Team:DTU-Denmark/substrate
Substrate
As the substrate utilization of Y. lipolytica is central to our project we have done an array of growth experiments. We have tested the growth on simple media to determine strengths and weaknesses in the catabolism of Y. lipolytica. We further expanded on the research by acquiring real waste streams and byproducts form organic industrial productions in the Nordic countries.
Introduction
The dimorphic, non-conventional yeast Yarrowia lipolytica, belonging to the Ascomyceta phylum, was first isolated in 1960s from lipid-rich materials, hence the name “lipolytica”. The organism was classified and reclassified a number of times, first as Candida lipolytica, then Endomycopsis lipolytica, Saccharomycopsis lipolytica and finally Yarrowia lipolytica1. Figure 1 shows Y. lipolytica cells under a microscope. The magnification factor is 100x.
In recent years Y. Lipolytica has received increased attention from researchers, as studies have found it to possess great potential for producing industrial enzymes and pharmaceutical proteins Biotechnological applications of Yarrowia lipolytica: Past, present and future. This potential is a result of several advantages Y. Lipolytica has over the conventional yeast S. cerevisiae. Y. Lipolytica prefer secreting proteins through the co-transcription pathway and does so very efficiently2, it does not exhibit hyperglycosylation as S. cerevisiae3. Y. Lipolytica has also been shown to exhibit excellent characteristics for the production of value-added chemicals such as a long range of organic acids and polyols Biotechnological applications of Yarrowia lipolytica: Past, present and future, and the recent introduction of several genome-scale models for Y. Lipolytica will most likely lead to more processes utilizing the chassis for production. Perhaps the most important advantage for using Y. Lipolytica over S. cerevisiae, to our project at least, is the broad substrate utilization range of Y. Lipolytica. Y. Lipolytica is known to naturally utilize alcohols (especially glycerol), acetate and hydrophobic substrates (eg. alkanes, fatty acids and oils) as carbon sources4. This has naturally lead to Y. Lipolytica becoming a model organism for several metabolic pathways, especially fatty acid transport and metabolism, and single cell oil (SCO) accumulation. Y. Lipolytica has even been shown to exhibit enhanced growth on mixed substrates Yarrowia lipolytica as an oleaginous cell factory platform for production of fatty acid-based biofuel and bioproducts, which renders it ideal for utilization of industrial waste streams due to the highly diverse content of these. These findings had us believe that we had found an excellent candidate chassis for our project. The table shows a comparison of the substrate range of Y. Lipolytica W29 and S. cerevisiae CEN.PK113-7D.
| Y. Lipolytica | S. cerevisiae | |
|---|---|---|
| Sediment from canola oil production | µ = 0.31 | None |
| Glycerol from Perstop | µ = 0.27 | None |
| Glycerol from Emmelev | µ = 0.45 | None |
| Glycerol from Daka | µ = 0.31 | None |
| Molasses from Dansukker | µ = 0.42* | µ = 0.47 |
* it should be noted that the molasses was autoclaved thus degrading some of the sucrose content. This growth might not be possible to replicate with untreated molasses.
As seen in the table Y. Lipolytica is able to grow on all the waste sources we tested, while S. cerevisiae is only able to grow on molasses.
Methods
Each growth experiment (for Y. Lipolytica and S. cerevisiae) is conducted according to the following:
Minimal medium is produced as stated by Mhairi Workman5 using a C-source concentration of 20 g/L was used all for growth experiments.
The cells were grown overnight in YPD medium, and prepared by spinning down and washed twice. The preculture was then used as inoculum for minimal medium (substituents) to a final concentration of 0,001 (OD600). The cultivations were carried out in a cytometer (brand) shaking 900 rpm at 30℃ (86℉). Cultures were grown, shaked and measured in 48 well microtitre plates (Cellstar). Measurements was carried out by a Hamilton Robot, (cpe201, Hamilton industries) connected to a BioTek spectrophotometer (See protocols here). OD600 Measurements were taken every 2 hours until the cultures reached stationary phase, and data was analysed using R-studio as is visualised in figure 2.
