Akeem Obe

Abstract
Oleaginous yeasts are known to accumulate lipids and that these lipids can be converted to biodiesel and other value-added renewable products using existing chemical reactions. The oleaginous yeasts species include Lipomyces starkeyi, Cryptococcus curvata, Rhodotorula glutinis, Rhodosporidium toruloides, Trichoosporon fermentas, and Yarrowia lipolytica. These species are known to accumulate lipids from 2572% of their dry weights. There are other microorganisms (microalgae, bacteria, molds) that are known to also accumulate lipids but oleaginous yeasts are advantageous because; their duplication times (time required for the yeast to double in size) are low, they are less affected by climate conditions, and their cultures are more easily scaled up from a laboratory setting into an industrial setting. In this research, Lipomyces starkeyi was grown using different mixtures of sugar (glucose and xylose) as carbon sources and urea as nitrogen source. Also the Carbon to Nitrogen ratio (C: N) was varied from 75 to 125 to test the effect of these conditions on cellular growth and sugar utilization. After data analysis, xylose samples had the highest cellular growth at 0.94 g/L but in analyzing the samples individually, sample 15 with a carbon source of glucose and a C: N ratio of 125 had the highest cellular growth at 1.58 g/L

Keywords: Lipomyces starkeyi. C: N. Glucose. Xylose. Value-added renewable

Introduction/Background
The increasing populations of the world and the need to obtain clean and efficient energy sources have made the search of alternative energy sources inevitable. Researchers and scientists have worked on the use of edible crop based resources including ethanol from corn and biodiesel from soybeans to compete with petroleum products obtained from non-renewable energy sources. The problem with the use of edible crop based resources is their competition with food sources used for human beings. Biomass is the only renewable resource that has the potential to compete with petroleum; over 1 billion tons of biomass is available each year in the US which can be converted into biofuels, bioproducts, and biopower (Robert et al.2005). In addition to reducing our foreign dependence on petroleum, utilizing renewable biomass feedstocks may provide new economic opportunities throughout the US. With considerable efforts put towards finding an alternative source of energy that would be cheap and efficient, scientists have identified micro-organisms with the ability to produce triacylglycerides and these can be extracted and converted to biodiesel and other value-added renewable products. The micro-organisms that are able to do that are referred to as “oleaginous micro-organisms” and they include; microalgae, fungi (yeasts and molds), and bacteria. Some oily yeasts like Cryptococcus curvata, Lipomyces starkeyi, Rhodotorula glutinis, and Trichosporon fermentans are known to accumulate lipids to levels greater than 20% of their cellular dry weights. Basically, lipids are accumulated in oleaginous yeasts as discrete fat globular deposits and can also be associated (less than 5%) with different cell organelles (Ageitos et al.2011). Yeasts can generate lipids from different carbon sources, even from lipids present in the culture media. They can in fact vary their lipid composition by replacing the fatty acids in the triglycerides with those present in the culture medium (Iassonova et al. 2008). Glucose is a cheap carbon source that has been used in growing Lipomyces starkeyi. Different carbon sources like sucrose, xylose, and cellulose hydrolysate have been tested with varying results. Objective: The objective of this research was to test various combinations of glucose and xylose at different C: N ratios to determine the cellular biomass growth and sugar utilization of the oleaginous yeast Lipomyces starkeyi.

Materials & Method
Organism & Cultural conditions. The organism used in this work was Lipomyces starkeyi ATCC 56304. A stock culture was prepared on YM agar ( yeast extract 3g/L, malt extract 3g/L, peptone 5g/L, glucose 10g/L, and agar 20g/L) using a petri dish and was stored in the refrigerator. Inoculum Preparation. A nutrient dense media was prepared using a yeast mold broth (glucose 10g/L, yeast extract 3g/L, malt extract 3g/L, and peptone, 5g/L) which was added to a 50mL flask of deionized water (0.5 g/L). The pH of the media was adjusted to 5 by adding 3M concentrated hydrochloric acid to the media. The resulting solution was autoclaved at 121°C for 15min. A loopful of Lipomyces starkeyi was then added to the flask, after which it was incubated in a refrigerator incubator shaker at 25°C and 200 RPM for 3 days. Growth Flask Preparation. 21 separate 1L flasks with nutrient media were prepared using Table 1. Glucose, xylose, and yeast extract (0.5 g/L) were used. The total sugar concentration used in each flask was 55g/L. The pH of the resultant solutions was adjusted to 5 using hydrochloric acid and these were autoclaved at 121°C for 15minutes. A 10% (v/v) inoculum with a concentration of 2.9E7 cells/mL was used to inoculate each flask for a total working volume of 50mL. 5mL sub-samples were taken after inoculation to measure initial biomass dry weight. The flasks were then placed in an incubator to grow at 25°C/ 200 RPM for 120 hours. After 120 hours, the flasks were harvested. # C/N Carbon source Urea (g/l) 1 75.0 Glucose 0.665 2 87.5 Glucose 0.577 3 100.0 Glucose 0.479 4 112.5 Glucose 0.424 5 125.0 Glucose 0.368 6 75.0 Xylose 0.665 7 87.5 Xylose 0.577 8 100.0 Xylose 0.479 9 112.5 Xylose 0.424 10 125.0 Xylose 0.368 11 75.0 Glucose + Xylose 0.665 12 87.5 Glucose + Xylose 0.577

13 14 15 16 17 18 19 20 21

100.0 112.5 125.0 75.0 125.0 125.0 87.5 75.0 75.0

Glucose + Xylose Glucose + Xylose Glucose + Xylose Glucose Glucose+ Xylose Glucose Xylose Xylose Glucose + Xylose

