Abstract: An oleaginous yeast strain, Cryptococcus curvatus NRRLY-1511 was used for the production of single cell oil (SCO) using a low cost cultivation medium containing beet molasses and corn gluten meal as carbon and nitrogen sources. Obtained results showed that 125 and 0.130 g L-1 showed to be the optimum concentrations for carbon and nitrogen, respectively. In addition, 28°C, 72 h, 5.5, 200 rpm were the favorable values of growth temperature, incubation period, pH value of cultivation medium and agitation speed, respectively. The extracted lipids were mainly 30.68% linoleic acid (C18:2), 22.66% oleic acid (C18:1) and 16.74% palmitic acid (C16:0). Furthermore, the GC analysis also showed that the total saturated fatty acids (n = 9) represented 41.96% while the value of the total unsaturated fatty acids (n = 6) was 58.04%. These results giving possibility to use such this yeast strain to produce SCO in a low cost medium from economic point of view.
INTRODUCTION
The concept of single cell oil (SCO) produced by lipid-producing (oleaginous) microorganisms as the supplementary sources of conventional oils and fats has attracted attention since the early 1980s. The majority of those lipids in these organisms are triacylglycerols (TAG), containing long-chain fatty acids that are comparable to conventional plant oils. It would be important to develop new oil resources by using microbes, which offers many advantages compared with traditional methods using animal fat and plant oils. Oleaginous yeasts are often considered for the production of single cell oil. The economics of these bioprocesses has become more favourable when zero or negative value waste substrates are utilized as carbon or nitrogen sources (Pan et al., 2009). The efficiency of oil biosynthesis by yeast and its composition depend on the genetic properties of the yeast strains, cultivation conditions and the composition of culture medium. Lipids are important storage compounds in plants, animal and fungi. Storage lipids are usually found within special organelles knows as lipid particles or lipid bodies. In yeast, these lipid bodies accumulate during stationary phase and they can constitute up to 70% of the total lipid content of the cell (Zweytick et al., 2000).
Triacylglycerols (TAGs) and steryl esters (STEs) are the most important storage lipids of eukaryotes cells such like yeast cells. TAG provides an energy source on one hand and a source of fatty acids for membrane phospholipid formation on the other hand. Mobilization of STE sets sterols free, which are also required for membrane proliferation, especially of the plasma membrane. In the yeast as in other eukaryotic cells, TAG and STE form the core of the so-called lipid particles which are surrounded by a phospholipid monolayer with a small amount of proteins embedded (Sorger et al., 2004).
The polyunsaturated fatty acids, linoleic (C18:2) and linolenic (C18:3) are the essential fatty acids. They are not synthesized by the human body and, therefore, must be obtained from the dietary intake of foods containing them. Linoleic acid aids in the prevention of platelet aggregation leading to blood clotting in the blood vessels as described by Renaud (1990). Linoleic acid, as essential fatty acid is able to reduce the incidence of cardiac arrest in rats induced by artery occlusion resulting in an increased blood flow to the heart, reduces serum cholesterol. They may prevent the development of cardiovascular disease (Hansen and Chiu, 2005). In addition, palmitic acid (C16:0) and oleic acid (C18:1) have a neutral effect on serum cholesterol, when the dietary intake of cholesterol is low.
The aim of this study was to determine the optimal conditions for oil biosynthesis by an oleaginous yeast strain, Cryptococcus curvatus NRRLY-1511 using a low cost cultivation medium containing beet molasses and corn gluten meal as carbon and nitrogen sources.
MATERIALS AND METHODS
Yeast Strain and Cultivation Media
The yeast strain used in this investigation was Cryptococcus curvatus
NRRLY-1511. The cultivation medium of Lindberg and Molin (1993) was used for
fermentation and cultivation of the yeast strain. Yeast extract malt broth (YMB)
medium was used for inoculum preparation and maintenance of the used yeast strain.
By-Products Used
Sugar Beet Molasses (SBM) that obtained from sugar beet factory of Belkas,
Dakahlia Governorate, Egypt was used as a sole carbon source. This by-product
containing about 48% total sugars before purification that became 23.3% after
purification. Corn gluten meal obtained from starch and glucose factory as a
by-product containing 2.22% nitrogen was used as a sole nitrogen source. Each
of these wastes was replaced with the other source in the cultivation medium
of Lindberg and Molin (1993) in the same ratio.
