Difference between revisions of "Team:DTU-Denmark/substrate"

 
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                   <h1>Substrate<p class="lead">As the substrate utilization of <i>Y. lipolytica</i> 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 <i>Y. lipolytica</i>. We further expanded on the research by acquiring real waste streams and byproducts form organic industrial productions in the Nordic countries.</p></h1>
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                   <h1>Substrate<p class="lead">As the substrate utilization of <i>Yarrowia lipolytica</i> is central to our project we have performed an array of growth experiments. We have tested the growth on simple media to determine strengths and weaknesses in the catabolism of <i>Y. lipolytica</i>. We further expanded our research by acquiring real waste streams and byproducts form organic industrial productions in the Nordic countries and screened <i>Y. lipolytica</i> growth for these substrates.</p></h1>
 
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                            <p>"Growth can be the result of many trials"</p>
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                            <small>Mohamad El Lakany, <cite title="Source Title">Mohamad's Mantra</cite></small>
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               <h2 class="h2">Introduction</h2>
 
               <h2 class="h2">Introduction</h2>
  
             
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                <p>"Growth can be the result of many trials"</p>
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                            <small>Mohamad El Lakany, <cite title="Source Title">Mohamad's Mantra</cite></small>
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               <p>
 
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                 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 <i>Candida lipolytica</i>, then <i>Endomycopsis lipolytica</i>, <i>Saccharomycopsis lipolytica</i> and finally <i>Yarrowia lipolytica</i><sup><a href="#references">1</a></sup>. The figure shows <i>Y. lipolytica</i> cells under a microscope. The magnification factor is 100x.
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                 The dimorphic, non-conventional yeast <i>Yarrowia lipolytica</i>, belonging to the Ascomycota phylum, was first isolated in the 1960s from lipid-rich materials, hence the name “lipolytica”. The organism was classified and reclassified a number of times, first as <i>Candida lipolytica</i>, then <i>Endomycopsis lipolytica</i>, <i>Saccharomycopsis lipolytica</i> and finally <i>Yarrowia lipolytica</i><sup><a href="#references">1</a></sup>. Figure 1 shows <i>Y. lipolytica</i> cells under a microscope.  
 
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                 <img id="Y.lMicro" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/5/57/T--DTU-Denmark--micro-Y.lip.png" alt="DESCRIPTION" width="400px">
 
                 <img id="Y.lMicro" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/5/57/T--DTU-Denmark--micro-Y.lip.png" alt="DESCRIPTION" width="400px">
                 <figcaption class="figure-caption">This figure shows <i>Y.lipolytica</i> in plactonic growth with 100x magnification</figcaption>
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                 <figcaption class="figure-caption"> <b>Figure 1:</b> <i>Y.lipolytica</i> in plactonic growth with 100x magnification.</figcaption>
 
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                In recent years <i> Y. Lipolytica</i> 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 <i> Y. Lipolytica</i> has over the conventional yeast <i> S. cerevisiae</i>. <i> Y. Lipolytica</i> prefer secreting proteins through the co-transcription pathway and does so very efficiently<sup><a href="#references">2</a></sup>, it does not exhibit hyperglycosylation as <i> S. cerevisiae</i><sup><a href="#references">3</a></sup>. <i> Y. Lipolytica</i> 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 <i> Y. Lipolytica</i> will most likely lead to more processes utilizing the chassis for production. Perhaps the most important advantage for using <i> Y. Lipolytica</i> over <i> S. cerevisiae</i>, to our project at least, is the broad substrate utilization range of <i> Y. Lipolytica</i>. <i> Y. Lipolytica</i> is known to naturally utilize alcohols (especially glycerol), acetate and hydrophobic substrates (eg. alkanes, fatty acids and oils) as carbon sources<sup><a href="#references">4</a></sup>. This has naturally lead to <i> Y. Lipolytica</i> becoming a model organism for several metabolic pathways, especially fatty acid transport and metabolism, and single cell oil (SCO) accumulation. <i> Y. Lipolytica</i> 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 <i> Y. Lipolytica</i> W29 and <i> S. cerevisiae</i> CEN.PK113-7D.
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                  In recent years, <i> Y. Lipolytica</i> has received increased attention from researchers, as studies have found it to possess great potential for producing industrial enzymes and pharmaceutical proteins. This potential is a result of several advantages that <i> Y. Lipolytica</i> has over the conventional yeast <i> S. cerevisiae</i>. <i> Y. Lipolytica</i> prefers secreting proteins through the co-transcription pathway and does so very efficiently<sup><a href="#references">2</a></sup> in addition, it does not exhibit hyperglycosylation as <i> S. cerevisiae</i> does<sup><a href="#references">3</a></sup>. <i> Y. Lipolytica</i> 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. The recent introduction of several genome-scale models for <i> Y. Lipolytica</i> will most likely lead to more processes utilizing the chassis for production. Perhaps, the most important advantage for using <i> Y. Lipolytica</i> over <i> S. cerevisiae</i>, to our project at least, is the broad substrate utilization range of <i> Y. Lipolytica</i>. <i> Y. Lipolytica</i> is known to naturally utilize alcohols (especially glycerol), acetate and hydrophobic substrates (eg. alkanes, fatty acids and oils) as carbon source <sup><a href="#references">4</a></sup>. This has naturally led to <i> Y. Lipolytica</i> becoming a model organism for several metabolic pathways, especially fatty acid transport, -metabolism, and single cell oil (SCO) accumulation. <i> Y. Lipolytica</i> has even been shown to exhibit enhanced growth on mixed substrates. <i>Yarrowia lipolytica</i> is an oleaginous cell factory platform for production of fatty acid-based biofuels and bioproducts. This renders it ideal for utilization of industrial waste streams due to their complex and variable content. These findings have us believe that we had found an excellent candidate chassis for our project. The table below shows a comparison of the substrate range of <i> Y. Lipolytica</i> W29 and <i> S. cerevisiae</i> CEN.PK113-7D.
 
