Optimization of Lipid Production by Schizochytrium limacinum Biomass Modified with Ethyl Methane Sulfonate and Grown on Waste Glycerol
Abstract
:1. Introduction
2. Materials and Methods
2.1. Experimental Design
2.2. Materials
2.2.1. Subsubsection
2.2.2. Growth Media and Chemical Reagents
2.3. Experimental Station
2.4. Experimental Procedure—Stage 1
2.4.1. Treatment of Algae with EMS
2.4.2. Incubation
2.4.3. Microalgae Screening
2.5. Optimization Design—Stage 2
2.5.1. Plackett–Burman Design
2.5.2. Central Composite Design (CCD)
2.5.3. Validation of Optimal Culture Conditions
2.6. Analytical Methods
2.7. Statistical Analysis
3. Results and Discussion
3.1. Stage 1
3.2. Stage 2
3.3. Stage 3
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Medium (Symbol) | Yeast Extract (g/dm3) | Peptone (g/dm3) | Glucose (g/dm3) | Glycerol (g/dm3) | Refined Glycerol (g/dm3) | Sea Water Made up to the Target vol. (dm3) |
---|---|---|---|---|---|---|
M1 | 5.0 | 5.0 | 15.0 | - | - | 1.0 (35 PSU) |
M2 | 0.5 | - | - | 15.0 | - | 1.0 (35 PSU) |
M3 | 5.0 | 5.0 | - | 15.0 | - | 1.0 (35 PSU) |
M4 | 20.0 | 20.0 | - | 100.0 | - | 1.0 (35 PSU) |
M5 | 20.0 | 20.0 | - | - | 204.4 | 1.0 (35 PSU) |
M6 | 20.0 | 20.0 | - | 136.3 | - | 1.0 (35 PSU) |
M7 | 20.0 | 20.0 | 100 | - | - | 1.0 (35 PSU) |
M8 | 45.0 | - | - | 249.0 | - | 1.0 (15 PSU) |
Code | Variable | Low (−) | High (+) |
---|---|---|---|
A | Initial cell concentrations in the culture (gDW/dm3) | 5 | 10 |
B | Volumetric air flow rate (dm3/min.) | 0.1 | 0.5 |
C | pH | 6 | 8 |
D | Glycerol level (g/dm3) | 100 | 200 |
E | Culture volume (dm3) | 1 | 2 |
F | Salinity (PSU) | 15 | 35 |
G | Turbine speed (rpm) | 400 | 800 |
H | Concentration of yeast extract (g/dm3) | 10 | 30 |
I | Temperature (°C) | 20 | 30 |
J | Oxygen concentration (%) | 20 | 35 |
Variant No. | A | B | C | D | E | F | G | H | I | J | d1 |
---|---|---|---|---|---|---|---|---|---|---|---|
1 | + | + | − | − | + | + | + | + | − | + | − |
2 | − | + | + | − | − | + | + | + | + | − | + |
3 | + | − | + | + | − | − | + | + | + | + | − |
4 | − | + | − | + | + | − | − | + | + | + | + |
5 | + | − | + | − | + | + | − | − | + | + | + |
6 | + | + | − | + | − | + | + | − | − | + | + |
7 | + | + | + | − | + | − | + | + | − | − | + |
8 | + | + | + | + | − | + | − | + | + | − | − |
9 | − | + | + | + | + | − | + | − | + | + | − |
10 | − | − | + | + | + | + | − | + | − | + | + |
11 | + | − | − | + | + | + | + | − | + | − | + |
12 | − | − | − | − | − | − | − | − | − | − | − |
Variable | Variable Code | Unit | −2 | −1 | 0 | 1 | 2 |
---|---|---|---|---|---|---|---|
Temperature | Z1 | °C | 15 | 20 | 25 | 30 | 35 |
Glycerol level | Z2 | g/dm3 | 50 | 100 | 150 | 200 | 250 |
Concentration of yeast extract | Z4 | g/dm3 | 10 | 30 | 50 | 70 | 90 |
Oxygen saturation | Z3 | % | 2.5 | 15 | 27.5 | 40 | 52.5 |
Strain Name | Variant | Survival Rate (%) | rDW (g/dm3·h) | rLIP (g/dm3·h) |
---|---|---|---|---|
