Stress tolerance and fermentation metrics of palm wine yeasts (Cyberlindnera fabianii, Candida tropicalis, Pichia sporocuriosa) supplemented with magnesium, vitamin E, and olive oil
Description
This dataset comprises experimental metrics from a study investigating natural supplements (magnesium ions, vitamin E, olive oil) to enhance stress tolerance in Nigerian palm wine yeasts (Cyberlindnera fabianii, Candida tropicalis, Pichia sporocuriosa) for bioethanol production. Data were generated under combined ethanol (25-30% v/v) and thermal stress (30-45°C) conditions. Key Contents: 1. Growth/Viability Metrics: - Optical density (OD₅₉₀) readings from spectrophotometry - Colony-forming units (CFU/mL) via plate counts at 10²-10⁸ dilutions - Transmittance percentages under stress conditions 2. Fermentation Performance: - Bioethanol yield (%) quantified by dichromate oxidation (OD₅₉₅) - Reducing sugar utilization (mg/dL) via DNSA method (OD₅₄₀) - Sugar-to-ethanol conversion efficiency 3. Experimental Variables: - Supplement concentrations: Mg²⁺ (10-50%), Vit E (4-20mg/L), Olive oil (1-5%) - Stress combinations: 25-30% ethanol × 30-45°C - Temporal data (Days 1-5 fermentation) Methodology: - Yeasts cultured in YEPD broth (100g/L glucose) with supplements - Stress tests: 72h exposure to ethanol/heat (triplicate trials) - Ethanol quantification: Tri-n-butyl phosphate extraction + K₂Cr₂O₇/H₂SO₄ oxidation - Sugar analysis: Dinitrosalicylic acid (DNSA) protocol - Statistical analysis: ANOVA with Tukey post-hoc (p<0.05) File Structure: - `Growth_Data.csv`: OD₅₉₀, transmittance%, CFU counts - `Ethanol_Metrics.xlsx`: Yield%, dichromate OD₅₉₅, process efficiency - `Sugar_Utilization.csv`: Reducing sugar (mg/dL), glucose standard curve data - `Experimental_Matrix.pdf`: Supplement combinations × stress conditions Potential Applications: - Optimizing industrial yeast fermentation under stress - Developing cost-effective bioethanol protocols - Training ML models for bioprocess prediction Ethical Compliance: Yeast isolates obtained from Nigeria Institute of Science Laboratory Technology (NISLT) under ethical guidelines (Amoa-Awua et al., 2007).
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Steps to reproduce
Steps to Reproduce Experimental Results Prerequisites: - Yeast isolates (C. fabianii, C. tropicalis, s P. sporocuriosa) - Chemicals: MgSO₄ (≥99%), D-α-tocopherol (vitamin E), food-grade olive oil - Equipment: UV-Vis spectrophotometer, incubator shaker, centrifuge 1. Culture Preparation a) Revive isolates on YEPD agar (28°C, 48h): Yeast Extract 10g/L, Peptone 20g/L, Dextrose 20g/L, Agar 20g/L b) Standardize inoculum to 0.5 McFarland (1.5×10⁸ cells/mL) in peptone water 2. Supplemented Media Setup** Prepare experimental groups in 50mL YEPD broth: Group 1: Control (no supplements) Group 2: Mg²⁺ (10-50% w/v MgSO₄•7H₂O) Group 3: Vitamin E (4-20mg/L filter-sterilized) Group 4: Olive oil (1-5% v/v autoclaved) Group 5: Combinations (e.g., 40% Mg²⁺ + 20mg/L Vit E + 5% olive oil) 3. Stress Application Apply stressors simultaneously: ethanol_concentration = [25, 30] % v/v temperature = [30, 35, 40, 45] °C incubation_time = 72hours shaking_speed = 120 rpm 4. Data Collection | Parameter| Method| Frequency | |---------------|------------|---------------| | Growth kinetics | OD₅₉₀ spectrophotometry | Every 12h | | Viability | Plate counts (CFU/mL, 10²-10⁸ dilutions) | Endpoint (72h) | | Ethanol yield | Dichromate oxidation (OD₅₉₅) | Daily (Day 1-5) | | Sugar utilization | DNSA assay (OD₅₄₀) | Daily (Day 1-5) | 5. Statistical Analysis R code example: library(agricolae) data <- read.csv(Fermentation_Metrics.csv) anova_model <- aov(Ethanol_Yield ~ Supplement Temperature, data=data) tukey_test <- HSD.test(anova_model, trt=c(Supplement","Temperature) 6. Validation - Replicate all experiments in triplicate (n=3) - Include positive controls (unstressed YEPD at 30°C) - Negative controls: Uninoculated media + stressors Critical Notes for Reproducibility 1. Strain Storage: Maintain isolates at -80°C in 25% glycerol 2. Vitamin E Handling: Add post-autoclaving (heat-labile) 3. Dichromate Reaction: - Precise timing: 10 min room temp before OD₅₉₅ reading - Standard curve range: 0-100g/L ethanol 4. DNSA Assay: - Boil samples with reagent exactly 5 min - Cool rapidly in ice bath Expected Outputs: - Dose-response curves (supplements vs. OD₅₉₀) - Time-series ethanol yield plots (like Fig 3.8) - ANOVA tables with F/p-values (p<0.05 significance) Troubleshooting Tips | Issue Solution | |-----------|--------------| | Low ethanol yield | Verify glucose concentration (must be 100g/L) | | Unstable OD readings | Vortex TBP/water mixtures ≥1 min before measurement | | High CFU variability | Use fresh YEPD plates (<2 weeks old) | | Precipitated dichromate | Warm reagent to 37°C and filter (0.22μm) | This protocol enables full replication in ≤3 weeks. For visualization templates (R/Python scripts for Figs 3.1-3.8), see the `Code_Scripts` folder in this repository.