Abstract: fuel for competitiveness in engine usage. Keywords: Sugars,

 Abstract: Alternative energy is the need of the hour in this warming world. Besides wind, hydroelectric energy very few exists for liquid fuels that are needed for running transportation vehicles. Bio-diesel is considered to be an alternative liquid fuel for such purposes. In this study, three oleaginous yeasts including Lipomyces starkeyi, Yarrowia lipolytica, and Metschnikowia pulcherrima were compared for their lipid productivity in different monosaccharides such as glucose, xylose, fructose and a mixture of glucose and xylose, xylose and fructose and glucose and fructose. Biomass is obtained from the cultured samples and the lipid was extracted for the three oleaginous yeasts which are further trans-esterified to produce bio-diesel. According to the experimental study, Lipomyces starkeyi had the highest lipid yield among the three yeast in both as individual media and in combined media. It was able to consume glucose and fructose as a carbon source for lipid accumulation with maximum yield. In this productivity of lipid has reached its highest yield during the stationary phase and finally attained the death phase due to the remobilization of lipids. Finally, bio-diesel is to be checked with conventional fuel for competitiveness in engine usage.

 

Keywords: Sugars, Lipomyces starkeyi, Yarrowia lipolytica, Metschnikowia pulcherrima

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I.                  Introduction

Biomass-based biofuel production represents a critical approach to face increased energy prices and probable reduction of crude oils reservoirs, for reducing greenhouse gas emissions, and to improve an ecological economy.  Microbial lipids are the one which is represented as valued feedstock for production of biodiesel, and also a potential elucidation for a bio-based economy3. In order to overcome increasing demand for biodiesel production, other oil sources have been discovered. Currently, processes have been developed to produce single cell oil (SCO) by using heterotrophic oleaginous microorganisms which have triggered substantial attention. The accumulation of lipids by these organisms mainly consisting of triacylglycerols (TAG), which form the storage fraction of the cell3.Microorganisms are generally considered to be oleaginous only when they undergo lipid accumulation of more than 20% of their cell dry weight2. Many oleaginous microorganisms can accumulate oils, especially TAGs, which are the main materials for biodiesel production. In general, future production of biodiesel is highly based on the microbial oils which are considered as one of the major potential feedstocks. The microbial oils cost must be reduced by exploring other carbon sources instead of glucose which is very important especially for the oils applied to biodiesel production.

Oleaginous yeasts accumulate 20-70% of their cell mass as intracellular lipids which are generally considered to be an alternative energy for the production of second generation fuels that has the capacity to produce biodiesel. When compared to filamentous fungi and algae, a yeast commonly has rapid growth rate and grow to higher cell density lipids produced by yeasts. Most of the bacteria produce only complex lipids, and a very few bacteria can produce oils which can be used as the feedstock for biodiesel production. Another reason for not using bacteria in the lipid production they have a very low yield of biomass productivity compared to certain other oleaginous microbes. Some yeasts strains, such as Rhodosporidium sp., Rhodotorula sp., lipomyces sp., Yarowia lipolytica, and Lipomyces starkeyi can accumulate intracellular lipids as high as 70% of their biomass dry weight10.

II. Materials and methods

A. Pre-culture

Yeast Malt medium was used as pre-culture to grow yeast cells. It contains glucose as carbon source 10 g/l, yeast extract 3 g/l, malt extract 3 g/l and peptone 5 g/l. To obtain solid medium, 16 g/l agar was added4. High-temperature moist heat sterilization method (Autoclave) was used for medium sterilization, 121 ºC temperature for 20 minutes at 15 lbs pressure.

B. Main culture

The semi-synthetic medium was prepared to grow oleaginous yeasts. Glucose, xylose, fructose and a mixture of glucose and fructose, glucose and xylose,  xylose and fructose were used as carbon sources with concentration 10 g/100ml of water.  The pH was maintained for the culture for various organisms to grow at a different temperature.

C. Yeasts used in the study

Lipomyces starkeyi, Yarrowia lipolytica, and Metchnikowia pulcherima were obtained from Microbial Type Culture Collection(MTCC), Chandigarh in a freeze-dried form which has pH value as 8, 6, 4 (it is approximately maintained at the pH of 7.56, 5.87, 3.89). Culture medium and the conditions in which the organisms are to be grown are mentioned in the manual given and culturing were as per the given specification.

