This study evaluated the pretreatment and the saccharification of the rice straw to integrate a most economic approach for the productions of the fermentable sugars.
Followed by the optimum operating condition of initial cell concentration and incubation temperature for ABE fermentation of the fermentable sugars by central composite design and response surface methodology under sterile or non-sterile conditions were investigated to seek a most productive and economic viability biotechnology to produce biobutanol.
The composition of the rice straw was determined as 38% cellulose, 35%
hemicelluloses, 7% Lignin, and 4% Ash. However, the different pretreatment procedures of the rice straw could vary the composition of the rice straw. Dilute acid pretreatment resulted in the reduction of hemicellulose whereas this was not appeared during the dilute base pretreatment. The removed hemicellulose remained in the acid hydrolysate was hydrolyzed to release xylose, glucose, and galactose. Meanwhile, the reduction of hemicelluose elevated the content of cellulose to more than 50%.
This pretreatment was related to the performance of the saccharification of the rice straw. In the saccharification experiments, the total sugar productivity and yield were proportional to the enzyme loadings. The higher enzyme loading was implemented, the higher total sugar productivity and yield were attained. Glucose was the main final product in the saccharification. During the saccharification, there was no lag for the appearance of glucose. However, galactose, xylose, and arabinose were appeared a longer lag than glucose. In fact, the occurrence of cellulose hydrolysis was in advance to that of hemicellulose. The modified Gompertz equation simulated the productions of glucose obtained the glucose production potential and the glucose production rate for NPRS, PRS, and MPRSH were 2.62 g and 3.14 g/d, 2.5 g and 3.81
g/d, and 2.94 g and 3.26 g/d, respectively. The glucose production potential was not profoundly affect by the dilute acid pretreatment. This was in consistent with the glucose yield. The glucose yield of 0.52 g glucose/g rice straw for NPRS was compatible to 0.50 and 0.58 g glucose/g rice straw for PRS and MPRSH, respectively.
However, the implementation of the dilute acid pretreatment to the rice straw resulted in the higher production rate compared to the untreated rice straw. It was consistent with the fact of the rate constants of the first-order kinetics. The values of k for NPRS, PRS, and MPRSH were 0.0024 h-1, 0.0027 h-1, and 0.0027 h-1, respectively.
There was no discrepancy between PRS and MPRSH. It suggests the activities of hydrolytic enzymes were not inhibited by the byproducts in the acid hydrolysate.
However, taking accounts of energy, chemical, and time cost of pretreatment, rice straw grinded without other chemical pretreatment revealed to be the most economical efficiency feedstock to use in saccharification to fermentable sugars. Thus, NPRS hydrolysate was used in the series of ABE fermentation studies.
Under various CCD designed conditions, initial cell concentration (X1) and incubation temperature (X2) combinations, ABE fermentation of NPRS hydrolysate could be a solventogenesis or acidogenesis dominant bioreaction, whether operated under sterile or non-sterile condition. (X1, X2) = (640 ± 57 mg/L, 35 ℃ ) were acidogenesis dominant, while other active runs were solventogenesis, such as (X1, X2)
= (1429±214 mg/L, 25℃), (1429±214 mg/L, 35℃), (1429±214 mg/L, 45℃), (2170±
214 mg/L, 28℃) etc. High incubation temperature, 42℃ and 45℃ lead to the inactivation of ABE fermentation. However, (X1, X2) = (808±74 mg/L, 28℃) were inactive under non-sterile condition, due to the contaminations and competition with other microbes.
in the B/A ratio and butanol yield, and a decrease in butanol productivity was obtained by decreasing the incubation temperature from 35 to 25℃, which was consistent with the results reported by Carnarius (1940). The trend of sugar consumption was consistent with the trend of butanol production. Glucose was an easy and instant carbon source for C. saccharoperbutylacetonicum N1-4 to utilize, while arabinose was rarely utilized. The sequence of sugar utilization was glucose, galactose, and then arabinose. Besides initial cell concentration and incubation temperature as variables, contaminated degree was the other important factor that affected butanol production under non-sterile condition. The inhibition and influence cause by contaminations could be restrained by elevate the initial cell concentration to over 2200 mg/L to make C. saccharoperbutylacetonicum N1-4 a dominant group in fermentation system.
The modified Gompertz equation predicted the butanol production potential (P), the butanol production rate (R), and delay time (I) for ABE fermentation. The P and R values for experimental runs conducted under sterile condition were 0.00-7.27 g/L and 0.27-4.82 g/L/d, respectively. The duration time of lag time was 35℃<28℃<25
℃, which reflected the incubation temperature of ABE fermentation was the main influential factor. As for experimental runs performed under non-sterile condition, P and R were 0.00-7.70 g/L and 0.11-3.21 g/L/d, respectively. Lag time was not only enlarged with decreasing temperature, but also with the deduction of initial cell concentration of C. saccharoperbutylacetonicum N1-4.
