3. MATERIALS AND METHODS
5.9 Enzyme loading studies
It is important to determine whether there are significant yield benefits from loadings higher than 25 FPU/g dry biomass or whether cellulose loadings less than 25 FPU/g dry biomass are sufficient. Sugar yields with lower enzyme loadings (1, 3, 5, 7, 10 and 15 FPU/g dry biomass) exist in literature (Kaar and Holtzapple, 2000; Chang et al., 2001).
After lime pretreatments of corn stover, Kaar and Holtzapple (2000) concluded that with 10 FPU/g dry biomass enzyme loading at 40 oC incubation temperature and 100 h period, the optimal reducing sugar yield was about 610 mg equivalent glucose/g dry biomass.
Palonen et al., (2004) investigated the wet air oxidation pretreated (at 200 oC) softwood enzymatic hydrolysis to reducing sugar using two cellulase mixtures (Celluclast and Multifect) with loadings corresponding to 5, 10, 30 FPU/g dry biomass. They concluded that using the highest enzyme load of 30 FPU/g dry biomass, under hydrolysis conditions of 40 oC, 20 g/L substrate concentration, and 24 h, maximum sugar yield of 257 mg/g dry biomass (55% conversion of polysaccharide) was achieved. The low sugar yield was partly caused by the low lignin removal during pretreatment (between 24–42 % of lignin in the softwood was removed). The enzyme loading results are illustrated in Figure 5.40 (150 oC, 1%H2O2, 10 bar air pressure, and 45 min optimized conditions) and in Figure 5.41 (120 oC 1%H2O2, and 30 min conditions); these figures contain some important features. First and foremost, higher reducing sugars yields were obtained at higher enzyme loadings (for example 25 FPU to 50 FPU/g dry biomass) in 4-d hydrolysis time.
137 Figure 5.38 – 4-d Effect of time and substrate concentration on sugars yield with
supplemental β-glucosidase. Pretreatment conditions: 120 oC, 1% H2O2, and 30 min. Enzyme hydrolysis conditions: 20 g/L (25 FPU cellulase, 15 IU β-glucosidase), 30 g/L (37.5 FPU cellulose, 22.5 IU β-glucosidase), 40 g/L (50 FPU cellulose, 30 IU β-glucosidase), 50 g/L (56.3 FPU/g dry biomass, 37.5 IU β-glucosidase/g dry biomass). 45 oC hydrolysis temperature, pH 4.8.
0 20 40 60 80 100 120
0 20 40 60 80 100 120
Reducing sugars yield (mg equivalent glucose/g dry biomass
Hydrolysis time (h)
20 30 40 50
Substrate concentration (g/L)
138 Figure 5.39 – 4-d Effect of time and substrate concentration on sugars yield with no
supplemental β-glucosidase. Pretreatment conditions: 120 oC, 1% H2O2, and 30 min. Enzyme hydrolysis conditions: 20 g/L (25 FPU cellulase, 15 IU β-glucosidase), 30 g/L (37.5 FPU cellulose, 22.5 IU β-glucosidase), 40 g /L (5 0 FPU cellulose, 3 0 IU β-glucosidase), 50 g/L (56.3 FPU/g dry biomass, 37.5 IU β-glucosidase/g dry biomass). 45 oC hydrolysis temperature, pH 4.8.
0 20 40 60 80 100 120 140 160 180
0 20 40 60 80 100 120
Reducing sugars yield (mg equivalent glucose/g dry biomass)
Hydrolysis time (h)
20 30 40 50
Substrate concentration (g/L)
139 The Figures (5.34 – 5.37) showed that 25 FPU/g dry biomass loading should be appropriate for the two pretreatment conditions considered. The 4-d reducing sugars yield at 50 FPU/g dry biomass for the 150 oC, 1%H2O2, 10 bar air pressure, and 30 min pretreatment condition was 365.62 mg equivalent glucose/g dry biomass while at 25 FPU/g dry biomass the yield was 335.35 mg equivalent glucose/g dry biomass. This was a difference of 8.3%, which can be considered minimal considering the economics of the process. For the 120 oC 1%H2O2, and 30 min pretreatment conditions, following the same trend, sugar yield at 25 FPU/g dry biomass was 11.4% less than sugar yield at 50 FPU/g dry biomass enzyme loading. It follows therefore that beyond 25 FPU/g dry biomass enzyme loading, the enzymatic hydrolysis becomes uneconomical as reducing sugars yields virtually did not change significantly. Cellulase loadings greater than 25 FPU/g dry biomass may have caused the cellulose sites to be saturated by the enzymes. Therefore, a cellulase loading of 25 FPU/g dry biomass is sufficient from a practical viewpoint because it represents the “shoulder” of the curve.
Also, we can conclude from these results that at high temperature of 150 oC 1%H2O2, longer time of 45 min, and 10 bar air pressure addition and at 25 FPU/g dry biomass enzymatic loading, reducing sugars yield (335.35 mg equivalent glucose/g dry biomass) increased than at 120 oC, 1%H2O2, and short time duration of 30 min(164.88 mg equivalent glucose/g dry biomass). Therefore, higher temperature, air pressure addition, and 1% H2O2 during pretreatment increased the reducing sugars yield.