D. Inoculation of yeast

For the purpose of preventing the contamination of substrate due to the microorganisms present as an external source, autoclaving is done before the culture was added to the substrate. Sample sterilization and growth medium will eliminate the other organisms growth that is tend to be harmful to the oleaginous yeast taken for culturing such as Lipomyces starkeyi, Yarrowia lipolytica, and Metchnikowia pulcherrima. The yeast strain was inoculated in the laminar airflow chamber to prevent the entry of other microorganisms and it was grown under an aerobic condition at 25°C in an orbital shaker at 150 rpm.

E. Culturing in orbital shaker

 Oleaginous yeasts used for the study were cultured in six different carbon source conditions. The semi-synthetic medium was prepared in 250ml flasks. After inoculating the cultures in the laminar flow chamber they are kept in the orbital shaker at 120 rpm and 28°C and it is rotated till it attains the death phase. Meanwhile, the OD value of the cultures was taken for every 24 hours in UV spectrophotometer to determine the growth of cultures in six carbon sources. After attaining the maximum OD value the cultures are taken out and centrifuged to obtain the biomass to extract the lipids.

F. Growth curve determination

Three oleaginous yeasts were cultured in Yeast malt medium and optical density (OD) was measured for every 24 hours in a Double reflection UV visible spectrophotometer (Perkin Elmer) at a wavelength of 590 nm. The standard solution was prepared from various sugars and it is used as a blank solution for the initial measurement of OD.For obtaining accurate optical density OD values between 0.1 to 1.The dilution was done several times for the cells to be suspended in the medium solution.

 

G. Cell density and cell dry weight analyses

The growth curve was analyzed for both control medium (Malt yeast liquid medium) and carbon source. Optical density value was noted for every 24 hrs and readings were taken for 8 days (24-182 hours) using UV- Visible spectrophotometer and growth curve was plotted based on the values obtained. Dried biomass of Lipomyces starkeyi, Yarrowia lipolytica and Metchnikowia pulcherrima grown in both synthetic medium and carbon source was obtained by filtering using whattman filter paper. The empty weight (W1) of filter paper was taken. After filtration, the paper was dried in a hot air oven at 100°C and then the weight (W2) of filter paper was taken. The weight difference (W2 – W1) gives the value of biomass dry weight.

H. Lipid extraction from biomass

THE FOLCH METHOD

The Folch method of lipid extraction involved the use of chloroform-methanol (2:1 by volume). The chloroform-methanol ratio was mixed thoroughly and the centrifuge was done at 9000  rpm for 5 mins at 25 °C.  The result of centrifuge mixture was formed in two separate layers in which chloroform is found in upper phase and methanol was obtained in lower phase. This method is generally regarded as one of the oldest wits in lipid extraction, which formed the basis for development of future extraction procedures with improvements (Folch et al., 1957)

BLIGH AND DYER METHOD

100 mg (lyophilized biomass) was blended with 3.75mL solvent mixture (Chloroform: Methanol: Distilled water = 1: 2: 0.75). Mixture homogenized with a magnetic stirrer at 300 rpm for 1 Hr. 1mL Chloroform + 0.5 mL distilled water was added to a mixed solvent to form two layers. After 10 min centrifugation at 1500 x g, the upper layer containing the mixture of methanol and distilled water was gently removed by Pasteur pipette. The lower layer containing chloroform was collected and then filtered with a fiber filter of 0.45µm. The sediment was re-extracted as the former steps and the residue was light colored. After evaporating, the filtered solvent by a Rota-evaporator, the lipids were obtained gravimetrically.

II. Materials and methods

A. Pre-culture

Yeast Malt medium was used as pre-culture to grow yeast cells. It contains glucose as carbon source 10 g/l, yeast extract 3 g/l, malt extract 3 g/l and peptone 5 g/l. To obtain solid medium, 16 g/l agar was added4. High-temperature moist heat sterilization method (Autoclave) was used for medium sterilization, 121 ºC temperature for 20 minutes at 15 lbs pressure.