During the growth experiments we cept to strains that were wild type og close to. This makes the results more general for the organism.
| Strains | Genotype | Comment/source |
|---|---|---|
| Y. Lipolytica | Wildtype | Parent strain to our laboratory bug, PO1f |
| S. cerevisiae | CEN.PK113-7D | Derived from parental strains ENY.WA-1A and MC996A, and is popular for use in systems biology studies |
Outline of proces
We performed growth experiments on an array of pure C-sources (seen in figure 3-4) to get a baseline of Y. Lipolytica growth patterns starting to determine the substrate range. In these experiments we observed the following growth rates or lack of growth.
| Y. Lipolytica | S. cerevisiae | |
|---|---|---|
| Glucose | µ = 0.24 | µ = 0.19 |
| Fructose | µ = 0.23 | µ = 0.426 |
| Glycerol | µ = 0.27 | None |
| Canola oil | µ = 0.08 | None |
| Sucrose | None | µ = 0.396 |
| Maltose | None | Growth7 |
| Xylose | None | None8 |
| Arabinose | None | None8 |
| Starch | None | None |
The interesting graphs repressenting these results can be seen in the figures 5-9:
Even though the pure carbon sources suggests that Y. Lipolytica exhibits excellent substrate utilization, we did not know if this translated into utilization of industrial waste streams. To investigate this, we had to get our hands on a few waste streams we could test. We contacted local industry that we knew had waste streams containing either sugars, glycerol og oily constituents. After many phone calls and long meetings, we received the following byproducts of organically based productions:
- Canola oil sediment
- Glycerol Perstorp Tech
- Glycerol Emmelev
- Glycerol Daka
- Molasses Dansukker
Canola oil sediment - Grønningaard
Grønningaard is a canola oil production facility situated on Zealand, Denmark. They produce 100 - 120 tons canola oil annually, and sell the remaining protein rich press cake for animal feed. The oil is derived by cold pressing organic rapeseeds. As cold pressing does not allow for filtering of the oil, small fibres remain in the oil. These fibres are removed by allowing the oil to sediment for 1 month and extracting the sediment. Besides the fibres from the plants and residual oil, the sediment contain, polyaromatic hydrocarbons in too high concentrations making the sediment unsuitable to be recycled in the process or used for animal feed, rendering it a “true waste” in the sense that it is only useful for generating heat through burning. The figure shows an overview of the process. The sediment constitutes 1-1.6% of the biomass of the product, amounting to 1 - 1.92 tons annually. These figures are based on the 4th. biggest producer in denmark Grønninggård. The largest with an estimated 80% market share is not willing to provide production numbers. (Personal communication)
During the experiments with this substrate we experienced a lot of problems with the od measurements because of the high content of plant fibers. By pressure filtering the sediment we extracted a sample that was transparent. From this sample we showed that Y. Lipolytica grows weary well on this waste stream. S. cerevisiae on the other hand is not able to utilize this carbon source as seen in the figures.
This show promising growth of Y. Lipolytica while it is clear that S. cerevisiae is suitable for fermentation based on canola oil sediments
Glycerol byproduct
In trying to find alternatives to fossil fuels the production of biodiesel have increased tremendously in the last two decades. Biodiesel is produced by ar base-catalyzed transesterification by a short chained alcohol and triacylglycerols derived from natural sources as seen below. This reaction produces 0.102 kg glycerol pr. liter biodiesel9. The increased production have resulted in a plummeting of glycerol prices making it a promising substrate for industrial fermentation.
