Biohydrogen and Methane Production from Sugarcane Leaves Pretreated by Deep Eutectic Solvents and Enzymatic Hydrolysis by Cellulolytic Consortia
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sugarcane Leaves Preparation
2.2. Deep Eutectic Solvents Preparations
2.3. Cellulolytic Consortium
2.4. Clostridium butyricum TISTR 1032 Prepartions
2.5. Anaerobic Sludge Preparations
2.6. The Optimization Factors Affecting the Pretreatment of Sugarcane Leaves
2.7. SHF for Two-Stage Hydrogen and Methane Production and One-Stage Methane Production from Pretreated Sugarcane Leaves
2.8. SSF for Two-Stage Hydrogen and Methane Production and One-Stage Methane Production from Pretreated Sugarcane Leaves
2.9. Enzyme acTIVITY Assay
2.10. Analytical Methods
2.11. Statistical Analysis
2.12. Calculations
3. Results
3.1. Effects of Molar Ratio and Type of Deep Eutectic Solvent (DES) on Sugarcane Leaves
3.2. Effects of Pretreatment Time and Pretreatment Temperature
3.3. Effect of the Substrate to DES Solution Ratio
3.4. Enzymatic Hydrolysis of DES Pretreated Sugarcane Leaves by the Cellulolytic Consortium
3.5. The Two-Stages Hydrogen and Methane Production by SHF and SSF
3.5.1. Hydrogen Production
3.5.2. Methane Production
3.6. One-Stage Methane Production by SHF and SSF
4. Discussion
4.1. The Effect of Molar Ratio and Type of Deep Eutectic Solvents (DESs) on Sugarcane Leaves
4.2. The Effect of Pretreatment Time and Pretreatment Temperature
4.3. Effects of the Substrate to DES Solution Ratio
4.4. The Enzymatic Hydrolysis of DES Pretreated Sugarcane Leaves by the Cellulolytic Consortium
4.5. The Two Stages of Hydrogen and Methane Production by SHF and SSF
4.5.1. Hydrogen Production
4.5.2. Methane Production
4.6. One-Stage Methane Production by SHF and SSF
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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DES | Molar Ratio | pH | Pretreatment | Composition of Residues | ||||
---|---|---|---|---|---|---|---|---|
Cellulose Recovery (%) | Hemicellulose Removal (%) | Lignin Removal (%) | Cellulose (%) | Hemicellulose (%) | Lignin (%) | |||
Untreated | - | - | - | - | - | 36.18 ± 0.73 f | 25.23 ± 0.02 a | 27.68 ± 0.35 a |
ChCl/MEA | 1:6 | 14.08 | 71.86 ± 0.37 c,d | 69.88 ± 2.93 b,c | 77.62 ± 0.79 a | 57.62 ± 0.30 b,c | 16.84 ± 1.64 d | 13.73 ± 0.48 e |
1:8 | 14.12 | 73.49 ± 0.53 c | 71.07 ± 0.54 b | 77.50 ± 0.33 a | 58.92 ± 0.42 a | 16.21 ± 0.30 d | 13.80 ± 0.30 e | |
1:10 | 14.18 | 70.96 ± 2.10 d | 68.21 ± 1.45 c | 76.41 ± 0.85 a | 55.33 ± 1.64 d | 17.28 ± 0.79 d | 14.07 ± 0.51 e | |