B. Main culture

The semi-synthetic medium was prepared to grow oleaginous yeasts. Glucose, xylose, fructose and a mixture of glucose and fructose, glucose and xylose,  xylose and fructose were used as carbon sources with concentration 10 g/100ml of water.  The pH was maintained for the culture for various organisms to grow at a different temperature.

C. Yeasts used in the study

Lipomyces starkeyi, Yarrowia lipolytica, and Metchnikowia pulcherima were obtained from Microbial Type Culture Collection(MTCC), Chandigarh in a freeze-dried form which has pH value as 8, 6, 4 (it is approximately maintained at the pH of 7.56, 5.87, 3.89). Culture medium and the conditions in which the organisms are to be grown are mentioned in the manual given and culturing were as per the given specification.

D. Inoculation of yeast

For the purpose of preventing the contamination of substrate due to the microorganisms present as an external source, autoclaving is done before the culture was added to the substrate. Sample sterilization and growth medium will eliminate the other organisms growth that is tend to be harmful to the oleaginous yeast taken for culturing such as Lipomyces starkeyi, Yarrowia lipolytica, and Metchnikowia pulcherrima. The yeast strain was inoculated in the laminar airflow chamber to prevent the entry of other microorganisms and it was grown under an aerobic condition at 25°C in an orbital shaker at 150 rpm.

E. Culturing in orbital shaker

 Oleaginous yeasts used for the study were cultured in six different carbon source conditions. The semi-synthetic medium was prepared in 250ml flasks. After inoculating the cultures in the laminar flow chamber they are kept in the orbital shaker at 120 rpm and 28°C and it is rotated till it attains the death phase. Meanwhile, the OD value of the cultures was taken for every 24 hours in UV spectrophotometer to determine the growth of cultures in six carbon sources. After attaining the maximum OD value the cultures are taken out and centrifuged to obtain the biomass to extract the lipids.

F. Growth curve determination

Three oleaginous yeasts were cultured in Yeast malt medium and optical density (OD) was measured for every 24 hours in a Double reflection UV visible spectrophotometer (Perkin Elmer) at a wavelength of 590 nm. The standard solution was prepared from various sugars and it is used as a blank solution for the initial measurement of OD.For obtaining accurate optical density OD values between 0.1 to 1.The dilution was done several times for the cells to be suspended in the medium solution.

 

G. Cell density and cell dry weight analyses

The growth curve was analyzed for both control medium (Malt yeast liquid medium) and carbon source. Optical density value was noted for every 24 hrs and readings were taken for 8 days (24-182 hours) using UV- Visible spectrophotometer and growth curve was plotted based on the values obtained. Dried biomass of Lipomyces starkeyi, Yarrowia lipolytica and Metchnikowia pulcherrima grown in both synthetic medium and carbon source was obtained by filtering using whattman filter paper. The empty weight (W1) of filter paper was taken. After filtration, the paper was dried in a hot air oven at 100°C and then the weight (W2) of filter paper was taken. The weight difference (W2 – W1) gives the value of biomass dry weight.

H. Lipid extraction from biomass

THE FOLCH METHOD

The Folch method of lipid extraction involved the use of chloroform-methanol (2:1 by volume). The chloroform-methanol ratio was mixed thoroughly and the centrifuge was done at 9000  rpm for 5 mins at 25 °C.  The result of centrifuge mixture was formed in two separate layers in which chloroform is found in upper phase and methanol was obtained in lower phase. This method is generally regarded as one of the oldest wits in lipid extraction, which formed the basis for development of future extraction procedures with improvements (Folch et al., 1957)

BLIGH AND DYER METHOD

100 mg (lyophilized biomass) was blended with 3.75mL solvent mixture (Chloroform: Methanol: Distilled water = 1: 2: 0.75). Mixture homogenized with a magnetic stirrer at 300 rpm for 1 Hr. 1mL Chloroform + 0.5 mL distilled water was added to a mixed solvent to form two layers. After 10 min centrifugation at 1500 x g, the upper layer containing the mixture of methanol and distilled water was gently removed by Pasteur pipette. The lower layer containing chloroform was collected and then filtered with a fiber filter of 0.45µm. The sediment was re-extracted as the former steps and the residue was light colored. After evaporating, the filtered solvent by a Rota-evaporator, the lipids were obtained gravimetrically.