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                 Each growth experiment (for <i> Y. Lipolytica </i> and <i>S. cerevisiae</i>) is conducted according to the following:  
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                 Each growth experiment (for <i> Y. Lipolytica </i> and <i>S. cerevisiae</i>) is conducted according to the following setup:  
 
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                 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.
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                 Minimal medium is produced as directed by Mhairi Workman<sup><a href="#references">5</a></sup> using 20g/L of a given carbon source for all the growth experiments.
  
 
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                 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&#8451; (86&#8457;). 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.
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                 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 (OD<sub>600</sub>) measured by Spectrophotometry (Shimadzu UV-1800). The cultivations were carried out in a cytomat (Thermo Scientific) shaking 900 rpm at 30 degree celsius. Cultures were grown, shaked and measured in a 48 well suspension culture plates (Cellstar, Greiner-bio-one). The measurements were carried out using a Hamilton Microlab Robot, (Hamilton Life science Robotics) connected to a plate spectrophotometer (BioTek Synergy 2).OD<sub>600</sub> measurements were taken every 2 hours until the cultures reached stationary phase. Data was then analysed and visualized using excel and R-studio Figure 2.
 
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                <figcaption class="figure-caption"> <b>Figure 2:</b> <b>A.</b> Overnight culture: strains of <i>Y. lipolytica</i> and <i>S. cerevisiae</i> are grown in YPD media overnight at 30&#8451; (86&#8457;)  to ensure balanced growth and comparable data.
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                  <b>B.</b> Washing- and inoculation steps: Cells are spinned down and washed to ensure removal of carbon-sources and other metabolites from the overnight-culturing. Washing and spinning step is repeated.
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                  Simple and complex substrates are inoculated with cells in 48 well suspension culture plates. The cells reaches final OD<sub>600</sub> 0.001
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                  <b>C.</b> Growth-experiment: Plates are incubated and shaken at 900 rpm in a cytometer and before measurement of OD in a spectrophotometer. Data are recorded and compiled in an excel sheet with two hours intervals. This process is assisted by using the Hamilton Microlab robot.
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                  <b>D.</b> Data analysis and -visualization step:</b> The data excel sheet (in step <b>C.</b>) are analyzed and visualized by plots using R-studio.</figcaption>
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              <p>During the growth experiments we kept to strains that were wild type or closely related. This makes the results more general for the organism.</p>
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               <h2 class="h2">Outline of proces</h2>
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               <h2 class="h2">Outline of Process</h2>
  
 
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                   <img id="CMp" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/4/42/T--DTU-Denmark--SamletKomplekse_medier_compri.jpg" alt="DESCRIPTION">
 
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                   <figcaption class="figure-caption">Picture of the waste products we recived</figcaption>
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                   <figcaption class="figure-caption"><b>Figure 3:</b> Picture of the waste products we received.</figcaption>
 
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                   <img id="MMp" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/5/56/T--DTU-Denmark--Samletsubstrater_smaavials_compri.JPG" alt="DESCRIPTION">
                   <figcaption class="figure-caption">Picture of the autoclaved C-source solutions</figcaption>
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                   <figcaption class="figure-caption"><b>Figure 4:</b> Picture of the autoclaved C-source solutions.</figcaption>
 
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               <p>
                 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.   
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                 We performed growth experiments on an array of pure C-sources (seen in figure 3-4) to get a baseline of <i> Y. Lipolytica </i> growth patterns emerged indicating the substrate range. In these experiments we observed the following growth rates or lack of growth.   
 
