Co-Cultivation of Penicillium sp. AKB-24 and Aspergillus nidulans AKB-25 as a Cost-Effective Method to Produce Cellulases for the Hydrolysis of Pearl Millet Stover
Abstract
:1. Introduction
2. Materials and Methods
2.1. Isolation and Identification of Fungi
2.2. FE-SEM Analysis of Selected Fungal Strains
2.3. Preparation of Substrates
2.4. Enzyme Production under SSF
2.5. Optimization of Cultural Conditions
2.6. Enzyme Production with Co-cultivation of Penicillium sp. AKB-24 and Aspergillus nidulans AKB-25
2.7. Saccharification Studies
2.8. Enzyme Assays
2.9. Statistical Analysis
3. Results and Discussion
3.1. Identification of Microorganisms
3.2. Agricultural Residues as the Carbon Sources for Enzyme Production
3.3. Time Course of Enzyme Production
3.4. Effect of Temperature and Initial pH on Enzyme Production
3.5. Effect of Initial Moisture Content on Enzyme Production
3.6. Effect of Nitrogen Sources
3.7. Effect of Surfactants
3.8. Improvement in Enzyme Production by Co-Cultivation
3.9. Saccharification by Crude Enzyme from Co-Culture
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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S. No. | Substrates | C % | N % | C/N Ratio | WRV (%) |
---|---|---|---|---|---|
1 | Sugarcane bagasse | 43.47 | 0.53 | 82.02 | 126.73 ± 3.17 a |
2 | Black gram | 42.59 | 0.98 | 43.46 | 145.15 ± 3.50 b |
3 | Corn stover | 43.93 | 0.74 | 59.36 | 160.12 ± 2.31 d |
4 | Pearl millet stover | 40.37 | 0.48 | 84.10 | 128.27 ± 3.23 a |
5 | Rice husk | 41.32 | 0.63 | 65.59 | 92.05 ± 3.60 e |
6 | Rice straw | 36.58 | 0.66 | 55.42 | 148.79 ± 3.61 b |
7 | Sunhemp | 43.27 | 0.54 | 80.13 | 133.24 ± 3.52 a |
8 | Sugarcane tops | 42.39 | 0.61 | 69.49 | 171.12 ± 4.19 c |
9 | Wheat bran | 44.12 | 1.95 | 22.63 | 174.94 ± 3.42 c |
10 | Wheat straw | 43.12 | 0.68 | 63.41 | 144.78 ± 3.09 b |
Variables | Endoglucanase (IU/gds) | FPase (FPU/gds) | β-Glucosidase (IU/gds) | Xylanase (IU/gds) |
---|---|---|---|---|
Incubation time, 4 days | 65.79 ± 3.19 | 1.58 ± 0.08 | 23.58 ± 1.30 | 1449.95 ± 81.63 |
Temperature, 30 °C | 65.79 ± 3.88 | 1.60 ± 0.09 | 23.93 ± 1.50 | 1472.17 ± 76.25 |
Initial pH, 8.0 | 79.00 ± 3.19 | 1.73 ± 0.10 | 27.50 ± 1.33 | 1832.25 ± 125.50 |
Moisture contents, 80.0% | 104.85 ± 6.98 | 2.06 ± 0.13 | 28.94 ± 1.78 | 1955.24 ± 126.30 |
Surfactants | Endoglucanase (IU/gds) | FPase (FPU/gds) | Xylanase (IU/gds) | ||||||
---|---|---|---|---|---|---|---|---|---|
Penicillium sp. AKB-24 | |||||||||