S. limacinum C | V1–reference strain | 100 | 0.046 ± 0.002 | 0.017 ± 0.02 |
S. limacinum E5 | V2–EMS (5 min) | 23 ± 4 | 0.051 ± 0.003 | 0.017 ± 0.02 |
S. limacinum E10 | V3–EMS (10 min) | 17 ± 7 | 0.045 ± 0.005 | 0.011 ± 0.01 |
S. limacinum E15 | V4–EMS (15 min) | 16 ± 3 | 0.059 ± 0.002 | 0.018 ± 0.02 |
S. limacinum E20 | V5–EMS (20 min) | 11 ± 2 | 0.054 ± 0.004 | 0.021 ± 0.03 |
S. limacinum E25 | V6–EMS (25 min) | 12 ± 5 | 0.039 ± 0.002 | 0.011 ± 0.02 |
S. limacinum E30 | V7–EMS (30 min) | 2 ± 1 | 0.031 ± 0.001 | 0.011 ± 0.03 |
Unit | S. limacinum E20 | S. limacinum C | |
---|---|---|---|
Dry weight | gDW/dm3 | 68.0 ± 0.3 | 47.0 ± 0.4 |
rDW | gDW/dm3·h | 0.47 ± 0.1 | 0.36 ± 0.1 |
rLIP | g/dm3·h | 0.21 ± 0.2 | 0.12 ± 0.1 |
Lipids | % DW | 48 ± 1.2 | 42 ± 0.9 |
Proteins | % DW | 15 ± 0.5 | 22 ± 0.8 |
Carbohydrates | % DW | 22 ± 0.3 | 23 ± 0.5 |
Ash | % DW | 15 ± 0.3 | 13 ± 0.2 |
C14:0 | % SCFA | 3.03 ± 0.5 | 2.19 ± 0.6 |
C16:0 | % SCFA | 61.02 ± 0.4 | 54.24 ± 0.4 |
C18:0 | % SCFA | 3.33 ± 0.3 | 2.87 ± 0.5 |
C22:5 | % SCFA | 5.23 ± 0.4 | 10.29 ± 0.9 |
C22:6 | % SCFA | 26.24 ± 1.1 | 31.23 ± 0.7 |
Culture | no. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Dry weight | gDW/dm3 | 72.2 | 73.1 | 75.2 | 74.3 | 68.3 | 31.8 | 22.3 | 73.4 | 75.1 | 52.0 | 67.5 | 74.0 |
Lipids | g/dm3 | 37.3 | 30.0 | 41.4 | 39.0 | 28.0 | 22.3 | 21.0 | 38.5 | 40.0 | 25.0 | 39.1 | 37.0 |
Variable | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|
Dry Cell Weight | A | B | C | D | E | F | G | H | I | J |
Effect | 10.37 | 14.20 | 19.93 | 23.23 | 17.37 | 19.57 | 12.53 | 20.97 | 42.43 | 23.10 |
F value | 10.28 | 19.29 | 38.01 | 51.63 | 28.85 | 36.62 | 15.03 | 42.05 | 172.23 | 51.04 |
p level | 0.192 | 0.143 | 0.102 | 0.088 | 0.117 | 0.104 | 0.161 | 0.097 | 0.048 | 0.089 |
Lipid Concentration | A | B | C | D | E | F | G | H | I | J |
Effect | 9.43 | 9.60 | 8.20 | 15.33 | 10.03 | 6.97 | 10.60 | 10.97 | 18.90 | 11.23 |
F value | 30.79 | 31.89 | 23.27 | 81.35 | 34.83 | 16.79 | 38.88 | 41.62 | 123.60 | 43.66 |
p level | 0.114 | 0.112 | 0.130 | 0.070 | 0.107 | 0.152 | 0.101 | 0.098 | 0.057 | 0.096 |
No ofExperiment | Variable Code | Dry Weight (gDW/dm3) | Lipid Concentration (g/dm3) | |||
---|---|---|---|---|---|---|
Z1 | Z2 | Z3 | Z4 | |||
1 | −1 | −1 | −1 | −1 | 74.6 | 33.0 |
2 | 1 | −1 | −1 | −1 | 82.9 | 48.8 |
3 | −1 | 1 | −1 | −1 | 75.0 | 49.0 |
4 | 1 | 1 | −1 | −1 | 82.3 | 45.2 |
5 | −1 | −1 | 1 | −1 | 75.0 | 31.8 |
6 | 1 | −1 | 1 | −1 | 82.5 | 46.4 |
7 | −1 | 1 | 1 | −1 | 80.0 | 47.3 |
8 | 1 | 1 | 1 | −1 | 83.2 | 44.0 |
9 | −1 | −1 | −1 | 1 | 75.1 | 33.3 |
10 | 1 | −1 | −1 | 1 | 81.1 | 42.2 |
11 | −1 | 1 | −1 | 1 | 79.1 | 46.0 |
12 | 1 | 1 | −1 | 1 | 84.2 | 49.0 |
13 | −1 | −1 | 1 | 1 | 75.0 | 47.0 |
14 | 1 | −1 | 1 | 1 | 82.9 | 47.5 |
15 | −1 | 1 | 1 | 1 | 80.4 | 47.8 |
16 | 1 | 1 | 1 | 1 | 82.2 | 46.2 |
17 | −2 | 0 | 0 | 0 | 75.0 | 45.0 |
18 | 2 | 0 | 0 | 0 | 82.5 | 37.2 |
19 | 0 | −2 | 0 | 0 | 82.4 | 45.7 |