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               <p> These results can be seen in the following pictures:</p>
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               <p> The graphs representing these results can be seen in the figures 5-9:</p>
             
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                   <img id="fruc" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/d/d1/T--DTU-Denmark--Fructose_y.lip.png" alt="DESCRIPTION">
 
                   <img id="fruc" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/d/d1/T--DTU-Denmark--Fructose_y.lip.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>Y. Lipolytica</i> growth on fructose</figcaption>
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                   <figcaption class="figure-caption"><b>Figure 5:</b> <i>Y. Lipolytica</i> growth on fructose.</figcaption>
 
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                   <img id="suc" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/1/1f/T--DTU-Denmark--Sucrose_y.lip.png" alt="DESCRIPTION">
 
                   <img id="suc" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/1/1f/T--DTU-Denmark--Sucrose_y.lip.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>Y. Lipolytica</i> do not grow on sucrose</figcaption>
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                   <figcaption class="figure-caption"><b>Figure 6:</b> <i>Y. Lipolytica</i> does not grow on sucrose.</figcaption>
 
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                   <img id="ole" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/2/28/T--DTU-Denmark--Canola_oil_y.lip.png" alt="DESCRIPTION">
 
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                   <figcaption class="figure-caption"><i>Y. Lipolytica</i> growth on Canola oil</figcaption>
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                   <figcaption class="figure-caption"><b>Figure 7:</b> <i>Y. Lipolytica</i> growth on Canola oil.</figcaption>
 
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                   <img id="gluY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/6/67/T--DTU-Denmark--Glucose_y.lip.png" alt="DESCRIPTION">
 
                   <img id="gluY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/6/67/T--DTU-Denmark--Glucose_y.lip.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>Y. Lipolytica</i> growth on fructose</figcaption>
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                   <figcaption class="figure-caption"><b>Figure 8:</b> <i>Y. Lipolytica</i> growth on glucose.</figcaption>
 
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                   <img id="glus" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/0/09/T--DTU-Denmark--Glucose_S.cer.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>S. cerevisiae</i> growth on glucose</figcaption>
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                   <figcaption class="figure-caption"><b>Figure 9:</b> <i>S. cerevisiae</i> growth on glucose.</figcaption>
 
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               <p><br>
                 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:
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                 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 or oily constituents. After many phone calls and long meetings, we received the following byproducts of organically based productions:
 
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               <h2 class="h2">Canola oil sediment - Grønningaard</h2>
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               <h2 class="h2">Industrial Byproduct Screenings</h2>
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              <h3 class="h3">Canola Oil Sediment - Grønningaard</h3>
 
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                 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 before extracting the sediment. Besides the fibres from the plants and residual oil, the sediment contains polyaromatic hydrocarbons in 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 incineration. Figure 10 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>
  
 +
              <center>
 
               <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="800px">
                 <figcaption class="figure-caption">Flow chart for the production of cold pressed canola oil</figcaption>
+
                 <figcaption class="figure-caption"><b>Figure 10:</b> Flow chart for the production of cold pressed canola oil.</figcaption>
 
               </figure>
 
               </figure>
 
+
              </center>
 
               <!-- The Modal with same picture-->
 
               <!-- The Modal with same picture-->
 
               <div id="GGprodModal" class="modal">
 
               <div id="GGprodModal" class="modal">
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               <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 using this substrate we experienced a lot of problems with the OD measurements because of the high content of plant fibers. Through pressure filtering, a transparent sample was extracted. We demonstrated that <i>Y. Lipolytica</i> grows very well on this waste stream. <i>S. cerevisiae</i> on the other hand is not able to utilize this carbon source as seen in figures 11-12.
 
               </p>
 
               </p>
  
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                 <figure class="figure">
 
                 <figure class="figure">
 
                   <img id="GGy" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/3/33/T--DTU-Denmark--Groenninggaard_waste_y.lip.png" alt="DESCRIPTION">
 
                   <img id="GGy" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/3/33/T--DTU-Denmark--Groenninggaard_waste_y.lip.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>Y. Lipolytica</i> growth on sediment from canola oil production</figcaption>
+
                   <figcaption class="figure-caption"><b>Figure 11:</b> <i>Y. Lipolytica</i> growth on sediment from canola oil production.</figcaption>
 
                 </figure>
 
                 </figure>
  
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                 <figure class="figure">
 