ChCl/G | 1:2 | 7.93 | 82.37 ± 0.71 b | 33.60 ± 1.16 e | 28.98 ± 1.23 d | 39.40 ± 0.34 e | 22.15 ± 0.45 b | 25.99 ± 0.45 b |
1:4 | 6.40 | 85.08 ± 0.81 a | 33.57 ± 1.67 e | 25.11 ± 0.61 e | 38.72 ± 0.53 e | 21.09 ± 0.53 b,c | 26.08 ± 0.21 b | |
1:6 | 6.60 | 82.14 ± 1.49 b | 37.35 ± 2.39 d | 29.41 ± 0.80 d | 39.12 ± 0.71 e | 20.81 ± 0.79 c | 25.72 ± 0.29 b | |
ChCl/G/AlCl3 | 1:2:0.33 | 0.40 | 65.65 ± 0.23 f | 87.00 ± 0.07 a | 68.57 ± 0.26 c | 59.28 ± 0.20 a | 8.19 ± 0.05 e | 21.72 ± 0.18 c |
1:4:0.33 | 0.38 | 66.61 ± 0.70 e,f | 88.54 ± 0.48 a | 70.27 ± 0.54 b | 58.74 ± 0.62 a,b | 7.05 ± 0.29 f | 20.06 ± 0.37 d | |
1:6:0.33 | 0.36 | 68.11 ± 0.81 e | 88.21 ± 0.22 a | 68.39 ± 0.19 c | 57.37 ± 0.68 c | 6.92 ± 0.13 f | 20.37 ± 0.13 d |
DES | Temp (°C)/Time (h) | Pretreatment | Composition of Residues | ||||
---|---|---|---|---|---|---|---|
Cellulose Recovery (%) | Hemicellulose Removal (%) | Lignin Removal (%) | Cellulose (%) | Hemicellulose (%) | Lignin (%) | ||
Untreated | 36.56 ± 0.77 k | 27.31 ± 0.03 a | 27.68 ± 0.35 a | ||||
ChCl/MEA (1:6) | 80/3 | 69.25 ± 0.97 g | 57.76 ± 0.72 h | 73.49 ± 0.86 i | 53.37 ± 0.75 j | 24.32 ± 0.41 b | 15.47 ± 0.50 b |
80/6 | 68.69 ± 1.51 g | 66.60 ± 1.90 g | 78.67 ± 0.52 h | 57.61 ± 1.27 h | 20.93 ± 1.19 c | 13.55 ± 0.33 c | |
80/9 | 68.37 ± 1.13 g | 70.23 ± 1.40 f | 80.81 ± 0.61 g | 60.93 ± 1.01 g | 19.82 ± 0.93 d | 12.95 ± 0.41 d | |
100/3 | 71.54 ± 0.30 f | 70.29 ± 0.30 f | 83.45 ± 0.02 e,f | 64.25 ± 0.27 f | 19.93 ± 0.20 d | 11.25 ± 0.01 e | |
100/6 | 72.81 ± 0.42 e | 75.05 ± 0.96 a,b | 85.96 ± 0.28 d | 67.52 ± 0.39 d | 17.28 ± 0.66 f,g | 9.86 ± 0.20 f | |
100/9 | 72.04 ± 0.40 e,f | 76.57 ± 0.19 a | 87.06 ± 0.01 c | 69.64 ± 0.39 b,c | 16.92 ± 0.14 g,h | 9.47 ± 0.01 f,g | |
120/3 | 78.75 ± 0.22 a | 70.64 ± 0.51 e,f | 86.73 ± 0.22 c | 70.17 ± 0.20 b | 19.54 ± 0.34 d | 8.95 ± 0.15 h,i | |
120/6 | 78.32 ± 0.38 a | 72.57 ± 0.37 d | 87.95 ± 0.53 b | 71.91 ± 0.34 a | 18.12 ± 0.24 e,f | 8.07 ± 0.35 j | |
120/9 | 78.80 ± 0.89 a | 74.54 ± 0.79 b,c | 89.18 ± 0.29 a | 71.33 ± 0.81 a | 17.22 ± 0.54 f,gh | 7.42 ± 0.20 k |
The Substrate to DES Solution Ratio | Pretreatment | Composition of Residues | ||||
---|---|---|---|---|---|---|
Cellulose Recovery (%) | Hemicellulose Removal (%) | Lignin Removal (%) | Cellulose (%) | Hemicellulose (%) | Lignin (%) | |
Untreated | 36.56 ± 0.77 d | 27.31 ± 0.03 a | 27.68 ± 0.35 a | |||
1:8 | 81.84 ± 2.23 b | 72.40 ± 0.68 c | 86.86 ± 0.16 b | 69.81 ± 0.62 c | 17.59 ± 0.43 b | 8.49 ± 0.10 b,c |
1:12 | 84.13 ± 0.77 a | 73.98 ± 0.42 b | 86.37 ± 0.36 b | 72.54 ± 0.67 b | 16.76 ± 0.27 c | 8.90 ± 0.23 b |
