPLC) ^[21]. The separation system consisted of a solvent delivery system (P2000 pump, Spectra-Physics, San Jose, CA) equipped with an autosampler (717, Waters Chromatography Division, Millipore Corp., Milford, MA), a refractive index detector (410 differential refractometer, Waters), a dual absorbance detector (2487, Waters), and a computer software based integration system (Chromquest 4.0, Spectra- Physics). Two ion moderated partition chromatography columns (Aminex HPX-87P with De-ashing and Carbo-P micro-guard cartridges, Aminex HPX 87H with Cation H micro-guard cartridge) were used. The Aminex HPX-87P column was maintained at 85 °C, and the sugars were eluted with Milli-Q filtered water at a flow rate of 0.6 mL/min. The Aminex HPX-87H column was maintained at 65 °C, and the sugars, organic acids, furfural, HMF, and ethanol were eluted with 10 mM HNO3 prepared using Milli-Q filtered water at a flow rate of 0.6 mL/min. Peaks were detected by refractive index or UV absorption (277 nm) and were identified and quantified by comparison to retention times of authentic standards (glucose, xylose, galactose, arabinose, furfural, HMF, acetic acid, succinic acid, and ethanol).

3. Results and Discussion

3.1. Effect of Alkaline Peroxide Concentration on Pretreatment and Enzymatic Saccharification

Initially, the effects of alkaline H₂O₂ level (0-5%, v/v) on the pretreatment of wheat straw 12.5%, w/v) at pH 11.5 and 35°C for 24 h were evaluated. The resultant glucose, xylose, arabinose, and total sugars yield in terms of mg per g of corn stover and sugarcane baggase after enzymatic saccharification using Saccharomyces cerevisiea enzyme at 37°C, pH 4.5, for 120 h is shown in Figure 1 and Figure 2. It was found that the sugar yield increased with increasing H₂O₂ up to 2.0% (v/v). There is not much difference between 2.0 and 5.0% (v/v) H₂O₂pretreatment on each individual sugar as well as total sugar yields. Thus, it was decided to use 2.0% (v/v) H₂O₂concentration for subsequent pretreatment studies.

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Figure 2. Effect of H₂O₂ level (0-5%, v/v) for the pretreatment (pH 11.5, 35°C, 24 h) of corn stover (12.5%, w/v) on its enzymatic saccharification (37°C, pH 4.5 , 120 h). The data presented are averages of two separate experiments

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Figure 3. Effect of H₂O₂ level (0-5%, v/v) for the pretreatment (pH 11.5, 35 °C, 24 h) of sugarcane baggase (12.5%, w/v) on its enzymatic saccharification (37 °C, pH 4.5, 120 h). The data presented are averages of two separate experiments

3.2. Effect of Duration of Alkaline Peroxide Pretreatment at Two Temperatures

The effects of duration of alkaline peroxide pretreatment on the enzymatic saccharification of wheat straw at 25 and 35°C were investigated. The results are presented in Figure 4 and Figure 5. It is evident that the effect of pretreatment time is more pronounced at 25°C than at 35°C. In case of corn stover the increase of formation of total sugars 80± 3 mg per g of corn stover by increasing the pretreatment time from 6 to 24 h at 25°C, whereas the increase of total sugars was only 30± 4 mg per g of corn stover within the same time period at 35^oC. On the other hand in case of sugarcane baggase the increase of formation of total sugars 75± 3 mg per g of sugarcane baggase by increasing the pretreatment time from 6 to 24 h at 25°C, whereas the increase of total sugars was only 25± 4 mg per g of sugarcane baggase within the same time period at 35°C. However, the longer is the pretreatment time; the better is the yield of sugars by enzymatic saccharification. Galactose was not detected in any of these hydrolyzates. The reason for this is not clear.

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Figure 4. Effect of duration of alkaline H₂O₂ pretreatment (2.0%, v/v; pH 11.5) of corn stover (12.5%, w/v) at two temperatures (25 and 35 ^oC) on its enzymatic saccharification (37 ^oC, pH 4.5, 6-24 h). The data presented are averages of two separate experiments

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Figure 5. Effect of duration of alkaline H₂O₂ pretreatment (2.0%, v/v; pH 11.5) of sugarcane baggase (12.5%, w/v) at two temperatures (25 and 35 °C) on its enzymatic saccharification (37 ^oC, pH 4.5, 6-24 h). The data presented are averages of two separate experiments

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Figure 6. Time course of enzymatic hydrolysis (37°C, pH 4.5) of alkaline H₂O₂ pretreated (pH 11.5, 35°C, 120 h) corn stover (12.5%, w/v) using Saccharomyces cerevisiea enzyme. The data presented are averages of two separate experiments

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Figure 7. Time course of enzymatic hydrolysis (37°C, pH 4.5) of alkaline H₂O₂ pretreated (pH 11.5, 35°C, 120 h) sugarcane baggase (12.5%, w/v) using Saccharomyces cerevisiea enzyme. The data presented are averages of two separate experiments

The time course of enzymatic hydrolysis of the alkaline H₂O₂ pretreated (2.0%, v/v; pH 11.5; 35°C, 120 h) of corn stover and sugarcane baggase is presented in Figure 6 and Figure 7. Most of the sugars were released within 24 h. However, the sugar yield increased very slowly up to 120 h, after which there was no increase in sugar concentration. This indicates that there is a long incubation time needed for getting the maximum sugar yield under the conditions used.