0.05% | 0.10% | 0.20% | 0.05% | 0.10% | 0.20% | 0.05% | 0.10% | 0.20% | |
T-20 | 108.82 ± 5.69 | 123.47 ± 6.03 b | 115.10 ± 5.47 | 2.47 ± 0.09 | 2.70 ± 0.10 b | 2.24 ± 0.10 | 2978.88 ± 109.02 | 3261.69 ± 151.66 b | 2855.71 ± 147.35 |
T-40 | 110.92 ± 5.71 | 125.57 ± 6.60 bc | 115.10 ± 4.48 | 2.50 ± 0.11 | 2.64 ± 0.12 b | 2.18 ± 0.12 | 2924.84 ± 141.26 | 3136.00 ± 162.13 b | 2541.48 ± 133.68 |
T-60 | 108.82 ± 4.32 | 123.47 ± 5.96 b | 113.01 ± 5.69 | 2.56 ± 0.11 | 2.79 ± 0.13 bc | 2.41 ± 0.13 | 3002.76 ± 140.82 | 3262.95 ± 162.82 b | 3049.27 ± 154.90 |
T-80 | 113.01 ± 5.81 | 129.75 ± 6.27 bc | 121.38 ± 4.24 | 2.68 ± 0.12 | 2.91 ± 0.15 bc | 2.78 ± 0.14 | 3002.76 ± 112.00 | 3335.85 ± 170.12 b | 3081.95 ± 146.39 |
T-x-100 | 117.19 ± 5.10 b | 113.01 ± 4.46 | 100.45 ± 4.90 | 2.70 ± 0.14 b | 2.54 ± 0.13 | 2.45 ± 0.045 | 2435.90 ± 94.75 a | 2185.77 ± 126.77 | 1690.55 ± 61.53 |
SDS | 125.57 ± 5.95 | 133.94 ± 7.19 c | 113.01 ± 3.76 | 2.63 ± 0.12 | 2.96 ± 0.14 c | 2.67 ± 0.13 | 3094.52 ± 160.29 | 3592.26 ± 170.99 c | 2379.34 ± 117.30 |
EDTA | 98.36 ± 4.61 a | 92.08 ± 3.48 | 85.80 ± 4.34 | 1.78 ± 0.11 d | 1.61 ± 0.09 | 1.56 ± 0.08 | 2469.83 ± 130.16 a | 2297.64 ± 81.79 | 2048.77 ± 88.71 |
Control | 106.73 ± 5.14 a | 2.08 ± 0.8 a | 2510.06 ± 126.75 a | ||||||
Aspergillus nidulans AKB-25 | |||||||||
T-x-100 | 152.14 ± 8.29 | 3.42 ± 0.18 | 2441.03 ± 144.99 |
Particulars | FPase, FPU/gds | Endoglucanase, IU/gds | |
---|---|---|---|
Carbon sources * | Wheat bran (WB) | 2.93 ± 0.10 | 137.19 ± 5.93 |
Black gram (BG) | 3.48 ± 0.14 | 155.89 ± 5.19 | |
WB:BG (1:1) | 3.64 ± 0.14 | 172.52 ± 6.80 | |
pH ** | 6 | 3.38 ± 0.12 | 166.29 ± 8.13 |
7 | 3.67 ± 0.17 | 174.60 ± 8.87 | |
8 | 3.83 ± 0.16 | 187.07 ± 9.09 | |
Moisture content *** | 77.5 | 3.84 ± 0.15 | 187.07 ± 8.19 |
80.0 | 4.04 ± 0.19 | 203.70 ± 9.41 |
Particulars | A. nidulans AKB-25 | Penicillium sp. AKB-24 | Co-culture of A. nidulans AKB-25 and Penicillium sp. AKB-24 |
---|---|---|---|
Optimal cultural parameters | |||
Carbon source | Black gram | Wheat bran | Black gram + Wheat bran |
Incubation time (days) | 4 | 7 | 7 |
Temperature (°C) | 30 | 30 | 30 |
pH | 8.0 | 7.0 | 8.0 |
Moisture content (%) | 80.0 | 77.5 | 80.0 |
Nitrogen source (%) | Ammonium sulphate (0.12% N) | Yeast extract (1.2% w/v) | Ammonium sulphate (0.6% N) + Yeast extract (0.6% w/v) |
Surfactant (%) | Triton-x-100 (0.05% w/v) | SDS (0.10% w/v) | Triton-x-100 (0.05% w/v) |
Enzyme activities | |||