                 <figure class="figure">
 
                   <img id="GGs" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/1/13/T--DTU-Denmark--Groenninggaard_waste_s.cer.png" alt="DESCRIPTION">
 
                   <img id="GGs" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/1/13/T--DTU-Denmark--Groenninggaard_waste_s.cer.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>S. cerevisiae</i> does not grow on sediment from canola oil production.</figcaption>
+
                   <figcaption class="figure-caption"><b>Figure 12:</b> <i>S. cerevisiae</i> does not grow on sediment from canola oil production.</figcaption>
 
                 </figure>
 
                 </figure>
  
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                 </div>
 
                 </div>
 
               </div>
 
               </div>
               <p>This show promising growth of <i>Y. Lipolytica</i> while it is clear that <i>S. cerevisiae</i> is suitable for fermentation based on canola oil sediments</p>
+
               <p>This shows promising growth of <i>Y. Lipolytica</i> while it is clear that <i>S. cerevisiae</i> is unsuitable for fermentation based on canola oil sediments</p>
  
 
             </div>
 
             </div>
  
             <div><a class="anchor" id="section-5"></a>
+
             <div>
               <h2 class="h2">Glycerol byproduct</h2>
+
               <h3 class="h3">Glycerol Byproduct</h3>
 
               <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>. The increased production have resulted in a plummeting of glycerol prices making it a promising substrate for industrial fermentation.
+
                 The push to find an alternative to fossil fuel has increased demand and production of biodiesel tremendously in the last two decades. Biodiesel is produced by a base-catalyzed transesterification by a short chained alcohol and triacylglycerols derived from natural sources as seen in figure 13. This reaction produces 0.102 kg glycerol pr. liter biodiesel<sup><a href="#references">9</a></sup>. The increased production has resulted in a plummeting of glycerol prices making it a promising substrate for industrial fermentation.
 
               </p>
 
               </p>
  
 
               <figure class="figure">
 
               <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" width = 400>
 
                 <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" width = 400>
                 <figcaption class="figure-caption">Flow chart for production of biodisel and glycerol waste.</figcaption>
+
                 <figcaption class="figure-caption"><b>Figure 13:</b> Flow chart for production of biodiesel and glycerol waste.</figcaption>
 
               </figure>
 
               </figure>
  
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               <h3 class="h3">Second generation biodiesel facility (DAKA)</h3>
+
               <h3 class="h3">Second Generation Biodiesel Facility (DAKA)</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)  
+
                 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 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 and raising the pH to 6 we were able to make <i> Y. Lipolytica </i> grow fairly well in spite of the relatively high salt levels as seen in Figure 14 (Personal communication).
 
               </p>
 
               </p>
  
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                 <figure class="figure">
 
                 <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">
 
                   <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>
+
                   <figcaption class="figure-caption"><b>Figure 14:</b> <i>Y. Lipolytica</i> growth on glycerol from second generation biodiesel.</figcaption>
 
                 </figure>
 
                 </figure>
  
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                 <figure class="figure">
 
                 <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">
 
                   <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>
+
                   <figcaption class="figure-caption"><b>Figure 15:</b> <i>S. cerevisiae</i> does not grow on glycerol.</figcaption>
 
                 </figure>
 
                 </figure>
  
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               </div>
 
               </div>
  
               <h3 class="h3">First generation glycerol/glycerin (Perstorp/ Emmelev) </h3>
+
               <h3 class="h3">First Generation Glycerol (Perstorp and Emmelev) </h3>
 
               <p>
 
               <p>
                 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 <i> Y. Lipolytica </i> as seen in the figure.
+
                 Perstop has two biodiesel production facilities, one located in Sweden and one in Norway. They produce high quality glycerin, that is sold as a component for chemical production. This has a purity of 95-100% the remainder of mostly water (Personal communication). This byproduct is also a great substrate for <i> Y. Lipolytica </i> as seen in Figure 16.
 
               </p>
 
               </p>
  
 +
              <div class = "col-md-12">
 
               <div class = "col-md-6">
 
               <div class = "col-md-6">
 
                 <figure class="figure">
 
                 <figure class="figure">
 
                   <img id="PTY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/a/ad/T--DTU-Denmark--Glycerol_%28Perstop_Tech%29_y.lip.png" alt="DESCRIPTION">
 
                   <img id="PTY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/a/ad/T--DTU-Denmark--Glycerol_%28Perstop_Tech%29_y.lip.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>Y. Lipolytica</i> growth on glycerol from first generation biodisel</figcaption>
+
                   <figcaption class="figure-caption"><b>Figure 16:</b> <i>Y. Lipolytica</i> growth on glycerol from first generation biodiesel.</figcaption>
 