3.3. Fermentation of Corn Stover and Sugarcane Baggase Hydrolyzates and Production of Ethanol

The results of fermentation of alkaline peroxide pretreated and enzyme saccharified corn stover and sugarcane baggase by the recombinant Saccaharomyces cerevisiea enzyme are summarized in Table 3. The yeast produced small amounts of succinic and acetic acids as byproducts in addition to ethanol. It is clear that the separate hydrolysis and fermentation (SHF) approach worked better than the simultaneous saccharification and fermentation (SSF) method with respect to ethanol yield. The maximum concentration of ethanol (per L) from corn stover (66.0 g) hydrolyzate by recombinant Saccaharomyces cerevisiea enzyme was 13.75 (0.38 g per g of available sugars in corn stover) by SHF. For SSF, the maximum concentration of ethanol was 11.2 (0.32 g per g of available sugars based on separate hydrolysis data). On the other hand the maximum concentration of ethanol (per L) from sugarcane baggase (66.0 g) hydrolyzate by recombinant Saccaharomyces cerevisiea enzyme was 14.20 (0.40 g per g of available sugars in sugarcane baggase) by SHF. For SSF, the maximum concentration of ethanol was 12.9 (0.35 g per g of available sugars based on separate hydrolysis data). Both SHF and SSF were completed within 48 h of fermentation even though the yield of ethanol was higher in the case of SHF than SSF. However, ethanol production by the above process is lower than the ethanol production from AFEX-pretreated corn stover by recombinant E.coli stains K011 and SL40 and by K. oxytoca strain P2, under P^H-controlled conditions ^[22].

Table 3. Ethanol production from corn stover and sugarcane baggase hydrolyzate by recombinant Saccharomyces cerevisiea enzyme at 37°C

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The medium contained hydrolyzates from 50 g of corn stover and sugarcane baggase per L. For pretreatment, corn stover and sugarcane baggase (12.56%, w/w) was treated with 2.0% H₂O₂ (v/v) at 35 °C for 24 h. Separate enzymatic saccharification was performed using Saccaharomyces cerevisiea at 37 °C and pH 4.5 for 120 h. Enzyme used 2 µL/50 gm of each raw materials. Fermentation experiments were performed at pH 6.5 for SHF and pH 6.0 for SSF. The data presented are averages of two separate experiments.

3.4. Effects of pH of Fermentation Process on Ethanol Concentration in Water

The study was carried out to determine the significant influences of pH on fermentation due to its effect on yeast growth and fermentation rate. The sample was fermented at different pH values from 3, 3.5, 4, 4.5 and 5 while the temperature was kept constant at 37°C to obtain maximum yield of bioethanol. The total bioethanol content in each sample was determined and recorded in the Table 4.

Table 4. The Effects of pH on Ethanol Concentration (%) in water

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Based on the results obtained, pH 4.5 showed the highest ethanol content in water which is 14.26 %, followed by pH 4.0 which is 11.94 %, then pH 3.5 at 10.4 % and pH 3.0 at 8.55 % in case of sugarcane bagasse. The lowest ethanol concentration in water was achieved at pH 5.0. The result reveals that ethanol concentration in water gradually increases along with the increases in pH and reaches a maximum percentage of ethanol production when pH is equals to 4.5 and later it start to declining. In case corn stover material the highest ethanol content in water was 13.75 at pH 4.5 followed by 11.32 at pH 4.0.

The maximum ethanol concentration in water at pH 4.5 reflects enzyme function in an environment ^[23] while the lower ethanol concentration in water at pH reflects lesser yeast activity. The maximum ethanol productivity was observed at pH of 4.2 to 4.5 ^[24]. Furthermore, the increase in ethanol concentration in water is more efficient with the increase in pH from 4.0-4.5 and also found that the optimum pH range for S.cerevisiae to be pH 4.5 ^[25].

In general, yeast is an acidophilic organism and as such, grows better under acidic condition. The optimum pH range for yeast growth can vary from pH 4.0 to 6.0, depending on the temperature, the presence of oxygen and strain of yeast. Optimum pH values are required for the activity of plasma membrane bound proteins, including enzymes and transport proteins ^[26]. During growth, it is important for the yeast to maintain a constant intercellular pH.

There are many enzymes functioning during within yeast cell during growth and its metabolism. Each enzyme works best at its optimal pH, which is acidic because of the acidophilic nature of the yeast itself. When the extracellular enzymes pH changes from the optimal level, the yeast cell required using energy to either pump in or pump out the hydrogen ions in order to maintain the optimum intercellular pH ^[27].

If the extracellular pH changes too much from the optimum pH range, it may too difficult for the cell to maintain constant intracellular pH and the enzyme may not function normally. Furthermore, if the enzymes are deactivated, the yeast cell will not be able to grow and make ethanol efficiently ^[26].This is the most likely explanation for the observed reduction in ethanol production when the initial medium pH was at 3.0. There were also low carbon dioxide productions at pH 3.0 because the low pH encourages the production of acid instead of alcohol ^[28].

However this study shows lowest ethanol concentration in water at pH 5. This may be due to the disability of the yeast strain to tolerate at pH 5. Different yeast strain has different pH range to activate and produce ethanol. There are other possibilities; the yeast that was used to conduct the experiment may be old. Old yeast will not carry out fermentation process efficiently compared to new yeast according to ^[29]. There is increased rate of ethanol production at pH 5. This statement is not applicable for this study since at pH 5 there is no ethanol production.

3.5. Effects of Temperature of Fermentation Process on Ethanol Concentration in Water

Temperature is one of the major factors that determine the ethanol production. Table 5 showed the ethanol concentration in water (%) that obtained at different temperature. Based on the result obtain, no ethanol concentration in water was observed at 25 and 30°C.

Table 5. Effects of temperature on ethanol concentration in water (%)

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