1:16 | 81.44 ± 1.21 b | 74.62 ± 0.41 b | 88.23 ± 0.83 a | 74.27 ± 1.11 a | 17.29 ± 0.28 b | 8.13 ± 0.57 c |
1:20 | 81.31 ± 1.11 b | 75.78 ± 0.45 a | 88.06 ± 0.48 a | 73.34 ± 1.00 a,b | 16.32 ± 0.30 c | 8.15 ± 0.33 c |
1:24 | 84.38 ± 0.15 a | 76.29 ± 0.10 a | 86.42 ± 0.09 b | 74.27 ± 0.13 a | 15.59 ± 0.07 d | 9.05 ± 0.06 b |
Substrate Loading (g-VSadded) | Reducing Sugar Concentration (g/L) | |||||||
---|---|---|---|---|---|---|---|---|
Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 | |
U-1 | 0.15 ± 0.02 a | 0.40 ± 0.01 e | 0.28 ± 0.01 e | 0.40 ± 0.04 d | 0.36 ± 0.01 e | 0.44 ± 0.01 d | 0.37 ± 0.01 f | 0.26 ± 0.24 f |
U-2 | 0.16 ± 0.02 a | 0.21 ± 0.01 g | 0.43 ± 0.00 e | 0.50 ± 0.00 d | 0.78 ± 0.05 d | 0.88 ± 0.05 c | 0.96 ± 0.02 d | 0.85 ± 0.04 e |
U-3 | 0.17 ± 0.02 a | 0.81 ± 0.03 c | 0.50 ± 0.02 d | 0.72 ± 0.02 c | 0.90 ± 0.02 c,d | 0.98 ± 0.00 c | 1.70 ± 0.01 d | 1.13 ± 0.01 d |
U-4 | 0.17 ± 0.02 a | 0.35 ± 0.02 f | 0.36 ± 0.00 f | 0.74 ± 0.02 c | 1.12 ± 0.20 b | 1.66 ± 0.33 a | 1.64 ± 0.29 c | 1.49 ± 0.28 c |
P-1 | 0.17 ± 0.02 a | 0.41 ± 0.02 e | 0.36 ± 0.03 f | 0.49 ± 0.02 d | 0.47 ± 0.09 e | 0.60 ± 0.14 d | 0.61 ± 0.11 e | 0.35 ± 0.07 f |
P-2 | 0.17 ± 0.02 a | 0.70 ± 0.01 d | 0.57 ± 0.02 c | 0.86 ± 0.15 b | 0.95 ± 0.11 c | 1.43 ± 0.02 b | 1.65 ± 0.06 c | 1.85 ± 0.15 b |
P-3 | 0.16 ± 0.00 a | 1.18 ± 0.03 a | 1.52 ± 0.01 a | 1.56 ± 0.02 a | 1.71 ± 0.02 a | 1.78 ± 0.02 a | 1.97 ± 0.08 b | 1.95 ± 0.07 b |
P-4 | 0.17 ± 0.02 a | 0.95 ± 0.03 b | 1.10 ± 0.03 b | 0.92 ± 0.03 b | 1.25 ± 0.03 b | 1.88 ± 0.01 a | 2.18 ± 0.02 a | 2.35 ± 0.02 a |
Substrate Loading (g-VSadded) | SSF-Two Stages Hydrogen Production | SHF-Two Stages Hydrogen Production | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Hydrogen Production (mL/L) | Hydrogen Production Rate (mL/L d) | Lag Phase (λ) (d) | Hydrogen Yield (mL/g-VSadded) | R2 | Hydrogen Production (mL/L) | Hydrogen Production Rate (mL/L d) | Lag Phase (λ) (d) | Hydrogen Yield (mL/g-VSadded) | R2 | |
U_1 | 21 ± 4 e | 1.8 ± 0.8 h | 0.0 | 1.0 ± 0.1 f | 0.9826 | 17 ± 1 e | 1.5 ± 0.2 h | 0.5 | 0.8 ± 0.1 f | 0.9844 |
U_2 | 66 ± 5 e | 10.0 ± 2.0 g,h | 2.0 | 1.7 ± 0.1 f | 0.9935 | 32 ± 15 e | 6.2 ± 6.4 g,h | 4.0 | 0.8 ± 0.4 f | 0.9814 |
U_3 | 133 ± 14 e | 22.0 ± 3.3 f,g | 2.0 | 2.2 ± 0.2 f | 0.9958 | 52 ± 10 e | 14.2 ± 5.2 g,h | 5.0 | 0.9 ± 0.2 f | 0.9892 |
U_4 | 229 ± 20 e | 34.7 ± 3.9 e,f | 2.0 | 2.9 ± 0.2 f | 0.9962 | 99 ± 81 e | 20.2 ± 15.4 f,g,h | 5.0 | 1.2 ± 1.0 f | 0.9885 |
P_1 | 152 ± 7.0 e | 23.0 ± 2.2 f,g | 0.5 | 7.6 ± 0.3 e | 0.9947 | 48 ± 19 e | 7.9 ± 3.6 g,h | 3.0 | 2.4 ± 1.0 f | 0.9902 |