                 </figure>
 
                 </figure>
  
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                 <figure class="figure">
 
                 <figure class="figure">
 
                   <img id="PTS" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/8/8e/T--DTU-Denmark--Glycerol_%28Perstop_Tech%29_s.cer.png" alt="DESCRIPTION">
 
                   <img id="PTS" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/8/8e/T--DTU-Denmark--Glycerol_%28Perstop_Tech%29_s.cer.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>S. cerevisiae</i> does not grow on glycerol.</figcaption>
+
                   <figcaption class="figure-caption"><b>Figure 17:</b> <i>S. cerevisiae</i> does not grow on glycerol.</figcaption>
 
                 </figure>
 
                 </figure>
  
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                 </div>
 
                 </div>
  
 +
              </div>
 
               </div>
 
               </div>
  
 
               <p>
 
               <p>
                 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 <i> Y. Lipolytica </i> as seen in the figure.
+
                 Emmelev A/S is a local oil mill, first generation biodiesel plant and a 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 components that inhibit the growth of <i> Y. Lipolytica </i> as seen in Figure 19.
 
               </p>
 
               </p>
 
+
             
 +
              <div class = "col-md-12">
 
               <div class = "col-md-6">
 
               <div class = "col-md-6">
 
                 <figure class="figure">
 
                 <figure class="figure">
 
                   <img id="EmY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/6/62/T--DTU-Denmark--Glycerol_%28Emmelev%29_y.lip.png" alt="DESCRIPTION">
 
                   <img id="EmY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/6/62/T--DTU-Denmark--Glycerol_%28Emmelev%29_y.lip.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>Y. Lipolytica</i> growth on glycerol from first generation biodisel</figcaption>
+
                   <figcaption class="figure-caption"><b>Figure 19:</b> <i>Y. Lipolytica</i> growth on glycerol from first generation biodiesel.</figcaption>
 
                 </figure>
 
                 </figure>
  
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                 <figure class="figure">
 
                 <figure class="figure">
 
                   <img id="EmS" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/f/f6/T--DTU-Denmark--Glycerol_%28Emmelev%29_S.cer.png" alt="DESCRIPTION">
 
                   <img id="EmS" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/f/f6/T--DTU-Denmark--Glycerol_%28Emmelev%29_S.cer.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>S. cerevisiae</i> does not grow on glycerol.</figcaption>
+
                   <figcaption class="figure-caption"><b>Figure 20:</b> <i>S. cerevisiae</i> does not grow on glycerol.</figcaption>
 
                 </figure>
 
                 </figure>
  
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                   <img class="modal-content" id="EmSImg">
 
                   <img class="modal-content" id="EmSImg">
 
                 </div>
 
                 </div>
 +
              </div>
 
               </div>
 
               </div>
  
               <p> In conclution <i>Y. Lipolytica</i> is the only sutable candidate for fermentation based on glycerol.</p>
+
               <p> In conclusion, <i>Y. Lipolytica</i> is the only suitable candidate for fermentation based on glycerol.</p>
  
 
             </div> <!-- Glycerol end-->
 
             </div> <!-- Glycerol end-->
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             <div><a class="anchor" id="section-6"></a>
+
             <div>
               <h2 class="h2">Molasses</h2>
+
               <h3 class="h3">Molasses</h3>
 
               <p>
 
               <p>
                 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.  
+
                 The process of creating refined sugar results in the waste product molasses. Molasses is a byproduct of the refining of sugarcane or sugar beets into sugar. It is brown in color and has a sweet flavor due to the high sucrose, glucose and fructose content. Therefore it is often used for prepacked meals and animal feed. Molasses is created when the juice from sugar canes is heated to boiling point. Sugars are extracted over two times as seen in Figure 21. Leaving molasses as a byproduct of this process. We think that it will be a quite useful substrate for fermentation.  
 
               </p>
 
               </p>
  
 
               <figure class="figure">
 
               <figure class="figure">
 
                 <img id="molasses" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/9/96/T--DTU-Denmark--Molasses_production.png" alt="DESCRIPTION">
 
                 <img id="molasses" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/9/96/T--DTU-Denmark--Molasses_production.png" alt="DESCRIPTION">
                 <figcaption class="figure-caption">Flow chart for production of refined sugar and molasses.</figcaption>
+
                 <figcaption class="figure-caption"><b>Figure 21:</b> Flow chart for production of refined sugar and molasses.</figcaption>
 
               </figure>
 
               </figure>
  
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               <p>
 
               <p>
                 As seen in the figure both <i> Y. Lipolytica </i> and <i> S. cerevisiae </i> grow on molasses. Normally <i> Y. Lipolytica </i> 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)  
+
                 As seen in the figure both <i> Y. Lipolytica </i> and <i> S. cerevisiae </i> grows on molasses. Normally <i> Y. Lipolytica </i> does not grow well on sucrose, but there is also a high content of glucose and fructose in molasses. On top of that we realised that sucrose degrades to fructose and glucose when autoclaved (Personal communication) that made fermentation more attractive.
 
