Chapter 7: Liquefaction of lignocellulosic biomass for the production of
7.3. Results and Discussion
7.3.4. Fermentation
water had been evaporated from the enzymatic hydrolysate (derived from 10% solid loading) without any sugars loss. The concentrated solution obtained after evaporation contains 198 g/L of glucose, 10 mg of cellobiose and 3 mg of xylose.
concentration in terms of mg/mL, but their overall concentration per total volume of hydrolysate remains constant. Prior to fermentation studies, pH of the pre-hydrolysate was adjusted to the cultivation pH of Pichia stipites (i.e.,5.5).
Table 7.4: Fermentation summary of pretreatment and enzymatic hydrolysis derived hydrolysates by P. stipitis and S. cerevisiae, respectively.
Parameters Ca(OH)2 Mg(OH)2 EDH-1 EDH-2 Ethanol concentration (g/L) 18.4 18.4 44.8 74.7 Ethanol yield (gP/gS) 0.45 0.45 0.47 0.46 Ethanol productivity (g/L/h) 0.25 0.25 3.7 2.9 Sugar consumption rate (g/L/h) 0.56 0.56 7.9 5.33 Max. Ethanol production Time (h) 72 72 12 30 Ethanol conversion efficiency (%) 88.3 88.3 92.4 91.5
A batch cultivation was carried out to evaluate the fermentation efficiency of Pichia stipitis on both pre-hydrolysates (PHF-1 and PHF-2), which are further referred as Mg-fermentation medium and Ca-fermentation medium. Summary of fermentation results are presented in Table 7.4. Even though without performing the over-liming process, a significant xylulosic ethanol conversion efficiency (88.3%) was attained during the fermentation of both pre-hydrolysates. Almost similar concentration (18.4 g/L and 18.4 g/L) of ethanol was produced with the ethanol yield of 0.45 gp/gs and productivity of 0.25 g/l/h (Figure 7.5a and 7.5b).
0 12 24 36 48 60 72 0
5 10 15 20 25 30 35 40
b
Glucose Xylose Ethanol
Time (h)
Sugars (g/L)
a
0 5 10 15 20
0 12 24 36 48 60 72
0 5 10 15 20 25 30 35 40
Glucose Xylose Ethanol
Time (h)
0 5 10 15 20
Ethanol (g/L)
Figure 7.5: Sugar consumption and ethanol production profiles of P. stipitis during the fermentation of pretreatment derived hydrolysate (a) PHF-1 and (b) PHF-2
According to ion exchange chromatography (IC) analysis, the concentration of SO4 2- ions present in the Mg fermentation medium is 5 times higher than that of Ca- fermentation medium. Even the presence of high concentration of SO4 2- ions did not affect the ethanol yield, productivity and sugar consumption rate. This could be attributed to the availability of Mg ions in the fermentation medium, which significantly influences the metabolic growth of a microbe. Typically, the efficiency of microbial conversion of substrate to product may be improved by altering Mg ions concentration so that more magnesium ions are available to the cells [192]. Especially in yeast based ethanolic fermentation, magnesium availability is important in governing the central pathways of carbohydrate catabolism. The previous literature reports ensure that the Mg ions influence the activation of key enzymes for the optimum flow of substrate to ethanol and it (Mg ions) stabilizes the cell membrane to protect yeast from chemical and physical stress [192].
In addition to this, the amount of fermentative inhibitors such as furfural, 5- HMF, acetic acid and formic acid present in both pre-hydrolysates did not exhibit detrimental effect on ethanol producing Pichia stipitis. Therefore, no lag phase was
observed during the fermentation of both the pre-hydrolysates. According to the Delgenes et al., (1996), 11.9- 15 g/L of acetic acid, 5 g/L of 5-HMF and 2 g/L of furfural are the inhibitory concentrations of P. stipitis and other yeast species [183] . In the present study, the amount of furfural, 5-HMF and acetic acid derived from pretreatment process is 3 times lower than that reported by Delgenes et al., (1996). This signifies the viability of current pretreatment process. Generally, when the acid-hydrolysate contains elevated levels of fermentative inhibitors then over-liming (Ca(OH)2) step can be performed to partially convert the toxic components into non-toxic forms for the enhancement of hydrolysate fermentability. The potential draw backs of over-liming process are sugar degradation due to hydroxide-catalysed degradation reactions, and formation of solid waste (calcium sulfate). Generally, solubility of calcium sulfate is very low at neutral and alkaline pH levels, therefore it forms solid waste (CaSO4) even during neutralization of acid hydrolysate. However, in the present study, over-liming process and solid waste formation are terminated, and Mg(OH)2 is suggested as a significant neutralizing agent for the acid hydrolysate and it also enhances the hydrolysate fermentability.
