Line 114: | Line 114: | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
− | + | ||
<p> | <p> | ||
* 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. | * 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. | ||
</p> | </p> | ||
− | + | ||
<p> | <p> | ||
As seen in the table <i> Y. Lipolytica </i> is able to grow on all the waste sources we tested, while <i>S. cerevisiae</i> is only able to grow on molasses. | As seen in the table <i> Y. Lipolytica </i> is able to grow on all the waste sources we tested, while <i>S. cerevisiae</i> is only able to grow on molasses. | ||
</p> | </p> | ||
− | + | ||
</div> <!-- /overview--> | </div> <!-- /overview--> | ||
<div><a class="anchor" id="section-2"></a> | <div><a class="anchor" id="section-2"></a> | ||
<h2 class="h2">Methods</h2> | <h2 class="h2">Methods</h2> | ||
− | + | ||
<p> | <p> | ||
Each growth experiment (for <i> Y. Lipolytica </i> and <i>S. cerevisiae</i>) is conducted according to the following: | Each growth experiment (for <i> Y. Lipolytica </i> and <i>S. cerevisiae</i>) is conducted according to the following: | ||
</p> | </p> | ||
− | + | ||
<p> | <p> | ||
Minimal medium is produced as stated by Mhairi Workman<sup><a href="#references">5</a></sup> using a C-source concentration of 20 g/L was used all for growth experiments. | Minimal medium is produced as stated by Mhairi Workman<sup><a href="#references">5</a></sup> using a C-source concentration of 20 g/L was used all for growth experiments. | ||
</p> | </p> | ||
− | + | ||
<p> | <p> | ||
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 degrees celsius. 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. | 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 degrees celsius. 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. | ||
</p> | </p> | ||
− | + | ||
<table class="table table-bordered"> | <table class="table table-bordered"> | ||
<thead> | <thead> | ||
Line 162: | Line 162: | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
− | + | ||
</div> | </div> | ||
Line 170: | Line 170: | ||
We performed growth experiments on an array of pure C-sources to get a baseline of <i> Y. Lipolytica </i> growth patterns starting to determine the substrate range. In these experiments we observed the following growth rates or lack of growth. | We performed growth experiments on an array of pure C-sources to get a baseline of <i> Y. Lipolytica </i> growth patterns starting to determine the substrate range. In these experiments we observed the following growth rates or lack of growth. | ||
</p> | </p> | ||
− | + | ||
<table class="table table-bordered"> | <table class="table table-bordered"> | ||
<thead> | <thead> | ||
Line 227: | Line 227: | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
− | + | ||
<p> | <p> | ||
Even though the pure carbon sources suggests that <i>Y. Lipolytica</i> 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: | Even though the pure carbon sources suggests that <i>Y. Lipolytica</i> 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: | ||
</p> | </p> | ||
− | + | ||
<ul> | <ul> | ||
<li>Canola oil sediment</li> | <li>Canola oil sediment</li> | ||
Line 247: | Line 247: | ||
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) | 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) | ||
</p> | </p> | ||
− | + | ||
<figure class="figure"> | <figure class="figure"> | ||
<img id="GGprod" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/d/df/T--DTU-Denmark--GGproduction.jpg" alt="DESCRIPTION" width="400px"> | <img id="GGprod" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/d/df/T--DTU-Denmark--GGproduction.jpg" alt="DESCRIPTION" width="400px"> | ||
Line 258: | Line 258: | ||
<img class="modal-content" id="GGprodImg"> | <img class="modal-content" id="GGprodImg"> | ||
</div> | </div> | ||
− | + | ||
<p> | <p> | ||
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 <i>Y. Lipolytica</i> grows weary well on this waste stream. <i>S. cerevisiae</i> on the other hand is not able to utilize this carbon source as seen in the figures. | 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 <i>Y. Lipolytica</i> grows weary well on this waste stream. <i>S. cerevisiae</i> on the other hand is not able to utilize this carbon source as seen in the figures. | ||
</p> | </p> | ||
− | + | ||
<div class = "col-md-6"> | <div class = "col-md-6"> | ||
<figure class="figure"> | <figure class="figure"> | ||
Line 291: | Line 291: | ||
</div> | </div> | ||
</div> | </div> | ||
− | + | ||
</div> | </div> | ||
<div><a class="anchor" id="section-5"></a> | <div><a class="anchor" id="section-5"></a> | ||
− | <h2 class="h2"> | + | <h2 class="h2">Glycerol byproduct</h2> |
<p> | <p> | ||