               </p>
 
               </p>
  
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                 <figure class="figure">
 
                 <figure class="figure">
 
                   <img id="molY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/d/d3/T--DTU-Denmark--Molasse_%28Dansukker%29_y.lip.png" alt="DESCRIPTION">
 
                   <img id="molY" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/d/d3/T--DTU-Denmark--Molasse_%28Dansukker%29_y.lip.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>Y. Lipolytica</i> growth on molasses from sugar production</figcaption>
+
                   <figcaption class="figure-caption"><b>Figure 22:</b> <i>Y. Lipolytica</i> growth on molasses from sugar production.</figcaption>
 
                 </figure>
 
                 </figure>
  
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                 <figure class="figure">
 
                 <figure class="figure">
 
                   <img id="molS" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/e/ef/T--DTU-Denmark--Molasse_%28Dansukker%29_S.cer.png" alt="DESCRIPTION">
 
                   <img id="molS" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/e/ef/T--DTU-Denmark--Molasse_%28Dansukker%29_S.cer.png" alt="DESCRIPTION">
                   <figcaption class="figure-caption"><i>S. cerevisiae</i> growth on molasses from sugar production</figcaption>
+
                   <figcaption class="figure-caption"><b>Figure 23:</b> <i>S. cerevisiae</i> growth on molasses from sugar production.</figcaption>
 
                 </figure>
 
                 </figure>
  
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               </div>
 
               </div>
  
               <p> Bowth organism might be suitable for growt on molasses, but because <i>S. cerevisiae</i> is able to utilize sucrose it might be the better choice for this biproduct.</p>
+
               <p> Both organisms might be suitable for growth on molasses as seen in figures 22-23, but because <i>S. cerevisiae</i> is able to utilize sucrose it might be the better choice for this byproduct.</p>
  
 
             </div> <!-- molasses end -->
 
             </div> <!-- molasses end -->
  
 +
 +
            <div><a class="anchor" id="section-5"></a>
 +
              <h2 class="h2">Discussion</h2>
 +
              <p>
 +
                From our experiments it is clear that <i>S. cerevisiae</i> and <i>Y. lipolytica</i> have very different substrate utilization ranges. <i>S. cerevisiae</i> is better at catabolizing simple sugars while <i>Y. lipolytica</i> is better at degrading lipids, its derivatives and other complex substrates. A lot of the sugar-based byproducts like molasses, are suitable for human consumption. A biobased production approach with these substrates competes with the increasing food demand. The lipid-based wastestreams like glycerol and oil sediments are not suitable for neither human nor animal consumption. As <i>Y. lipolytica</i> displays good growth on these wastestreams, it is well-suited for biobased production systems. However, it should be noted that these wastestreams cannot be utilized for all possible compounds of interest. More specifically, a lot of problems need to be overcome in order to produce compounds such as pharmaceuticals that have to adhere to specific regulations. Based on our interviews with industry representatives, bulk compounds might be a more viable option. Still, new challenges arise when switching to productions based on oily carbon sources. Amongst others the cleaning procedure for the fermentation tanks must be adapted. This was pointed out at our meeting with <a href = “https://2016.igem.org/Team:DTU-Denmark/HP/Gold#section-3”>Novozymes</a> and can be read in the <a href =”https://static.igem.org/mediawiki/2016/a/a6/T--DTU-Denmark--InterviewwithGernotNovozymes.pdf”>resume</a>.             
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               <li><a href="#section-1">Introduction</a></li>
 
               <li><a href="#section-1">Introduction</a></li>
 
               <li><a href="#section-2">Methods</a></li>
 
               <li><a href="#section-2">Methods</a></li>
               <li><a href="#section-3">Outline of proces</a></li>
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               <li><a href="#section-3">Outline of process</a></li>
               <li><a href="#section-4">Canola oil sediment</a></li>
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               <li><a href="#section-4">Industrial Byproduct Screenings</a></li>
               <li><a href="#section-5">Glycerol byproduct</a></li>
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               <li><a href="#section-5">Discussion</a></li>
              <li><a href="#section-6">Molasses</a></li>
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              <li><a href="#references">References</a></li>
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Substrate

As the substrate utilization of Yarrowia lipolytica is central to our project we have performed 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 our research by acquiring real waste streams and byproducts form organic industrial productions in the Nordic countries and screened Y. lipolytica growth for these substrates.


