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.

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.

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

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:

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).

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.

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.

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).

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.

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.

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.

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.

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

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