0.479 0.424 0.368 0.665 0.368 0.368 0.577 0.665 0.665

Table1: Data for Growth Flask Preparation Urea concentration Determination: The concentrations of urea to be used for the different sugar concentrations were determined from their respective C: N ratios. An example of how this was done for glucose at C: N of 75 is show below;

( (

) )

( )

( )

( (

) )

( )

( ( Solving for Y

) )

Biomass Dry Weigh Determination: The initial and final samples were centrifuged using a setting of t = 15 minutes at a speed of 10. After centrifuging, 1ml of supernatant in each flask was removed and placed into eppendorf tubes. Both the initial and final samples were washed with 5ml of 1 % potassium chloride and vortexed until yeast pellet was suspended in each flask after which they were centrifuged using the same settings as previously stated. After this, the supernatant was removed from each flask and a 2ml of deionized water was added and vortexed once again. The remaining solutions in the flasks were then poured out into tins which were dried overnight at 80°C in an oven. The final weights of the tins were recorded and the differences between the initial and final samples were used to determine the dry weight accumulation. The yeast growth was reported as a concentration (g/L). Sugar Concentration Determination: Supernatant from eppendorf tubes were diluted 10fold and analyzed for glucose, xylose as major sugars using a Phenomenex RPM monosaccharide column (300 mm 7.8mm; Phenomenex, CA) in a Shimadzu CBM-20A HPLC system connected to a Shimadzu RID-10A refractive index detector. Experimental Design: Performed a one factor response surface methodology design testing two factors. The numerical factor was the carbon to nitrogen ratio which varied from 75 to 125 (+1, -1) having a mean of 100 (0) and two non-central points of 87.5 and 112.5 (+0.5, -0.5). The categorical factor was the ratio of glucose: xylose which varied from 100:0, 75:25, and 0:100, respectively. The design then created 21 treatments where each sugar ratio had 7 treatments that included replicates (n=2) for each your +1,-1 and then 0, +0.5, -0.5 were performed without replication.

Table 2: Experimental Design Layout Run C:N Sugars Ratio 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 75.0 112.5 100.0 75.0 100.0 87.5 125.0 125.0 112.5 100.0 87.5 125.0 75.0 125.0 125.0 75.0 112.5 125.0 75.0 75.0 87.5 Xylose Xylose/Glucose Xylose/Glucose Glucose Xylose Glucose Xylose/Glucose Glucose Xylose Glucose Xylose/Glucose Xylose Xylose Xylose/Glucose Glucose Xylose/Glucose Glucose Xylose Glucose Xylose/Glucose Xylose

Results
The effects of the carbon source (glucose, xylose, and glucose + xylose) and the carbon to nitrogen ratios were to be examined in this experiment. Effect of carbon sources on Lipomyces starkeyi growth: In this experiment 3 different carbon sources were used (glucose, xylose, and a 75:25 mixture of glucose & xylose). From the bar graph presented below, it can be seen that overall xylose samples had the best cellular growth of 0.94 g/L when compared with glucose, and glucose + xylose samples. Error bars were also created to determine the extent at which samples with the same treatment differed. It can be denoted also from the graph that xylose samples differed less when compared to glucose and glucose + xylose mixtures. When the 21 samples are analyzed individually, sample 15 (carbon source = glucose; C: N = 125) had the highest cellular growth of 1.58 g/L as shown in graph 2

1.4 Cellular Growth (g/L)

1.2
1.0 0.8 0.6 0.4 0.2 0.0 glucose xylose glu/xyl
Cellular Growth

B

A

B

Figure 1: Cellular growth of Lipomyces starkeyi using different carbon sources

1.5 Cellular growth (g/L) 1.0 0.5 0.0 1 3 5 7 9 11 13 15 17 19 21 Samples

Figure 2: Cellular growth of Lipomyces starkeyi for each treatment sample (n=21). Effect of carbon sources & C: N Ratio on cellular growth: Using the graph below that shows the interaction between carbon sources and the C: N ratio, it can be noted that that at a C: N ratio of 75, xylose had the highest cellular growth at 1.18 g/L while the glucose:xylose mixture had the least at 0.125 g/L. In xylose samples, it can be noted that as the C: N ratios increased from 75 to 125 that cellular growth drastically reduced (Figure 3). This may suggest that as the C: an N ratios increase, the nitrogen which is used for growth by Lipomyces starkeyi is quickly depleted resulting in decreased cellular growth. The trend is different for glucose and glucose + xylose samples. Shown in figure 3, as the C: N ratio increases from 75 to 125, the cellular growth gradually increases to an optimum of 0.68 g/L.

Figure 3: Interaction between Cell growth (g/L), Carbon sources, & C: N Ratio

Conclusion
In this experiment, xylose samples had the highest cellular growth at 0.94 g/L but in analyzing the samples individually, sample 15 with a carbon source of glucose and a C: N ratio of 125 had the highest cellular growth at 1.58 g/L. To further my research, lower C: N ratios with high nitrogen content will be used in order to ensure an increased

growth in Lipomyces starkeyi. Also, other sugar sources will be tested in order to determine their utilization rates in Lipomyces starkeyi.

References
  Ageitos, J. et al. (2011) Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol 90:1219-1227 Iassonova DR, Hammond EG, Beattie SE (2008) Oxidative stability of polyunsaturated triacylglycerols encapsulated in oleaginous yeast. J Am Oil Chem Soc 85:711-716  Robert, D. et al. (2005) Biomass as a Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion-Ton Annual Supply

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