## 3 Results and Discussion
### Optimization of Fermentation Processes
#### 3.2.2 RSM Optimization of Enzymatic Hydrolysis  
The regression equation (Equation S2b) was used to determine factor treatment levels for glucose solubilization by enzymatic hydrolysis. Statistical significance of factors and interactions between factors on glucose yield is shown in Table S4b. No treatment factor or interaction of factors significantly impacted glucose yield. Surface plots for evaluating factor interaction effects on glucose values are shown in Figure S3e. Optimum conditions for enzymatic hydrolysis of pretreated vetiver biomass were determined to be a treatment pH of 2.0, temperature of 30 \({}^{\circ}\)C, and time of 5.5 days with \(\beta\)-glucosidase loading at 120 pNPGU/g cellulose. The prediction of glucose liberation under these parameters is 13.125 g/L. After carrying out the optimized conditions for enzymatic hydrolysis of pretreated vetiver biomass 19.197 \(\pm\) 0.090 g/L glucose was achieved, a 146.3% yield. The effects of pretreatment time on desirable sugar and inhibitor concentrations mimicked trends observed by temperature with respect to formation of furfural and degradation of 5-hydroxymethylfurfural. Increasing treatment time leads to increased formation of xylose degradation products [60]. Monomeric carbohydrate concentrations were observed to increase with increasing length of treatment. A greater recovery of sugars has been observed with longer dilute acid pretreatment times [60]. Relationships were positive between biomass loading and each of the four response variables (Figure S3a-d). Out of the responses, monosaccharide and acetic acid formation were most significantly affected. The relationship between monosaccharide production and biomass loading was linear with an R2 value equal to 1. Optimal biomass loading of 5-15% was reported by [PERSON] et al. [61]. Although biomass loading above 6.44% (_m/v_) achieves greater sugar yield, greater concentrations in inhibitor response variables was observed. Acetic acid is a weak acid that disrupts fermentation by causing an imbalance in the proton motive force as acids cross the cellular membrane [59]. ATP depletion results from the energy requiring process of proton efflux and renders the cells unviable [59]. Hemicellulose contains acetyl groups, which are hydrolyzed simultaneously as xylose is liberated from xylan [62]. Thus, the correlation between increasing sugar and acetic acid formation can be explained. Dilute acid pretreatment of sugarcane with varying solid loading showed similar results [63]. The pretreated vetiver biomass was used for RSM optimization of glucose recovery from cellulose by enzymatic saccharification. There were no significant interactions between treatment factors in this study (Table S4b and Figure S3c). It was observed that as pH increased, glucose recovery decreased. A low pH of 2.5 was deemed optimal. Cellulose and \(\beta\)-glucosidaseate activities are known to be supported by a wide pH range [64]. Hydrolysis temperature was optimal at 30 \({}^{\circ}\)C, with a severe decrease in glucose recovery at temperatures exceeding 30 \({}^{\circ}\)C. Similar results were observed in the enzymatic saccharification of rice straw, with an optimum hydrolysis temperature of 35.4 \({}^{\circ}\)C [65]. Time of enzymatic hydrolysis was optimum at 5.5 days. Above or below the optimized time, there was a significant decrease in glucose recovery, this was also observed for the enzymatic hydrolysis of sugarcane tops and maize starch [63; 66]. There was a positive correlation between \(\beta\)-glucosidase loading and glucose yield. \(\beta\)-glucosidaseate was responsible for maximum glucose recovery, predicted at 13.124 g/L, at 120 pNPGU/g cellulose. The lack of cellulase activity and cellulolytic role of \(\beta\)-glucosidaseate was also observed for the enzymatic hydrolysis of softwood [67]. A similar threshold for \(\beta\)-glucosidase loading and activity was also observed [67]. Recovery of glucose from the cellulosic fraction of vetiver exceeded the predicted concentrations under the optimized conditions.