Endoglucanase (IU/gds) | 152.14 ± 8.29 | 133.94 ± 7.19 | 203.70 ± 9.41 [(+33.88%) *, (+52.08%) **] |
FPase (FPU/gds) | 3.42 ± 0.18 | 2.96 ± 0.14 | 4.04 ± 0.19 [(+18.12%) *, (+36.48%) **] |
Exoglucanase (IU/gds) | 3.34 ± 0.14 | 2.48 ± 0.09 | 3.70 ± 0.13 [(+10.77%) *, (+49.19%) **] |
β-Glucosidase (IU/gds) | 44.05 ± 2.17 | 5.88 ± 0.26 | 35.28 ± 1.15 [(−19.90%) *, (+500%) **] |
Xylanase (IU/gds) | 2441.03 ± 144.99 | 3592.26 ± 170.99 | 3674.94 ± 183.12 [(+50.54%) *] |
Amylase (IU/gds) | 37.41 ± 1.95 | 194.90 ± 11.05 | 83.25 ± 4.30 [(+122.53%) *, (−57.28%) **] |
Crude enzyme from co-cultures of A. nidulans AKB-25 and Penicillium sp. AKB-24 | ||||
Enzyme activity (IU/mL) | 0.42 | |||
Enzyme dosing (FPU/g) | 5 (11.90 mL) * | 10 (23.80 mL) * | 15 (35.71 mL) * | 20 (47.61 mL) * |
Hydrolysis time (h) | ||||
12 | 259.24 ± 8.92 | 323.55 ± 16.50 | 406.52 ± 18.05 | 420.13 ± 15.84 |
24 | 336.08 ± 14.25 | 396.25 ± 13.79 | 476.11 ± 20.57 | 490.55 ± 24.92 |
36 | 389.96 ± 16.11 | 460.37 ± 19.98 | 532.54 ± 17.25 | 547.62 ± 21.25 |
48 | 419.49 ± 16.32 | 514.66 ± 22.23 | 575.27 ± 22.72 | 582.85 ± 25.35 |
60 | 446.17 ± 13.30 | 537.16 ± 21.11 | 602.39 ± 29.34 | 606.92 ± 22.09 |
72 | 455.64 ± 16.58 | 550.36 ± 26.75 | 612.45 ± 24.17 | 616.75 ± 27.57 |
84 | 458.82 ± 15.31 | 554.15 ± 24.05 | 615.38 ± 28.92 | 618.44 ± 27.77 |
Crude enzyme from A. nidulans AKB-25 | ||||
Enzyme activity (IU/mL) | 0.36 | |||
Enzyme dosing (FPU/g) | 5 (13.88 mL) * | 10 (27.77 mL) * | 15 (41.66 mL) * | 20 (55.55 mL) * |
Hydrolysis time (h) | ||||
72 | 451.72 ± 14.44 | 556.14 ± 25.85 | 613.17 ± 20.42 | 610.35 ± 24.56 |
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Kumar, A.; Gautam, A.; Dutt, D. Co-Cultivation of Penicillium sp. AKB-24 and Aspergillus nidulans AKB-25 as a Cost-Effective Method to Produce Cellulases for the Hydrolysis of Pearl Millet Stover. Fermentation 2016, 2, 12. https://doi.org/10.3390/fermentation2020012
Kumar A, Gautam A, Dutt D. Co-Cultivation of Penicillium sp. AKB-24 and Aspergillus nidulans AKB-25 as a Cost-Effective Method to Produce Cellulases for the Hydrolysis of Pearl Millet Stover. Fermentation. 2016; 2(2):12. https://doi.org/10.3390/fermentation2020012
Chicago/Turabian StyleKumar, Amit, Archana Gautam, and Dharm Dutt. 2016. "Co-Cultivation of Penicillium sp. AKB-24 and Aspergillus nidulans AKB-25 as a Cost-Effective Method to Produce Cellulases for the Hydrolysis of Pearl Millet Stover" Fermentation 2, no. 2: 12. https://doi.org/10.3390/fermentation2020012