Bioethanol Production from Saw Dust through Simultaneous Saccharification and Fermentation

With the industrialization revolution, petroleum demand was increases also (Saxena et al., 2009). Petroleum and diesel are considered non-renewable resources and will be scarce in near future. In recent times, there has been great concern in the production of substitute energy sources (Nwakaire et al., 2013). Bioethanol was being widely recognized as a promising renewable and environment friendly source of energy. Meeting the demands of bioethanol depends upon a regular supply of its primary raw material, i.e. biomass. Biomass has been considered as a major source of energy and it provides 10-14% of energy worldwide (Saxena et al., 2009).

A major component of naturally-occurring biomass is cellulose. Lignocellulosic material consists of six carbon sugar that has been used for bio-ethanol production (Nadeem et al., 2013). In nature cellulose is found in relation with other components, e.g. hemicellulose, lignin and pectin in an average of 4:3:3 but the exact percentage of this component varies from source to source (Sun and Cheng, 2002). Three major unit processes convert lignocelluloses to bioethanol: pretreatment of raw material, pretreated raw material into fermentable sugar by using enzymatic hydrolysis and fermentation of sugars into bioethanol (Alvira et al., 2010).

Pretreatment can be done by various methods like physical, chemical and biological (Irfan et al., 2016). Pretreatment is required in order to efficiently hydrolyze the fibrous cellulose into monomeric sugar because of the hard nature of lignocellulosic biomass. The pretreated material are saccharified and fermented by using separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) (Abo-state et al., 2014). Saccharification is done by cellulases and hemicellulases enzymes. Trichoderma species have capability to produce cellulolytic enzymes with efficient enzymatic activity (Oinonen and Suominen, 2002). Fermentation is done by bacteria and yeast commonly used yeast such as Saccharomyces cerevisiae. In this work research has been made to compare the efficient bioethanol production from Trichoderma viride, Trichoderma koninjii and Trichoderma harzianum.

Saw dust used in this research was purchased from local market of Lahore city. The sawdust was washed, sun-dried followed by oven drying at 70oC till constant weight and packed into zipper bags for further use.

Fungal strains of Trichoderma viride, Trichoderma koninji and Trichoderma harzianum were obtained from a culture bank of Institute of Agriculture Science, University of the Punjab, new campus Lahore, Pakistan. The strains were sustained on potato dextrose agar slants and preserved at 4˚C for further use. Sacchromyces cervisae was obtained from Food and Biotechnology Research Center (FBRC), PCSIR and maintained on PDA then preserved at 4˚C.

Pretreatment of sawdust was performed as described by Irfan et al., (2011). Briefly ten grams of sawdust was soaked in 100ml of 2.5% NaOH for 2 h at room temperature. Then sample was subjected to steam in an autoclave at 121˚C for 60 minutes. The solid material was washed with distilled water to get pH 7.

Simultaneous saccharification and fermentation strategy was applied for ethanol production. The medium comprised of (%) 0.2 MgSO4, 0.3 K2HPO4, 0.5 (NH4)2SO4, 0.3 Peptone, 0.3 yeast extract and 3% alkali pretreated sawdust as a carbon source. The medium was sterilized at 121oC for 15min at 15psi. After sterilization, the medium was inoculated with 1ml suspension of Saccharomyces cervisiae and 1ml of T. viride, T. koninji and T. harzianum in respective flasks and incubated at 30oC for 7 days with shaking speed of 120rpm. After finishing of fermentation time, the ethanol produced was estimated. This strategy was applied in sterilized and unsterilized conditions.

Cellulose of treated and untreated samples was measured by the method as described by Gopal and Ranjhan (1980). The lignin content of treated and untreated biomass was measured (Milagres, 1994). Ash and Moisture contents were measured by AOAC (2005) methods. Reducing sugar was determined by Miller (1959) method. The amount of ethanol was estimated calorimetrically (Captui et al., 1968). The ethanol yield was measured by using the formula as described by Yoswathana and Phuriphipat (2010).

Ethanol yield=Ethanol measured in sample/Theoretical ethanol

All the data collected was statistically analyzed using Microsoft excel program and values presented were the mean of triplicates.

In this study, saw dust was treated with 2.5% NaOH and further used for saccharification process. Untreated saw dust contains 42% cellulose and 12% lignin while alkali treated (2.5% NaOH) saw dust 22% cellulose and 10% lignin (Table 1). One research showed that increase in alkalinity of treating saw dust resulted lignin reduction (Kim et al., 2012). Lignin content decreased to 27.1%, 25.5% and 24.6% in the saw dust while increase in NaOH concentration of 0.5%, 1.0% and 2.0% respectively (Kim et al., 2012).

Table 1: Composition of raw material.

After pretreatment, saw dust was saccharified using T. harzianum, T. koninji and T. viride at 35˚C for 48h. Results (Figure 1) indicated that the maximum sugars was produced from saw dust by T. viride (10.17%) followed by T. harzianum (9.19%) and T. koninji (6.91%) after 48h of incubation at 35oC. Further increase in time period resulted decline in sugar production. Previous study shows that T. viride had ability to convert cellulose into glucose (Li et al., 2010). The reducing sugar was maximum (55.27 mg/g) by saccharification of rice straw after 9 days of incubation at 27˚C (Mishra et al., 2013).

After saccharification, ethanol production was conducted through separate hydrolysis and fermentation. Results (Figure 2) revealed that maximum ethanol production was obtained from T. koninji (2.44%) hydrolyzates followed by T. viride (1.80%) and T. harzianum (1.30%) after 7 days of fermentation at 30˚C. After this, ethanol percentage started to decrease due to contamination of ethanol into undesired products. In one research shows that T. viride produce maximum ethanol (17.54mg/ml of substrate) after 4 days of fermentation (Mishra et al., 2013). Ayeni et al., (2016) reported that alkaline peroxide oxidation pretreatment of shae tree sawdust yields 12.73g/L ethanol after 96h of fermentation by Saccharomyces cervisae. Rathna et al., (2014) reported that saw dust had potential for ethanol production in submerged fermentation by Saccharomyces cervisae under shaking conditions.

Ethanol production was also checked in simultaneous saccharification and fermentation. From the results (Figure 3), it was clearly showed that maximum ethanol production was observed by T. viride + Saccharomycese cervisae (3.88%) followed by T. harzianum+ Saccharomycese cervisae (0.75%) and T. koninji + Saccharomycese cervisae (0.53%) after 12 day of fermentation at 30˚C. Frias-Sanchez et al., (2017) reported maximum ethanol yield (17.1 g/L) in separate hydrolysis and fermentation of pine sawdust treated with nitric acid followed by sodium hydroxide pretreatment. Trevorah and Othman (2015) pretreated sawdust from Australian timber mills with 7% NaOH and reported maximum ethanol yield of 30.6% after 24h through simultaneous saccharification and fermentation with commercial enzymes and Saccharomyces cervisae. Kim et al., (2013) reported ethanol yield of 81.7% in fed-batch simultaneous saccharification and fermentation of dilute sulphuric acid treated poplar sawdust.

Results of this study concluded that sodium hydroxide pretreatment effectively delignify the biomass. T. viride proved to be potent fungal strain for better saccharification and in simultaneous saccharification and fermentation process for the production of bioethanol.