20 | 0 | 2 | 0 | 0 | 84.5 | 43.7 |
21 | 0 | 0 | −2 | 0 | 84.1 | 47.8 |
22 | 0 | 0 | 2 | 0 | 83.4 | 46.4 |
23 | 0 | 0 | 0 | −2 | 83.9 | 50.1 |
24 | 0 | 0 | 0 | 2 | 83.5 | 48.7 |
25 | 0 | 0 | 0 | 0 | 82.7 | 48.8 |
26 | 0 | 0 | 0 | 0 | 81.6 | 47.9 |
27 | 0 | 0 | 0 | 0 | 82.1 | 48.0 |
28 | 0 | 0 | 0 | 0 | 81.9 | 48.5 |
29 | 0 | 0 | 0 | 0 | 81.0 | 48.0 |
30 | 0 | 0 | 0 | 0 | 80.4 | 47.0 |
Variable | Dry Weight (gDW/dm3) | Lipid Concentration (g/dm3) | ||||
---|---|---|---|---|---|---|
Estimate | F Value | p-Value | Estimate | F Value | p-Value | |
Z0 | 81.61 | 48.03 | ||||
Z1-Temperature | 2.588 | 38.552 | 0.000 | 0.771 | 0.800 | 0.385 |
Z2-Glycerol level | 0.896 | 4.621 | 0.048 | 1.688 | 3.835 | 0.069 |
Z3-Oxygen saturation | 0.229 | 0.302 | 0.590 | 0.363 | 0.177 | 0.680 |
Z4-Yeast extract | 0.154 | 0.137 | 0.717 | 0.446 | 0.268 | 0.612 |
Z1 Z2 | −0.769 | 2.269 | 0.153 | −2.844 | 7.260 | 0.017 |
Z1 Z3 | −0.394 | 0.595 | 0.452 | −0.856 | 0.658 | 0.430 |
Z1 Z4 | −0.344 | 0.454 | 0.511 | −0.781 | 0.548 | 0.471 |
Z2 Z3 | 0.219 | 0.184 | 0.674 | −1.206 | 1.306 | 0.271 |
Z2 Z4 | 0.394 | 0.595 | 0.452 | −0.406 | 0.148 | 0.706 |
Z3 Z4 | −0.306 | 0.360 | 0.557 | 1.531 | 2.105 | 0.167 |
Z12 | −1.166 | 8.941 | 0.009 | −1.991 | 6.098 | 0.026 |
Z22 | 0.009 | 0.001 | 0.981 | −1.091 | 1.831 | 0.196 |
Z32 | 0.084 | 0.047 | 0.832 | −0.491 | 0.370 | 0.552 |
Z42 | 0.072 | 0.034 | 0.856 | 0.084 | 0.011 | 0.918 |
Culture Conditions | Dry Weight (gDW/dm3) | rDW (gDW/dm3xh) | |
---|---|---|---|
Optimal values for dry biomass production | Predicted value | 83.4 | - |
Experimental value | 84.0 ± 0.11 | 0.66 | |
Error (%) | +0.6 | - | |
Culture Conditions | Lipid Concentration (g/dm3) | rLIP (g/dm3xh) | |
Optimal values for lipid accumulation | Predicted value | 54.1 | - |
Experimental value | 54.8 ± 0.1 | 0.38 | |
Error (%) | +1.2 | - |
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Talbierz, S.; Dębowski, M.; Kujawska, N.; Kazimierowicz, J.; Zieliński, M. Optimization of Lipid Production by Schizochytrium limacinum Biomass Modified with Ethyl Methane Sulfonate and Grown on Waste Glycerol. Int. J. Environ. Res. Public Health 2022, 19, 3108. https://doi.org/10.3390/ijerph19053108
Talbierz S, Dębowski M, Kujawska N, Kazimierowicz J, Zieliński M. Optimization of Lipid Production by Schizochytrium limacinum Biomass Modified with Ethyl Methane Sulfonate and Grown on Waste Glycerol. International Journal of Environmental Research and Public Health. 2022; 19(5):3108. https://doi.org/10.3390/ijerph19053108
Chicago/Turabian StyleTalbierz, Szymon, Marcin Dębowski, Natalia Kujawska, Joanna Kazimierowicz, and Marcin Zieliński. 2022. "Optimization of Lipid Production by Schizochytrium limacinum Biomass Modified with Ethyl Methane Sulfonate and Grown on Waste Glycerol" International Journal of Environmental Research and Public Health 19, no. 5: 3108. https://doi.org/10.3390/ijerph19053108