Cross-Linked Enzyme Aggregate (CLEA) Preparation from Waste Activated Sludge
Abstract
1. Introduction
2. Materials and Methods
2.1. Materials
2.2. CLEA Preparation
2.2.1. General Processes
2.2.2. Chemical Precipitation of Soluble Enzymes
2.2.3. Cross-Linking of Precipitated Enzymes
2.3. CLEA Preparation from Crude AS Enzyme Extracts
2.3.1. Enzyme Cross-Linking with Glutaraldehyde
2.3.2. Enzyme Cross-Linking with Dextran Aldehyde
2.4. Enzyme Activity Assays
2.5. Fourier-Transform Infrared Spectroscopy
2.6. CLEA Morphology
2.7. Hydrolysis of Organic Materials by CLEA
2.7.1. Substrate Preparation
- Gamma (γ) irradiation: diluted WAS was exposed to a cesium (Cs) 137 radiator (Gammacell 3000 ELAN, Best Theratronics Ltd., Canada). The WAS suspension (450 mL) was placed in a 500 mL HDPE bottle and was exposed to the radiator for 10 h at room temperature; the total radiation dose was 1944 Gy at a dose rate of 3.24 Gy/min.
- Sonication: diluted WAS was treated by sonication probe (VCX130, Sonics & Materials Inc., Newtown, CT, USA) for 10 min duration at 0 °C, 40% amplitude, in pulse cycle mode with 1 min on followed by 1 min off.
2.7.2. Hydrolysis of Organic Substrates
2.7.3. Indicators of the Progress of Enzymatic Hydrolysis
- Soluble Total Organic Carbon
- Reducing Sugar and Tyrosine
2.7.4. Progress Curve of Enzymatic Hydrolysis
- CTotal for sTOC was derived from the specific VS content (0.989 g/g TS) and the element composition ratio of wheat flour (C:H:O:N:S = 1:2.004:0.625:0.249:0.01) reported by [41], and it was equivalent to 0.568 g/g TS.
- CTotal for reducing sugar was derived by dividing the total carbohydrate content in the flour (0.771 g/g TS, which was assumed to be in the form of starch) by the coefficient, 0.9 [42], and it was equivalent to 0.857 g/g TS.
- CTotal for tyrosine was estimated based on the value reported by Siddiqi et al. [43], and it was 5.6 mg/g TS.
2.8. Statistical Analysis
3. Results and Discussion
3.1. Cross-Linking AS Enzymes with Glutaraldehyde
3.2. Cross-Linking AS Enzymes with Dextran Aldehyde
3.2.1. Ammonium Sulfate Precipitation
3.2.2. Acetone Precipitation
3.3. Fourier-Transform Infrared Spectroscopy Analysis
3.4. Hydrolysis Kinetics of Wheat Flour
3.5. Hydrolysis Kinetics of γ-Irradiated WAS and Sonicated WAS
4. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A. Fourier-Transform Infrared Spectroscopy
Class and Groups | Assignments | Band/Peak Position (Wavenumber, cm−1) | |
---|---|---|---|
Free Enzyme | CLEA | ||
Amide | N-H stretching | 3271 | 3271 |
Aliphatic; amine salt | C-H stretching; N-H stretching | 2863–2927 | 2860–2926 |
Amide | C=O stretching, N-H bending and C-N stretching | 1628 | 1634 |
Imine (Schiff’s Base) | C=N stretching | - | 1634 |
Amide | N-H bending and C-N stretching | 1505 | 1520 |
Amide/amine | N-H Bending and C-N stretching, along with C-H and N-H deformation | 1245 | 1235 |
Appendix B. Statistical Validation Protocol
Appendix C. Morphology of AS CLEAs
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Chemical Parameter, Model Coefficient and Statistics | CLEA Dose Rate (CLEA Suspension: Substrate) | |
---|---|---|
1:100 v/v | 1:10 v/v | |
sTOC: | ||
C0 (mg/g TS) | 13.6 ± 0.22 | 15.5 ±2.16 |
C∞ (mg/g TS) | 58.0 ±3.78 | 66.9 ± 5.08 |
k (h−1) | 0.114 ± 0.014 | 0.263 ± 0.059 |
Equation | C = 13.60 + 44.42 (1-e−0.114t) | C = 15.51 + 51.39 (1-e−0.263t) |
R2 | >0.99 | 0.96 |
p-value for normality test | >0.05 | >0.05 |
Pass normality test (alpha = 0.05) | Yes | Yes |
Reducing sugar: | ||
C0 (mg/g TS) | 7.19 ± 0.12 | 4.05 ± 0.45 |
C∞ (mg/g TS) | 42.4 ± 2.31 | 59.9 ± 9.39 |
k (h−1) | 0.086 ± 0.008 | 0.102 ± 0.024 |
Equation | C = 7.19 + 35.24 (1-e−0.086t) * | C = 4.05 + 55.82 (1-e−0.102t) |
R2 | >0.99 | 0.99 |
p-value for normality test | >0.05 | >0.05 |
Pass normality test (alpha = 0.05) | Yes | Yes |
Tyrosine: | ||
C0 (mg/g TS) | 0.92 ± 0.02 | 1.34 ± 0.02 |
C∞ (mg/g TS) | 4.47 ± 0.34 | 5.97 ± 0.08 |
k (h−1) | 0.125 ± 0.018 | 0.287 ± 0.010 |
Equation | C = 0.92 + 3.55 (1-e−0.125t) | C = 1.34 + 4.63 (1-e−0.287t) ** |
R2 | >0.99 | >0.99 |
p-value for normality test | <0.001 | >0.05 |
Pass normality test (alpha = 0.05) | No *** | Yes |
Sludge Type | γ-Irradiated WAS | Sonicated WAS |
---|---|---|
C0 (mg/g TS) | 5.61 ± 0.18 | 7.08 ± 0.06 |
C∞ (mg/g TS) | 8.21 ± 0.23 | 10.27 ± 0.12 |
k (h−1) | 0.521 ± 0.131 | 0.342 ± 0.031 |
Equation | C = 5.61 + 2.60 (1-e−0.521t) * | C = 7.08 + 3.19 (1-e−0.342t) ** |
R2 | 0.91 | >0.99 |
p-value for normality test | >0.05 | >0.05 |
Pass normality test (alpha = 0.05) | Yes | Yes |
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Liu, Z.; Smith, S.R. Cross-Linked Enzyme Aggregate (CLEA) Preparation from Waste Activated Sludge. Microorganisms 2023, 11, 1902. https://doi.org/10.3390/microorganisms11081902
Liu Z, Smith SR. Cross-Linked Enzyme Aggregate (CLEA) Preparation from Waste Activated Sludge. Microorganisms. 2023; 11(8):1902. https://doi.org/10.3390/microorganisms11081902
Chicago/Turabian StyleLiu, Ziyi, and Stephen R. Smith. 2023. "Cross-Linked Enzyme Aggregate (CLEA) Preparation from Waste Activated Sludge" Microorganisms 11, no. 8: 1902. https://doi.org/10.3390/microorganisms11081902
APA StyleLiu, Z., & Smith, S. R. (2023). Cross-Linked Enzyme Aggregate (CLEA) Preparation from Waste Activated Sludge. Microorganisms, 11(8), 1902. https://doi.org/10.3390/microorganisms11081902