III. Results and Discussion

A.Growth curve analysis:

Growth curve analysis is done for all three yeast of six different samples. The OD values were noted down for every 24 hours. The organisms tend to maintain the stationary phase at 144,168 and 182nd hours and it attains the death after a particular time. In the graph shown below, the increase in OD reading represents the growth of new cells. The graph shows distinctive lag phase, exponential or growth phase, and stationary phase. The stationary phase is desired for lipid production as there are no new cells produced while, all the consumption by the microorganism is converted and stored as lipid in the existing cells. On account of this, the species, lipomyces starkeyi, is found to have reached the stationary phase earlier than the other two species, (OD reading of 0.95 after 8 days of inoculation).

 

Lipomyces starkeyi

The results of the L.starkeyi growth are shown in Figure 2. From the figure, it can be seen that the there are two maximum OD values are obtained. In fructose alone media and in the combined media of glucose and fructose gave maximum values of 0.95 and 0.9 respectively.

 Figure.1 Growth rate Vs OD reading at 590 nm tracing growth curve of different monosaccharides of L.starkeyi  for a period of 182 days

 

Yarrowia lipolytica

The results of the Yarrowia lipolytica growth are shown in figure 3. From the figure, it can be seen that the there are two maximum OD values are obtained. In Glucose alone media and in the combined media of glucose and xylose gave maximum values of 0.85 and 0.8 respectively.

Figure 2  Growth rate Vs OD reading at 590 nm tracing growth curve of different monosaccharides of Y.lipolytica  for a period of 182 days

 

 

 

Metschnikowia pulcherrima

The results of the Metschnikowia pulcherrima growth are shown in figure 4. From the figure, it can be seen that the there are two maximum OD values are obtained. In Glucose alone media and in the combined media of glucose and fructose gave maximum values of 0.85  and 0.8 respectively.

 

Figure 3 Growth rate Vs OD reading at 590 nm tracing growth curve of different monosaccharides of M.pulcherrima  for a period of 182 days

B.Biomass extraction

The biomass was extracted from the three oleaginous yeast of six different carbon samples to determine the yeast which can produce a higher amount of lipid.

 Lipomyces starkeyi

Biomass dry weight results for Lipomyces starkeyi are shown in figure 5 and the corresponding graph is shown in figure 4. From the figure, it is interpreted that maximum growth occurred in media supplied with fructose and combined media of glucose and fructose has 18.42 g/l and 22.32 g/l respectively.

 

Figure 4 Biomass obtained for various sugars in Lipomyces starkeyi

 

Yarrowia lipolytica

Biomass dry weight results for Yarrowia lipolytica are shown in figure 6 and the corresponding graph is shown in figure 4.5. From the figure, it is interpreted that maximum growth occurred in media supplied with Glucose and combined media of glucose and Xylose has 12 g/l and 10 g/l respectively.

 

Figure 5 biomass obtained for various sugars in Yarrowia lipolytica

Metschnikowia pulcherrima

Biomass dry weight results for Metschnikowia pulcherrima are shown in figure 7 and the corresponding graph is shown in figure 6. From the figure, it is interpreted that maximum growth occurred in media supplied with Glucose and combined media of glucose and fructose has 10 g/l and 8.2 g/l respectively.

 

Figure 6 Biomass obtained for various sugars in Metschnikowia pulcherrima

C. Cell dry weight and lipid weight comparison

All yeasts were cultured in six different sugar conditions and harvested at the early stationary growth phase according to measured optical density data. Lipomyces starkeyi was able to grow at a maximum rate in each monosaccharide mainly preferred to grow in glucose as individual media and glucose and fructose as combined media among the six combinations which directly influenced the cell biomass. L.Starkeyi shows the higher growth rate and obtained higher biomass when compared with other two yeasts and it also tends to produce a higher amount of lipids among the three oleaginous yeasts.

Lipomyces starkeyi

Lipid yields from L.starkeyi show maximum Lipids were from media with fructose as a sole carbon source and in combined media with glucose and fructose. The values are shown in figure 8 and figure 7 that the maximum yield from sole and combined media reached 75 % and 80 % respectively.