Second generation biodiesel facility (DAKA)
The Danish branch of DAKA refines waste streams from the feedstock industry (such as meat and agricultural industry), turning it into products such as fertilizers, animal feed and biodiesel. The biodiesel production is based on animal tallow and fats from the danish meat industry. The glycerol derived from from this production, has a high salt content and a particularly low pH, and therefore requires several purification steps before it can be used in the chemical industry. By adding NaOH raising the pH to 6 we able to make Y. Lipolytica grow fairly well despite of the relatively high salt levels. (Personal communication)
First generation glycerol/glycerin (Perstorp/ Emmelev)
Perstop has two biodiesel production facilities. One located in Sweden and one in Norway. They produce high quality glycerin and glycerol, that is sold as a component for chemical production. This has a purity of og 98% and the rest is mostly water (Personal communication). This byproduct is also a great substrate for Y. Lipolytica as seen in the figure.
Glycerin from first generation biodiesel facility Emmelev A/S. Emmelev A/S is a local oil mill, first gen. biodiesel plant and glycerin destillor located on the second biggest island in Denmark, Fyn. The glycerin is distilled to 80% purity and sold to the chemical industry (Personal communication) . This is fairly high quality and there are no content inhibiting the growth of Y. Lipolytica as seen in the figure.
In conclution Y. Lipolytica is the only sutable candidate for fermentation based on glycerol.
Molasses
The process of creating refined sugar results in the waste product molasses that is quite applicable. Molasses, the byproduct of the refining of sugarcane or sugar beets into sugar, has a brown color and is sweet flavor do to the high sucrose, glucose and fructose content. There for it is often used prepared meals or animal feed. Molasses is created when the juice from sugar canes is boiled and the crystallized sugar is removed twice as seen in the figure. As molasses ultimately is a byproduct it will be quite useful to use as a substrate for fermentation.
As seen in the figure both Y. Lipolytica and S. cerevisiae grow on molasses. Normally Y. Lipolytica does not grow well on sucrose, but there is a high content of glucose and fructose as well. On top of that we realised that sucrose degrades to fructose and glucose when autoclaved. (Personal communication)
Bowth organism might be suitable for growt on molasses, but because S. cerevisiae is able to utilize sucrose it might be the better choice for this biproduct.
References
- Barth, G. and Gaillardin, C. (1997), Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiology Reviews, 19: 219–237. doi:10.1111/j.1574-6976.1997.tb00299.x
- María Domínguez, Jonathan D. Wasserman, Matthew Freeman, Multiple functions of the EGF receptor in Drosophila eye development, Current Biology, Volume 8, Issue 19, 24 September 1998, Pages 1039-1048, ISSN 0960-9822, http://dx.doi.org/10.1016/S0960-9822(98)70441-5.
- E. V. Shusta, R. T. Raines(1998). ncreasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. http://www.nature.com/nbt/journal/v16/n8/pdf/nbt0898-773.pdf
- Barth, G. (2013). Yarrowia lipolytica Genetics, Genomics, and Physiology. http://www.springer.com/us/book/9783642383199
- Mhairi Workman, Philippe Holt (2013). Comparing cellular performance of Yarrowia lipolytica during growth on glucose and glycerol in submerged cultivations
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- Jansen, M. L. A., Daran-Lapujade, P., de Winde, J. H., Piper, M. D. W., & Pronk, J. T. (2004). Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity. Applied and Environmental Microbiology, 70(4), 1956–1963. http://doi.org/10.1128/AEM.70.4.1956-1963.2004
- Wisselink, H. W., Toirkens, M. J., del Rosario Franco Berriel, M., Winkler, A. A., van Dijken, J. P., Pronk, J. T., & van Maris, A. J. A. (2007). Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of l-Arabinose . Applied and Environmental Microbiology, 73(15), 4881–4891. http://doi.org/10.1128/AEM.00177-07
- Syed Shams Yazdani, Ramon Gonzalez, Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry, Current Opinion in Biotechnology, Volume 18, Issue 3, June 2007, Pages 213-219, ISSN 0958-1669, http://dx.doi.org/10.1016/j.copbio.2007.05.002.
