Yeast Responses to Humic Substances
Introduction
Currently, numerous chemical and biological methods are known for determining physiologically active plant compounds such as vitamins, auxins, gibberellins, and others. However, methods for assessing the biological activity of humic substances remain poorly studied, despite their importance for chemists, physicians, and biochemists.
Chemical methods for identifying humic substances are complex due to the heterogeneity of these compounds, and their biological activity does not always correlate with chemical structure. Therefore, the only objective evaluation criterion remains biological testing. Existing methods based on measuring barley root growth or algae are labor-intensive and not always reproducible.
To solve this problem, it was necessary to select a sensitive, simple, and reproducible biotest suitable for serial studies. Reactions associated with different phases of cell growth - elongation and division - were considered.
Yeast Test
The idea of using a microbiological test to evaluate biocatalytic substances in peat was proposed by Bottomley (1915) but was not developed. We studied Saccharomyces cerevisiae yeast and found that potassium humate from leonardite accelerates their reproduction and fermentation.
For testing, we used Zygosaccharomyces ellipsoideus, Debaryomyces disporus, Saccharomyces ellipsoideus, and yeast from fermenting wort. The Reader nutrient medium (composition: glucose - 20 g/l, KH₂PO₄ - 1 g/l, MgSO₄·7H₂O - 0.7 g/l, (NH₄)₂SO₄ - 3 g/l) was supplemented with vitamins (H, B₁, B₆, Bс, inositol). Potassium humate was added at a concentration of 0.1%.
Results
Table 1. Yeast responsiveness to potassium humate (Reader medium with distilled water)
| Experimental scheme | Saccharomyces ellipsoideus | Debaryomyces disporus | Zygosaccharomyces ellipsoideus | Wine yeast |
|---|---|---|---|---|
| Control | 1.6 | 0.7 | 0.7 | 0.8 |
| Control + potassium humate + B₆, PP | --- | --- | --- | 18.5 |
| Medium + vitamins (H, B₁, B₆, PP) | --- | --- | --- | 4.7 |
| Medium + vitamins + potassium humate | 22 | 5.5 | 13.5 | 85 |
Table 2. Yeast responsiveness to potassium humate (Reader medium with tap water)
| Experimental scheme | Zygosaccharomyces ellipsoideus | Debaryomyces disporus | Wine yeast | Saccharomyces ellipsoideus |
|---|---|---|---|---|
| Control | 8.9 | 1.9 | 11.5 | 4 |
| Control + potassium humate | 14.0 | 7.0 | 71.0 | 14 |
| Medium + vitamins (H, B₁, B₆, PP) | 23.0 | 78.0 | 56.0 | 53 |
| Medium + vitamins + potassium humate | 295.0 | 104.0 | 104.0 | 103 |
Table 3. Mutual influence of vitamins and potassium humate
| Nutrient medium variant | Cell count (×10³/ml) |
|---|---|
| Control | 1.2 |
| Medium + B₁ | 3.0 |
| Medium + B₁ + potassium humate | 28 |
| Medium + B₆ + PP | 5.0 |
| Medium + B₆ + PP + potassium humate | 83 |
Table 4. Sensitivity of Saccharomyces ellipsoideus to potassium humate concentrations
| Potassium humate concentration (%) | Cell count (×10³/ml) |
|---|---|
| 0 (control) | 0.8 |
| 0.001 | 8.5 |
| 0.01 | 107 |
| 0.04 | 215 |
Table 5. Yeast growth depending on incubation time
| Incubation period | Cell count (×10³/ml) |
|---|---|
| 4 days | 66 |
| 7 days | 117 |
| 14 days | 215 |
Conclusions
- Humic substances (potassium humate) stimulate yeast reproduction, especially in the presence of vitamins B₁, B₆, Bс, H, and inositol.
- The best results were obtained using Saccharomyces ellipsoideus on Reader medium with the addition of vitamins and potassium humate (0.02%).
- The optimal evaluation period is 4 days of incubation at 28°C.
- The yeast test is sensitive, reproducible, and suitable for assessing the biological activity of humic substances.
Conclusion
The conducted studies confirmed that humic substances have a pronounced stimulating effect on yeast. The method can be used to search for and isolate physiologically active compounds from peat and other natural sources.