Introduction

"Growth can be the result of many trials"

Mohamad El Lakany, Mohamad's Mantra

The dimorphic, non-conventional yeast Yarrowia lipolytica, belonging to the Ascomycota phylum, was first isolated in the 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.

DESCRIPTION
Figure 1: Y.lipolytica in plactonic growth with 100x magnification.

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. This potential is a result of several advantages that Y. Lipolytica has over the conventional yeast S. cerevisiae. Y. Lipolytica prefers secreting proteins through the co-transcription pathway and does so very efficiently2 in addition, it does not exhibit hyperglycosylation as S. cerevisiae does3. 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. 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 source 4. This has naturally led to Y. Lipolytica becoming a model organism for several metabolic pathways, especially fatty acid transport, -metabolism, and single cell oil (SCO) accumulation. Y. Lipolytica has even been shown to exhibit enhanced growth on mixed substrates. Yarrowia lipolytica is an oleaginous cell factory platform for production of fatty acid-based biofuels and bioproducts. This renders it ideal for utilization of industrial waste streams due to their complex and variable content. These findings have us believe that we had found an excellent candidate chassis for our project. The table below 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 setup:

Minimal medium is produced as directed by Mhairi Workman5 using 20g/L of a given carbon source for all the 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) measured by Spectrophotometry (Shimadzu UV-1800). The cultivations were carried out in a cytomat (Thermo Scientific) shaking 900 rpm at 30 degree celsius. Cultures were grown, shaked and measured in a 48 well suspension culture plates (Cellstar, Greiner-bio-one). The measurements were carried out using a Hamilton Microlab Robot, (Hamilton Life science Robotics) connected to a plate spectrophotometer (BioTek Synergy 2).OD600 measurements were taken every 2 hours until the cultures reached stationary phase. Data was then analysed and visualized using excel and R-studio Figure 2.

DESCRIPTION
Figure 2: A. Overnight culture: strains of Y. lipolytica and S. cerevisiae are grown in YPD media overnight at 30℃ (86℉) to ensure balanced growth and comparable data. B. Washing- and inoculation steps: Cells are spinned down and washed to ensure removal of carbon-sources and other metabolites from the overnight-culturing. Washing and spinning step is repeated. Simple and complex substrates are inoculated with cells in 48 well suspension culture plates. The cells reaches final OD600 0.001 C. Growth-experiment: Plates are incubated and shaken at 900 rpm in a cytometer and before measurement of OD in a spectrophotometer. Data are recorded and compiled in an excel sheet with two hours intervals. This process is assisted by using the Hamilton Microlab robot. D. Data analysis and -visualization step: The data excel sheet (in step C.) are analyzed and visualized by plots using R-studio.

During the growth experiments we kept to strains that were wild type or closely related. 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 Process

DESCRIPTION
Figure 3: Picture of the waste products we received.
DESCRIPTION
Figure 4: Picture of the autoclaved C-source solutions.

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 emerged indicating 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 graphs representing these results can be seen in the figures 5-9:

DESCRIPTION
Figure 5: Y. Lipolytica growth on fructose.
DESCRIPTION
Figure 6: Y. Lipolytica does not grow on sucrose.
DESCRIPTION
Figure 7: Y. Lipolytica growth on Canola oil.
DESCRIPTION
Figure 8: Y. Lipolytica growth on glucose.
DESCRIPTION
Figure 9: S. cerevisiae growth on glucose.


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 or 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

Industrial Byproduct Screenings

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 before extracting the sediment. Besides the fibres from the plants and residual oil, the sediment contains polyaromatic hydrocarbons in 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 incineration. Figure 10 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).

DESCRIPTION
Figure 10: Flow chart for the production of cold pressed canola oil.

During the experiments using this substrate we experienced a lot of problems with the OD measurements because of the high content of plant fibers. Through pressure filtering, a transparent sample was extracted. We demonstrated that Y. Lipolytica grows very well on this waste stream. S. cerevisiae on the other hand is not able to utilize this carbon source as seen in figures 11-12.

DESCRIPTION
Figure 11: Y. Lipolytica growth on sediment from canola oil production.
DESCRIPTION
Figure 12: S. cerevisiae does not grow on sediment from canola oil production.

