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Introduction

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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 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.

DESCRIPTION — This figure shows *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 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

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Section 7