P_2 | 821 ± 48 d | 85.9 ± 5.1 d | 4.0 | 20.5 ± 1.2 c | 0.9956 | 682 ± 154 d | 40.9 ± 10.7 e | 4.0 | 17.0 ± 3.8 d | 0.9916 |
P_3 | 1840 ± 201 c | 211.2 ± 14.7 b | 4.0 | 30.7 ± 3.3 b | 0.9978 | 1700 ± 136 c | 112.4 ± 7.2 c | 5.0 | 28.1 ± 1.9 b | 0.9962 |
P_4 | 3187 ± 209 a | 317.5 ± 20.5 a | 5.0 | 39.8 ± 2.6 a | 0.9941 | 2135 ± 315 b | 197.2 ± 20.5 b | 7.0 | 26.7 ± 3.9 a | 0.9952 |
Substrates Loading (g-VSadded) | SSF Two-Stages Methane Production | SHF Two-Stages Methane Production | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Methane Production (mL/L) | Methane Production Rate (mL/L d) | Lag Phase (λ) (d) | Methane Yield (mL/g-VSadded) | R2 | Methane Production (mL/L) | Methane Production Rate (mL/L d) | Lag Phase (λ) (d) | Methane Yield (mL/g-VSadded) | R2 | |
U_1 | 1448 ± 354 f | 29.9 ± 10.4 e | 14 | 115.9 ± 28.3 c,d,e,f | 0.9931 | 1468 ± 441 f | 33.3 ± 13.9 d,e | 13 | 117.4 ± 35.3 c,d,e,f | 0.9921 |
U_2 | 2633 ± 390 c,d,e | 64.7 ± 9.7 c,d,e | 11 | 105.3 ± 15.6 d,e,f | 0.9968 | 1974 ± 545 e,f | 43.1 ± 15.6 d,e | 12 | 91.1 ± 21.4 e,f | 0.9972 |
U_3 | 3009 ± 516 b,c | 63.1 ± 15.6 c,d,e | 12 | 80.3 ± 13.8 e,f | 0.9949 | 2847 ± 530 b,c,d | 53.8 ± 3.1 d,e | 10 | 75.9 ± 14.1 e,f | 0.9950 |
U_4 | 3311 ± 806 b,c | 76.8 ± 18.8 b,c,d,e | 19 | 66.2 ± 16.1 e,f | 0.9954 | 3097 ± 244 b,c | 70.1 ± 3.6 b,c,d,e | 18 | 61.9 ± 4.9 f | 0.9926 |
P_1 | 2654 ± 277 c,d,e | 101.8 ± 27.7 b,c | 6 | 212.3 ± 22.2 a | 0.9950 | 2077 ± 308 d,e,f | 68.4 ± 25.4 b,c,d,e | 6 | 166.1 ± 24.6 a,b,c,d | 0.9873 |
P_2 | 3402 ± 760 b,c | 109.7 ± 68.4 b,c | 3 | 178.5 ± 72.0 a,b,c | 0.9660 | 3644 ± 473 b | 77.5 ± 25.3 b,c,d,e | 16 | 201.2 ± 113.8 a,b | 0.9795 |
P_3 | 5179 ± 291 a | 160.1 ± 15.2 a | 1 | 138.1 ± 7.8 b,c,d,e | 0.9751 | 3221 ± 417 b,c | 113.8 ± 30.9 b | 1 | 85.9 ± 11.1 e,f | 0.9797 |
P_4 | 5923 ± 251 a | 159.3 ± 19.0 a | 1 | 118.5 ± 5.0 c,d,e,f | 0.9828 | 3583 ± 128 b | 79.3 ± 9.8 b,c,d | 2 | 71.7 ± 2.6 e,f | 0.9778 |
Substrates Loading (g-VSadded) | SSF One-Stage Methane Production | SHF One-Stage Methane Production | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Methane Production (mL/L) | Methane Production Rate (mL/L d) | Lag Phase (λ) (d) | Methane Yield (mL/g-VSadded) | R2 | Methane Production (mL/L) | Methane Production Rate (ml/L d) | Lag Phase (λ) (d) | Methane Yield (mL/g-VSadded) | R2 | |
U_1 | 1632 ± 481 f,g | 27.4 ± 14.0 e | 9 | 130.5 ± 38.5 a,b,c | 0.9848 | 1597 ± 12 f,g | 21.5 ± 6.3 e | 10 | 127.8 ± 33.5 a,b,c | 0.9918 |
U_2 | 2015 ± 370 e,f,g | 30.0 ± 7.0 d,e | 9 | 80.6 ± 14.8 | 0.9816 | 2179 ± 405 e,f,g | 33.4 ± 5.1 d,e | 18 | 87.2 ± 16.2 d,e,f,g | 0.9938 |
