For ethanol production, wheat straw media was prepared and pre-treated with alkali to facilitate the hydrolysis and fermentation process as per earlier reported method as shown in Fig. 1. The steam and alkali pre-treatment of wheat straw demonstrated an increase in glucose production from 0.184 mg/ml to 0.223 mg/ml compared to untreated wheat straw after 24 hours of co-culturing (Fig S1). Alkali pre-treatment has been observed to increase the degradation of hemicellulose and lignin. Increase in degradation is due to the removal of acetate group from the hemicellulose, increasing the access of hydrolytic enzymes to cellulose [26]. In addition to this, alkali addition results in lignin solubilization which also enhance the enzyme susceptibility [27]. Matrix of cellulose and lignin are held together by hemicellulose chains in a lignocellulosic complex. During pre-treatment, the lignin-cellulose matrix breaks down and reduce the crystallinity by enhancing the proportion of the amorphous phase [28].
After pre-treatment of the wheat straw media, the media was inoculated with B. licheniformis to produce reducing sugars by enzymatic hydrolysis. Later, media was inoculated with S. cerevisiae for co-culturing to produce bioethanol via SSF. Time point for the inoculation of S. cerevisiae for co-culturing was optimized by inoculating the S. cerevisiae at 0th hour, 24th hour and 48th hour after the inoculation of B. licheniformis. It was observed that inoculation at 48th hour has produced highest bioethanol in comparison to other time points (shown in figure S2). Owing to sufficient time provided for B. licheniformis to produce reducing sugars helps in enhancing the ethanol production. Enzymatic hydrolysis plays an important role in enhancing the production of reducing sugars and would further increase the ethanol production. For the better enzymatic activity, environmental conditions play an important role. pH and temperature are the most important factors and have been optimized to improve enzymatic activity as well as growth of micro-organisms. In this study, pH was optimized for co-culturing by performing SSF at different pH (5,6,7 and 8) at 37oC. in Fig. 2, higher production of reducing sugars as well as ethanol demonstrates the suitable pH required for the SSF. The ethanol production was increase from 1.04 ± 0.15% to 2.46 ± 0.18% v/v, when the pH was changed from 5 to 7 after 96 hours of SSF. However, further increase in pH to 8 has decreased the ethanol production to 2.18 ± 0.034% v/v (Fig. 2A). In situ analysis of ethanol and glucose was carried out up to 96 hours of SSF as shown in Fig. 2B. Decrease in glucose concentration with increase in ethanol production demonstrated the conversion carried out by S. cerevisiae under anaerobic conditions. Ethanol concentration in wheat straw medium has increased from ⁓0.55% v/ v after 24 hours to ⁓2.46% v/v at pH 7. Gupta et al have also demonstrated that B. licheniformis demonstrated > 90% cellulase activity at pH 7 in case of water soluble carboxymethylcellulose (CMC) [29].
Substrate concentration in wheat straw medium plays an important role in improving the growth as well as production of enzymes in microbes. To optimize the substrate concentration, 2.5, 5.0, 7.5 and 10.0 mg/ml of substrates concentrations were prepared to perform SSF. Ethanol production increases from ⁓2.91% to 4.29 ± 0.27% v/v (96 hours SSF) after increasing substrate concentration from 2.5 to 5.0 mg/ml as shown in Fig. 3A. However, further increase in substrate concentration to 7.5 and 10 mg/ml has decreased the ethanol production to 3.79 ± 0.014% and 3.27 ± 0.007 % v/v, respectively. Glucose estimation at different substrate concentration demonstrates a decrease in glucose production when the substrate concentration was increased from 5 mg/ml to 7.5 and 10.0 mg/ml as shown in Fig. 3B. The decrease in ethanol production could be due to catabolite repression that tends to lower the yield of glucose, thereby, lowering the ethanol production [30].
Apart from wheat straw, a nitrogen source was added in the form of ammonium nitrate to prepare wheat straw medium. Nitrogen source is essential to boost up the production of proteins/enzymes required for the intra-cellular processes during the growth and division of the micro-organisms. Ammonium nitrate was used as a nitrogen source for SSF as it has been observed to support higher cellulase synthesis by micro-organisms in comparison to other nitrogen sources [31]. For bioethanol production, nitrogen source was optimized by performing SSF at 0.03, 0.06, 0.09 and 0.12 M concentrations of ammonium nitrate in wheat straw medium. Bioethanol production increased from 4 ± 0.014 % to 5.67 ± 0.28%, on increasing nitrogen source concentration from 0.03 M to 0.06 M as shown in Fig. 4A. Whereas further increase in nitrogen source concentration has declined the bioethanol production to 4.76 ± 0.34% and 3.14 ± 0.23% v/v for 0.09 M and 0.12 M, respectively. Decline in bioethanol production could be due to catabolite repression, which has decreased the cellulase production and have resulted in lower glucose production. Similar behaviour has been reported by Hernandez and coworkers while studying the effect of nitrogen source effect on ethanol production in S. cerevisiae [32].
Optimization of co-culture time, substrate concentration, pH of wheat straw medium and nitrogen source concentration demonstrated an increase in bioethanol production. For better control over the SSF process and the process parameters, the process was scaled up to 1.5 litre fermenter level to further improve the bioethanol production. SSF process was carried out by employing the optimized parameters in BIO-AGE 3 litres fermenter. Bioethanol produced was characterized through gas chromatography, which demonstrated a sharp increase in ethanol production from ⁓5.67% to ⁓18.64% v/v (Shown in figure S6). Studies by Mosier et al have demonstrated similar rise in ethanol production after scaling up o fermenter level [21].