7.3.4.2. Cellulosic ethanol production from enzymatic hydrolysate
Generally, lignocellulosic biomass contain different types of organic acids which are structurally incorporated within their respective polymers. For example, hemicellulose contains acetylated xylan back bone and three-dimensional methoxylated lignin contains various types of organic acids such as vanillic acid, p- coumaric acid, ferulic acid and cinnamic acid [34]. These organic acids in lignocellulosic biomass can alter the pH of the enzymatic hydrolysis medium (which optimally proceed at 4.8‒5.5) and creates unfavourable conditions for enzyme action.
Thus, enzymatic hydrolysis was conducted at high citrate buffer strength (50 mM) to
restrict the pH change [86]. In addition, use of 50 mM citrate buffer fundamentally hampered the growth metabolism of glucose fermenting microorganism [86]. This resulted in low ethanol yield and productivity. Moreover, enzymatic hydrolysis in 50 mM citrate buffer strength is commercially not feasible. However, in the present study, significant percent of acetic acid and lignin were removed successfully during dilute acid pretreatment and delignification step, respectively.
0 6 12 18 24
0 20 40 60 80 100
b
Time (h)
Glucose (g/L)
a
0 10 20 30 40 50
0 6 12 18 24 30
0 30 60 90 120 150 180
Time (h)
0 10 20 30 40 50 60 70 80
Ethanol (g/L)
Figure 7.6: Glucose consumption and ethanol production profiles of S. cerevisiae during the fermentation of enzymatic hydrolysis derived hydrolysate (a) CDH-1 and (b) CDH-2
Thus, by considering the aforementioned facts, enzymatic hydrolysis was conducted at low citrate buffer strength i. e., 0.5 mM, which is 100 times lower than that of standard citrate buffer strength. As expected, significant ethanol production of 44.8 g/L and 74.7g/L of was achieved by utilizing the glucose concentration of 95 g/L and 160 g/L, respectively (Figure 7.6a and 7.6b). Thus, the glucose to ethanol conversion efficiency and ethanol yield were found to be above 91% and 0.46 gp/gs, respectively. As compared to EDH-1 fermentation (95 g/L glucose), sugars consumption rate and ethanol productivity was found to be lower during the fermentation of EDH-2. During the fermentation of EDH-1 sugars consumption rate
and ethanol productivity was observed to be 7.9 g/L/h and 3.7 g/L/h, respectively.
Whereas, 5.3 g/L/h sugar consumption rate and 2.5 g/l/h ethanol productivity were attained during the EDH-2 fermentation (Table 7.4). Reduction of sugar consumption rate and ethanol productivity could be due to high concentration of ethanol production (74.7 g/L) during the EDH-2 fermentation. According to the literature reports, when the ethanol concentration of fermentation medium reached above 55 g/L, it initiates the product inhibition effect [90]. As evident from Table 7.4, until the ethanol concentration reaches 58.7 g/L in 18 h, sugar consumption rate and ethanol productivity was found to be 6.8 g/L/h and 3.2 g/L/h, respectively. Thereafter, sugar consumption rate and ethanol productivity deceased to 5.3 g/L/h and 2.4 g/L/h, respectively, which could be due to ethanol inhibition effect. However, in the present study, 74.7 g/L ethanol production was attained, which is equivalent to 9.4% (v/v) (based on ethanol density). Usually, a typical range of industrial titer of bioethanol production from edible sources such as corn (USA), sugarcane (Brazil) and sugar beet (EU) have been found to be 8-12% [2,12]. However, in the present study, around 9.1% (v/v) of bioethanol production was achieved by using inedible agriculture waste.
7.3.5. Determination of optimum liquefaction process for sorghum biomass