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Team:Alberta/Growth
From 2011.igem.org
Growth
The objective of the growth experiments was to test the ability of Neurospora crassa to grow on a diverse selection of cellulosic waste products. Overall, there were two visions for this ambitious project. One was to create a large scale industry application for the MycoDiesel system. The other was to design a smaller scale system for home and/or community applications.
For industrial applications, we elected to test growth on wheat straw, sawdust and sawmill sludge (solid and liquid forms) because they reflected Albertan industries (agricultural and forestry) that produced large amounts of waste biomass. For the home and community system, we tested grass clippings and used coffee grounds. As a benchmark, we tested the growth of Neurospora on the classic Potato-Dextrose media (PD), the standard media used in the early research on Neurospora. The fungus grows very rapidly of this media. Moreover, the use of PDM allows for a good comparison of growth on cellulosic feedstock with a carbohydrate/starch feedstock. For a control, Vogel's Sucrose Trace Elements Biotin (VSuTB) media was prepared as given on the fungal genetic stock center (http://www.fgsc.net/methods/vogels.html). VSuTB is the basic complete media for Neurospora used in current research.
Results:
Through experimentation, we found that Neurospora was able to grow appreciably on wheat straw, liquid sawmill sludge, and grass clippings. The solid sawmill sludge, sawdust, and brewed coffee grounds did not produce significant amounts of growth. The solid sawmill sludge likely did not have the necessary level of moisture to facilitate growth of the fungus. The sawdust likely contained a high lignin content, effectively slowing growth, as Neurospora mainly degrades cellulose. 1 Lastly, the brewed coffee grounds were a mixture of different type of coffee from a local coffee shop. The varying combination of coffee may have contributed to the poor growth. Furthermore, coffee is normally acidic, thus the pH of the media may have been detrimental for Neurospora growth.2
From these preliminary results, we focused our testing on only one type of feedstock for each of two applications of our system. We selected wheat straw for the industrial angle and grass clippings for the home/community approach.
The next series of experiments tested the rate of growth of Neurospora on the two feedstocks. VSuTB and PD served as our control and benchmark media. Race tubes were used to assess the rate of growth via hyphal extension on each media. A race tube is, essentially, a long glass cylinder half filled with solid media and placed horizontally. Neurospora is a filamentous fungus, and will grow hyphae in all directions. In a race tube, the direction of growth is restricted. Thus, when we inoculate one end of the tube, the hyphae will grow towards the other end. The rate of growth is then proportional to the distance down the tube that the hyphae covered over time.
The following video shows the hyphal extension of wildtype (74A Oakridge) Neurospora crassa in race tubes. The video represents images captured over 7 days at 1 hour intervals.
The different media in each race tube is as follows:
| Media | Comments |
|---|---|
| Potato | Potato media without added glucose, to test the growth on starch only |
| Potato dextrose | PD media as a benchmark for maximum growth rate |
| Wheat straw, fertilizer, biotin and trace elements | Wheat straw media with household fertilizer for Nitrogen and Phosphorous. Biotin and trace elements added to ensure all essential nutrients are provided. |
| Wheat straw, and fertilizer | Wheat straw media with household fertilizer for Nitrogen and Phosphorous. |
| Wheat straw | Only wheat straw. |
| VSuTB | Vogel's Sucrose Trace Elements Biotin, a complete media for fungal growth |
| Grass clippings, fertilizer, biotin, and trace elements | Grass clippings media with household fertilizer for Nitrogen and Phosphorous. Biotin and trace elements added to ensure all essential nutrients are provided |
| Grass clippings, and fertilizer | Grass clippings media with household fertilizer for Nitrogen and Phosphorous. |
| Grass clippings | Only grass clippings |
As one can see, the PD media definitely showed the strongest growth, which was expected as the benchmark. However, what was interesting was that when the wheat straw was supplemented with fertilizer, biotin and trace elements, and when the grass clippings were supplemented with fertilizer, the growth rates were fairly growth rate on VSuTB. This result is promising a larger scale growth of Neurospora on cellulosic feedstock for implementation in the MycoDiesel system.
References
- Tian, C., Beeson, W. T., Lavarone, A. T., Sun, J., Marletta, M. A., Cate, J. H. D., and N. Louis Glass. Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa. PNAS. http://www.pnas.org/cgi/doi/10.1073/pnas.0906810106.
- Murthy,P.S., Naidu, M. M., and Pullabhatla Srinivas. Production of α-amylase under solid-state fermentation utilizing coffee waste. J Chem Technol Biotechnol; 84: 1246-1249 (2009)