A deeper and precise investigation of the individual and interactive effect of initial cell concentration (X1) and incubation temperature (X2) on butanol productivity (Y1), yield (Y2), and modified Gompertz equation predicted butanol production rate (Y3) and a determination of optimized conditions were achieved by full factorial central composite design and response surface methodology (CCD-RSM). For experimental runs conducted under sterile condition, X2 and X22 terms were the main factors
determined Y1, Y2, and Y3. The peak value of 0.06 g/L/d of Y1, 0.22 of Y2, and 0.17 g/L/d of Y3 were obtained under the combination conditions of (X1, X2) = (1960 mg/L, 32.3℃), (2010 mg/L, 26.3℃), and (2330 mg/L, 30.5℃), respectively; Nevertheless, for experimental runs conducted under non-sterile condition, X2, X1, and X1X2 terms were the main factors determined Y1, Y2, and Y3, respectively. The peak value of 0.06 g/L/d of Y1, 0.32 of Y2, and 0.16 g/L/d of Y3 were obtained under the combination conditions of (X1, X2) = (2330 mg/L, 26.4℃), (2330 mg/L, 25.0℃), and (2330 mg/L, 25.0℃), respectively.
To the final conclusion, ABE fermentation of C. saccharoperbutylacetonicum Nl-4 by using synthetic NPRS hydrolysate under non-sterile condition was found to be a feasible and viable biotechnology to produce biofuels, which reduced cost by recycling agricultural waste, and declined the energy cost and time by skipping the sterilization.
Based on this study, it was suggested that ABE fermentation could be conducted under non-sterile condition, when inoculated with high initial cell concentration (2330 mg/L) and low incubation temperature (25℃) of C. saccharoperbutylacetonicum Nl-4 at pH 5.42. For future prospects, it recommended that optimum pH value for ABE fermentation of NPRS hydrolysate should be investigated through CCD-RSM model, since many researchers believed pH is a factor required for triggering the onset of solventogenesis. Although, it has been reported that pH of 4.5 is the optimal pH for butanol production using C. acetobutylicum (Li et al., 2011). Still, the optimum pH value for solventogenesis appears to vary quite widely depending on the particulate strain and experimental conditions. In general, Clostridia could grow and produce solvents under the pH range of mild-acid, while most other bacteria need to grow in the
other bacteria. By building up the optimum conditions of initial cell concentration, incubation temperature, and pH for non-sterile ABE fermentation, the pilot scale experiment could be conducted eventually.
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Appendix A Metabolic product (maximum solvents/acids > 1)
(1) Experimental runs under sterile condition
0
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
35℃, Cell conc. = 1429±214 mg/L (Run 10)
25℃, Cell conc.= 1429±214 mg/L (Run 1)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
0
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
(Run 11) (Run 7)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
28℃, Cell conc. = 808±74 mg/L (Run 4)
Figure A-1 The profiles of ABE fermentation products with maximum solvents/acids ratio > 1 for all experimental runs in A experimental group.
(2) Experimental runs under non-sterile condition
0
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
35℃, Cell conc. = 1429±214 mg/L (Run5)
35℃, Cell conc. = 1429±214 mg/L (Run 9)
0
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
(c) 25℃, Cell conc.= 1429±214 mg/L (Run 1)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
35℃, Cell conc. = 2331+28 mg/L (Run 11)
Figure A-2 The profiles of ABE fermentation products with maximum solvents/acids ratio >1 for all experimental runs in B experimental group.
Appendix B Metabolic product (maximum solvents/acids ratio < 1)
(1) Experimental runs under sterile condition
0
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
35℃, Cell conc. = 640±57 mg/L (Run 3)
Figure B-1 The profile of ABE fermentation products with maximum solvents/acids ratio < 1 for all experimental runs in A experimental group.
(2) Experimental runs under non-sterile condition
0
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
(a) 35℃, Cell conc. =1429±214 mg/L (Run 10)
(b) 35℃, Cell conc. = 640±57 mg/L (Run 3)
Figure B-2 The profile of ABE fermentation products with maximum solvents/acids ratio
< 1 for all experimental runs in B experimental group.
Appendix C Inactive runs with no metabolic products
(1) Experimental runs under sterile condition
0
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
(c) 42℃, Cell conc. = 2170±157 mg/L (Run 8)
Figure C-1 The profile of metabolic products of inactive runs in A experimental group
(2) Experimental runs under non-sterile condition
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
Concentration of solvents/acetic acid (g/L) Concentration of butyric acid (g/L)
time (d)
42℃, Cell conc. = 808±74 mg/L (Run 6)
42℃, Cell conc. = 2170±157 mg/L (Run 8)
Figure C-2 The profile of metabolic products of inactive runs in B experimental group.
Appendix D Total sugar, pH, and cell concentration, and
Solvent/acid ratio, cell concentration, pH Total sugar concentration (g/L)
Time (d)
Solvent/acid ratio, cell concentration, pH Total sugar concentration (g/L)
Time (d)
Solvent/acid ratio, cell concentration, pH Total sugar concentration (g/L)
Time (d)
Solvent/acid ratio, cell concentration, pH Total sugar concentration (g/L)
Time (d)
Solvent/acid ratio, cell concentration, pH Total sugar concentration (g/L)
Solvent/acid ratio, cell concentration, pH Total sugar concentration (g/L)