This shows promising growth of Y. Lipolytica while it is clear that S. cerevisiae is unsuitable for fermentation based on canola oil sediments

Glycerol Byproduct

The push to find an alternative to fossil fuel has increased demand and production of biodiesel tremendously in the last two decades. Biodiesel is produced by a base-catalyzed transesterification by a short chained alcohol and triacylglycerols derived from natural sources as seen in figure 13. This reaction produces 0.102 kg glycerol pr. liter biodiesel9. The increased production has resulted in a plummeting of glycerol prices making it a promising substrate for industrial fermentation.

DESCRIPTION
Figure 13: Flow chart for production of biodiesel and glycerol waste.

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 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 and raising the pH to 6 we were able to make Y. Lipolytica grow fairly well in spite of the relatively high salt levels as seen in Figure 14 (Personal communication).

DESCRIPTION
Figure 14: Y. Lipolytica growth on glycerol from second generation biodiesel.
DESCRIPTION
Figure 15: S. cerevisiae does not grow on glycerol.

First Generation Glycerol (Perstorp and Emmelev)

Perstop has two biodiesel production facilities, one located in Sweden and one in Norway. They produce high quality glycerin, that is sold as a component for chemical production. This has a purity of 95-100% the remainder of mostly water (Personal communication). This byproduct is also a great substrate for Y. Lipolytica as seen in Figure 16.

DESCRIPTION
Figure 16: Y. Lipolytica growth on glycerol from first generation biodiesel.
DESCRIPTION
Figure 17: S. cerevisiae does not grow on glycerol.

Emmelev A/S is a local oil mill, first generation biodiesel plant and a 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 components that inhibit the growth of Y. Lipolytica as seen in Figure 19.

DESCRIPTION
Figure 19: Y. Lipolytica growth on glycerol from first generation biodiesel.
DESCRIPTION
Figure 20: S. cerevisiae does not grow on glycerol.

In conclusion, Y. Lipolytica is the only suitable candidate for fermentation based on glycerol.

Molasses

The process of creating refined sugar results in the waste product molasses. Molasses is a byproduct of the refining of sugarcane or sugar beets into sugar. It is brown in color and has a sweet flavor due to the high sucrose, glucose and fructose content. Therefore it is often used for prepacked meals and animal feed. Molasses is created when the juice from sugar canes is heated to boiling point. Sugars are extracted over two times as seen in Figure 21. Leaving molasses as a byproduct of this process. We think that it will be a quite useful substrate for fermentation.

DESCRIPTION
Figure 21: Flow chart for production of refined sugar and molasses.

As seen in the figure both Y. Lipolytica and S. cerevisiae grows on molasses. Normally Y. Lipolytica does not grow well on sucrose, but there is also a high content of glucose and fructose in molasses. On top of that we realised that sucrose degrades to fructose and glucose when autoclaved (Personal communication) that made fermentation more attractive.

DESCRIPTION
Figure 22: Y. Lipolytica growth on molasses from sugar production.
DESCRIPTION
Figure 23: S. cerevisiae growth on molasses from sugar production.

Both organisms might be suitable for growth on molasses as seen in figures 22-23, but because S. cerevisiae is able to utilize sucrose it might be the better choice for this byproduct.

Discussion

From our experiments it is clear that S. cerevisiae and Y. lipolytica have very different substrate utilization ranges. S. cerevisiae is better at catabolizing simple sugars while Y. lipolytica is better at degrading lipids, its derivatives and other complex substrates. A lot of the sugar-based byproducts like molasses, are suitable for human consumption. A biobased production approach with these substrates competes with the increasing food demand. The lipid-based wastestreams like glycerol and oil sediments are not suitable for neither human nor animal consumption. As Y. lipolytica displays good growth on these wastestreams, it is well-suited for biobased production systems. However, it should be noted that these wastestreams cannot be utilized for all possible compounds of interest. More specifically, a lot of problems need to be overcome in order to produce compounds such as pharmaceuticals that have to adhere to specific regulations. Based on our interviews with industry representatives, bulk compounds might be a more viable option. Still, new challenges arise when switching to productions based on oily carbon sources. Amongst others the cleaning procedure for the fermentation tanks must be adapted. This was pointed out at our meeting with Novozymes and can be read in the resume.

References

  1. 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
  2. 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.
  3. 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
  4. Barth, G. (2013). Yarrowia lipolytica Genetics, Genomics, and Physiology. http://www.springer.com/us/book/9783642383199
  5. Mhairi Workman, Philippe Holt (2013). Comparing cellular performance of Yarrowia lipolytica during growth on glucose and glycerol in submerged cultivations
  6. H. Shafaghat, G.D. Najafpour. Growth Kinetics and Ethanol Productivity of Saccharomyces cerevisiae PTCC 24860 on Varius Carbon Sources. ISSN 1818-4952
  7. 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
  8. 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
  9. 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.

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