Figure 7 Comparison of biomass dry weight and lipids in Lipomyces starkeyi

Yarrowia lipolytica

Lipid yields from Y. lipolytica shows maximum Lipids were from media with Glucose as a sole carbon source and in combined media, with fructose and Xylose the values are shown in figure 9 and figure 8  that the maximum yield from sole and combined media reached 50 % and 50 % respectively

Figure 8 Comparison of biomass dry weight and lipids in Lipomyces starkeyi

Metschnikowia pulcherrima

Lipid yields from M. pulcherrima shows maximum Lipids were from media with Glucose as sole carbon source and in combined media, with fructose and Xylose the values are shown in figure 10 and figure 9  that the maximum yield from sole and combined media reached 40 % and 38 % respectively

Figure 9 Comparison of biomass dry weight and lipids in Metschnikowia pulcherrima

D. FTIR analysis for lipid confirmation

Fourier Transform Infra-red Spectrophotometer is a qualitative analysis which is used to find the types of fatty acids esters present in the sample obtained from the lipid extraction. All three oleaginous yeasts liberate the unsaturated fatty acid like methyl linoleate by 90 %. Fatty acids have OH and CO groups, which show characteristic peaks at 3367 cm-1 and1408 cm-1 respectively. The unsaturated bonds representing the carbon-carbon double bonds are present in 2993 & 2881 cm-1. Hence they have confirmed as long chain unsaturated fatty acids. Figures 4.8 & 4.9 shows the presence of lipid confirmed matching the peaks of COOH and C=C groups by FITR spectrophotometer.The following are the results of Lipid confirmation of various yeasts.

Figure 10 FTIR spectrum for lipid sample for L.starkeyi

Figure 11 FTIR spectrum for lipid sample for L.Yarowia

Figure 12 FTIR spectrum for lipid sample for M.pulcherrima

      From the Figure 11 indicates the mixture of various fatty acids such as methyl linoleate, Butyl Stearate, Ethyl palmitate, Ethyl myristate and Ethyl linoleate present in the sample found by FTIR.

   

 

 

 

 

 

Figure 11 Various fatty acids found by FTIR library

S.NO

FTIR RESULTS

L. starkeyi

Y. lipolytica

M. pulcherrima

 
1

Methyl linoleate natural

 
90.7%

 
90%

 
89.21%

 
2

Butyl Stearate

 
88.76%

 
88.42%

 
87.0%

 
3

Ethyl Palmitate

 
87.89%

 
87.62%

 
86.0%

 
4

Ethyl Myristate

 
80.84%

 
80.23%

 
78.33%

 
5

Ethyl linoleate

 
69.4%

 
68.41%

 
64.89%

 

Figure 11 represents the fatty acid compounds found using IR library FLUKA. The best match found for the lipids were various methyl and ethyl esters of unsaturated fatty acids which are common components of triglycerides.

Figure 12 FTIR Analysis of lipids

S.No

FTIR Results

Compound

Ester Types

1

Methyl linoleate natural

Methyl esters of linoleic acid

 
 
Unsaturated fatty acids

2

Ethyl Palmitate

Ethyl esters of palmitic acid

3

Ethyl Myristate

Ethyl esters of myristic acid

4

Ethyl Linoleate

Ethyl esters of linoleic acid

 

IV. Conclusion

From the study, it was found that oleaginous yeast Lipomyces starkeyi has highest biomass yield of about 18.42 g/L in fructose as an individual media and 22.32 g/L in glucose and fructose as combined media. Similarly, Yarrowia lipolytica and Metschnikowia pulcherrima has the biomass yield of about 10g/L and 10 g/L in glucose as individual media and 10g/L and 8.2g/L in glucose and xylose and in glucose and fructose as combined media. The biomass was extracted further for the lipid yield from three oleaginous yeast. The yield of lipid from the media in Lipomyces starkeyi is found to be 15.6 g/L in Fructose and 12.6 g/L in Glucose and fructose and in Yarrowia lipolytica and Metschnikowia pulcherrima the yield of lipid is found to be 6g/L and 4 g/Lin glucose and 6g/L and 3.28g/L in glucose and xylose and in glucose and fructose. From the above investigation, it is concluded that the yeast Lipomyces starkeyi is found to have the highest lipid yield among the three oleaginous yeast used for the study and it can further used for the production of biodiesel through various transesterification methods.