Compared to published works on enzymatic hydrolysis of woody materials, the optimal enzyme loading under the specified conditions in this study was higher (25 FPU/ g dry biomass) than some of the values in literature (Kaar and Holtzapple, 2000; Chang et al., 2001. On the other hand, the performance at 150 oC, 1% H2O2, 10 bar, and 45 min pretreatment and subsequent enzymatic hydrolysis was higher than that reported by Palonen et al., (2004). The raw materials used and operating conditions in these studies must have contributed to these discrepancies.
140 Figure 5.40 – 4-d Effect of enzyme loading on sugar yields. Pretreatment conditions: 150
oC, 1% H2O2, 10 bar, 45 min. Enzyme hydrolysis conditions: 5 UI β-glucosidase/g dry biomass, 45 oC hydrolysis temperature, pH 4.8, 40 g/L substrate concentration.
0 50 100 150 200 250 300 350 400
0 10 20 30 40 50 60
Reducing sugars yield (mg equivalent glucose/g dry biomass)
Cellulase loading (FPU/g dry biomass)
2h 24h 72h 96h
141 Figure 5.41 – 4-d Effect of enzyme loading on sugar yields. Pretreatment conditions: 120
oC, 1% H2O2, and 30 min. Enzyme hydrolysis conditions: 5 UI β-glucosidase/g dry biomass, 45 oC hydrolysis temperature, pH 4.8, 40 g/L substrate concentration.
0 20 40 60 80 100 120 140 160 180 200
0 10 20 30 40 50 60
Reducing sugars yield (mg equivalent glucose/g dry biomass)
Cellulase loading (FPU/g dry biomass)
2h 24h 72h 96h
142 5.10 Hydrolysis studies of untreated and washed only biomass
It can be noted from Figure 5.42 that it was necessary to treat the raw material before enzymatic saccharification. Pretreatment is said to cause a disruption in the lignocellulosic matrix thereby making the enzymes more accessible to substrates. In this study, sugar yields of the pretreated sawdust were significantly higher than untreated sawdust. Figure 5.42 shows treated biomass reducing sugars concentration of 358.45 mg equivalent glucose/g dry biomass to untreated material of 17.73 mg equivalent glucose/g dry biomass. This is a 20-fold increase in reducing sugars produced from the treated to the untreated biomass. The maximum amount of reducing sugar yield in this study under the conditions specified was 42.4% lower than that reported for poplar wood (Chang et al., 2001). The large difference in value may have been caused by raw material composition, pretreatment conditions, efficiency of the different enzymes used, enzyme concentration, and reaction period.
Enzymatic digestibility of shea tree sawdust was boosted by pretreatment.
Oxidative lime pretreated sawdust enzyme hydrolysis was also enhanced with high pretreatment temperature (150 oC) and a combination of air and hydrogen peroxide addition. Higher temperature was more favourable because of more delignification, which resulted in more extensive enzymatic hydrolysis. However, around 170 oC and above polysaccharide degradation began to occur. 4-d enzymatic hydrolysis period with lower hydrolysis temperature (45 oC) produced higher sugar yields than 3-d at 50 oC hydrolysis temperature. The digestibility of the oxidative lime pretreated biomass depended on the cellulase as well as a little of β-glucosidase loadings. Moderate cellulase loading (25 FPU/g dry biomass) and withou t β-glucosidase supplements gave comparable sugar yields to cellulase loading with β-glucosidase supplements. 40 g/L substrate concentration was considered as optimum substrate loading. Below this substrate loading, sugar yields decreased and above it the sugar yields also decreased.
143 5.11 Simultaneous saccharification and fermentation
After 96 h fermentation, the quantity of ethanol obtained (g/L) from each of pretreatment conditions at 2% effective cellulose loading was 9.71 g/L for pretreatment at 150oC, 1%H2O2, 10 bar air pressure, and 45 min. Pretreatment at 120 oC, 1%H2O2, 30 min produced 7.35 g/L. At increased effective cellulose loading of 3%, ethanol obtained did not significantly increase for the two pretreatment conditions. The percent theoretical ethanol yields (based on cellulose conversion) for the two pretreatment conditions were higher at 2% effective cellulose loading than at 3% effective cellulose loading.
Ethanol concentration tended to be higher at 3% substrate loading, but at this loading, the % theoretical ethanol yields were much lower than that of 2% substrate loading. This implies that more of the cellulose was converted at 2% loading than at 3%
substrate loading. The high ethanol yield at 3% loading showed that more reducing sugar was produced by enzymatic hydrolysis and were probably quickly assimilated by yeast for cell growth and ethanol production than at 2% loading.
Cellulose conversion at 2% substrate loading should be more appropriate for the SSF under the conditions considered. However, more of the substrate will be needed for the fermentation process.
144 Figure 5.42 – 4-d Effect of time and substrate concentration on sugars yield for untreated and treated biomass. Concentrations in prime notation indicate enzymatic hydrolysis of pretreated samples. Pretreatment conditions: 150 oC, 1%
H2O2, 10 bar, 45 min. Enzyme hydrolysis conditions: 20 g/L (25 FPU cellulase, 15 IU glucosidase), 30 g/L (37.5 FPU cellulose, 22.5 IU β-glucosidase), 40 g/L (50 FPU cellulose, 30 IU β-β-glucosidase), 50 g/L (56.3 FPU/g dry biomass, 37.5 IU β-glucosidase/g dry biomass). 45 oC hydrolysis temperature, pH 4.8.
0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00
20 30 40 50 20' 30' 40' 50'
Reducing sugars yield (mg equivalent glucose/g dry biomass)
Substrate concentration (g/L) 2h 24h 72h 96h
145 CHAPTER SIX