− | + | 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 biodiesel<sup><a href="#references">9</a></sup>. In 2011 the production of biodiesel reached over 21 billion liters resulting in an increased production of glycerol<sup><a href="#references">10</a></sup>. The increased production have resulted in a plummeting of glycerol prices making it a promising substrate for industrial fermentation<sup><a href="#references">11</a></sup>. | |
</p> | </p> | ||
− | < | + | |
− | < | + | <figure class="figure"> |
− | + | <img id="Glycero" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/e/e7/T--DTU-Denmark--Glycerol_production.png" alt="DESCRIPTION"> | |
− | + | <figcaption class="figure-caption">Flow chart for production of biodisel and glycerol waste.</figcaption> | |
+ | </figure> | ||
+ | |||
+ | <!-- The Modal with same picture--> | ||
+ | <div id="GlyceroModal" class="modal"> | ||
+ | <span class="close Glycero">×</span> | ||
+ | <img class="modal-content" id="GlyceroImg"> | ||
+ | </div> | ||
− | + | ||
− | + | <h3 class="h3">Second generation biodiesel facility (DAKA)</h3> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <h3 class="h3"> | + | |
<p> | <p> | ||
− | + | 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 <i> Y. Lipolytica </i> grow fairly well despite of the relatively high salt levels. (Personal communication) | |
− | + | ||
− | + | ||
</p> | </p> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
+ | <div class = "col-md-6"> | ||
+ | <figure class="figure"> | ||
+ | <img id="DakaY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/b/b3/T--DTU-Denmark--Glycerol_%28Daka%29_y.lip.png" alt="DESCRIPTION"> | ||
+ | <figcaption class="figure-caption"><i>Y. Lipolytica</i> growth on glycerol from second generation biodisel</figcaption> | ||
+ | </figure> | ||
− | </div> | + | <!-- The Modal with same picture--> |
+ | <div id="DakaYModal" class="modal"> | ||
+ | <span class="close DakaY">×</span> | ||
+ | <img class="modal-content" id="DakaYImg"> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <div class = "col-md-6"> | ||
+ | |||
+ | <figure class="figure"> | ||
+ | <img id="DakaS" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/f/f8/T--DTU-Denmark--Glycerol_%28Daka%29_s.cer.png" alt="DESCRIPTION"> | ||
+ | <figcaption class="figure-caption"><i>S. cerevisiae</i> does not grow on glycerol.</figcaption> | ||
+ | </figure> | ||
+ | |||
+ | |||
+ | <!-- The Modal with same picture--> | ||
+ | <div id="DakaSModal" class="modal"> | ||
+ | <span class="close DakaS">×</span> | ||
+ | <img class="modal-content" id="DakaSImg"> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | </div> <!-- Glycerol end--> | ||
+ | |||
+ | |||
+ | |||
+ | |||
<div><a class="anchor" id="section-6"></a> | <div><a class="anchor" id="section-6"></a> | ||
<h2 class="h2">Section 6</h2> | <h2 class="h2">Section 6</h2> | ||
Line 354: | Line 378: | ||
<li>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</li> | <li>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</li> | ||
<li>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</li> | <li>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</li> | ||
− | <li></li> | + | <li>(Yazdani and Gonzalez, 2007)</li> |
− | <li></li> | + | <li> (Avinash, 2014)</li> |
− | <li></li> | + | <li> (Ashby et al., 2008)</li> |
<li></li> | <li></li> | ||
<li></li> | <li></li> |
Revision as of 10:50, 19 October 2016
Introduction
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Someone famous in Source Title
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. The figure 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 degrees celsius. 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.
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 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 |
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.
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. In 2011 the production of biodiesel reached over 21 billion liters resulting in an increased production of glycerol10. The increased production have resulted in a plummeting of glycerol prices making it a promising substrate for industrial fermentation11.
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)
Section 6
Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
Section 7
Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
References
- Barth and Gaillardin, 1996
- (Domínguez et al., 1998)
- (Shusta et al. 1998)
- Barth 2013
- (Mhairi Workman, Philippe Holt, 2013)
- H. Shafaghat, G.D. Najafpour. Growth Kinetics and Ethanol Productivity of Saccharomyces cerevisiae PTCC 24860 on Varius Carbon Sources. ISSN 1818-4952
- 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
- (Yazdani and Gonzalez, 2007)
- (Avinash, 2014)
- (Ashby et al., 2008)