U_3 | 2735 ± 186 c,d,e | 42.6 ± 3.1 c,d,e | 16 | 76.3 ± 5.5 d,e,f,g | 0.9907 | 1941 ± 558 e,f,g | 29.2 ± 11.3 e | 16 | 51.7 ± 14.9 e,f,g | 0.9629 |
U_4 | 2988 ± 112 b,c,d | 46.8 ± 4.9 c,d,e | 12 | 59.8 ± 2.2 e,f,g | 0.9940 | 2304 ± 304 d,e,f | 35.9 ± 7.5 c,d,e | 15 | 46.1 ± 6.1 g | 0.9867 |
P_1 | 2029 ± 104 e,f,g | 41.8 ± 3.7 c,d,e | 4 | 162.4 ± 8.3 a | 0.9948 | 1853 ± 242 f,g | 34.5 ± 7.9 d,e | 9 | 148.3 ± 19.3 a,b | 0.9948 |
P_2 | 2155 ± 449 e,f,g | 59.3 ± 31.2 b,c | 2 | 113.0 ± 44.9 b,c,d | 0.9905 | 1478 ± 796 g | 44.6 ± 17.7 c,d,e | 2 | 74.7 ± 34.3 d,e,f,g | 0.9600 |
P_3 | 3474 ± 613 a,b,c | 79.8 ± 15.8 b | 1 | 92.6 ± 16.3 c,d,e,f | 0.9875 | 3723 ± 340 a,b | 60.3 ± 13.4 b,c | 12 | 99.3 ± 9.1 c,d,e | 0.9965 |
P_4 | 4067 ± 319 a | 120.5 ± 9.5 a | 4 | 81.3 ± 6.4 d,e,f,g | 0.9936 | 3349 ± 415 a,b,c | 54.7 ± 16.6 c,d | 3 | 67.0 ± 8.3 e,f,g | 0.9793 |
Substrate | Pretreatment Method | Pretreatment Conditions | Fermentation Mode | Methane Yield | References |
---|---|---|---|---|---|
Sugarcane leaves | Ammonium fiber explosion (AFEX) pretreatment | 80–120 °C, 60 min | N/A-One-stage | 336 mL/g-VSadded | [58] |
Sodium hydroxide (6% NaOH) pretreatment | 25 °C, 3 days | N/A-One-stage | 287 mL/g-TSadded | [57] | |
Potassium hydroxide (KOH) pretreatment | 170 °C, 60 min | N/A-One-stage | 205 mL/g-TSadded | [59] | |
Liquid hot water (LHW) pretreatment | 190 °C, 60 min | N/A-One-stage | 162 mL/g-TSadded | [59] | |
Dilute acid (DA) pretreatment | 170 °C, 15 min | N/A-One-stage | 156 mL/g-TSadded | [59] | |
DES Pretreatment | 120 °C, 3 h | SSF-Two-stage | 118 mL/g-VSadded | This study | |
DES Pretreatment | 120 °C, 3 h | SSF-One-stage | 81 mL/g-VSadded | This study |
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Miftah, A.K.; Sittijunda, S.; Imai, T.; Salakkam, A.; Reungsang, A. Biohydrogen and Methane Production from Sugarcane Leaves Pretreated by Deep Eutectic Solvents and Enzymatic Hydrolysis by Cellulolytic Consortia. Fermentation 2022, 8, 396. https://doi.org/10.3390/fermentation8080396
Miftah AK, Sittijunda S, Imai T, Salakkam A, Reungsang A. Biohydrogen and Methane Production from Sugarcane Leaves Pretreated by Deep Eutectic Solvents and Enzymatic Hydrolysis by Cellulolytic Consortia. Fermentation. 2022; 8(8):396. https://doi.org/10.3390/fermentation8080396
Chicago/Turabian StyleMiftah, Apik Khautsart, Sureewan Sittijunda, Tsuyoshi Imai, Apilak Salakkam, and Alissara Reungsang. 2022. "Biohydrogen and Methane Production from Sugarcane Leaves Pretreated by Deep Eutectic Solvents and Enzymatic Hydrolysis by Cellulolytic Consortia" Fermentation 8, no. 8: 396. https://doi.org/10.3390/fermentation8080396