Preparation of the Industrial By-Products
Sample of Sugar Beet Molasses (SBM) was prepared as carbon source by diluting
with water in an equal volume using the method of Pandey and Agarwal (1993)
with little modification. H2SO4 solution was used to reduce
the pH value to reach 3.0. Sample was boiled at 100°C for 1 h then maintained
at room temperature for 24 h, centrifuged at 3000 rpm and filtered. Filtrated
solution was used to determine the total sugars to be used as a sole carbon
source. Nitrogenous by-product namely corn gluten meal, was oven dried at 105°C
for 2 h and milled. About 7.5 g of waste was add to 100 mL of 1.5% H2SO4
and autoclaved for 45 min then filtered. The obtained supernatant was attained
at pH 6, being used as a sole nitrogen source.
Preparation of the Inoculum
The inoculum used in the experiments was prepared in Erlenmeyer flasks using
YMB medium. The cultivation conditions were carried out at 28°C; pH 5; 72
h and shake speed was 200 rpm. The inoculum size of 3% v/v (5x105cfu
mL-1) was added to the tested cultivation medium.
Medium Optimization
Fermentation was carried out in 250 mL capacity Erlenmeyer flasks containing
100 mL of the previously mentioned fermentation medium and initial pH was adjusted
to 6.0 using a pH meter before autoclaving at 121°C for 20 min then inoculated
with 3% v/v (5x105 cfu mL-1) of cells suspension of the
examined yeast in sterile distilled water. The culture was incubated at the
growth temperature on a rotary shaker at 200 rpm for the required fermentation
period using LAB-line instrument, Inc., plaza, Mel Rose, Park, ILL. 60160.
Four agro-industrial by-products as carbon sources namely sugar beet molasses, sugar cane molasses, potatoes peel and tomatoes peel were individually added to the basal cultivation medium at a concentration of 10% as glucose to study the effect of each carbon source on the production of microbial oil. Yeast strain was grown on the oil production medium provided with different concentrations of sugar beet molasses ranging from 75-175 g L-1 were used to select the optimal concentration.
Determination of Yeast Dry Weight
Yeast dry weight (single cell protein) determination was performed by harvesting
culture samples, centrifuged at 5000 rpm, washed twice with distilled water
and dried at 60°C to constant weight (Granger et al., 1993). The
growth yield efficiency (economic coefficient) was calculated according to the
following equation:
The productivity of oil produced (conversion coefficient) was also calculated according to the following equation:
Determination of Total Sugars
Total sugars were determined according to the method of Herbert et al.
(1971) as follow: Into a thick walled tube, 1 mL of the tested sample was pipetted
and well mixed with 1 mL of 5% phenol solution and then 5 mL of concentrated
sulphuric acid were directly added on the surface of a liquid with shaking.
The tubes were allowed to stand in a water bath at 25°C for 20 min before
reading the density of obtained colour at 490 nm using a Jenway model 6305 UV/visible
range spectrophotometer. The standard was carried out by the same process using
glucose. The total sugars was expressed as mg glucose/mL using the equation
of Y = 0.0274 x+0.024 with R2 = 0.9586.
Single Cell Oil Extraction
Single cell oil extraction was carried out according to the method of Granger
et al. (1992) as follows: Yeast cells were separated by centrifugation
at 6000 rpm (Type 16000, sponnung 220 V, German Democratic Republic) for 15
min and dried at 60°C to constant weight. The dried cells were then milled
for 20 min with carbonium powder and extracted using a condenser unit at 60-70°C
then filtered using a filter paper Whatman No. 1 and oven dried at 70°C.
The single cell oil yield efficiency was also calculated according to the following
equation:
Extraction of Fatty Acids
The method of extraction for fatty acids determination of obtained single
cell oil was carried out according to the method recommended by Radwan (1978).
Extraction was obtained by adding 2.5 mL of 1% sulfuric acid in anhydrous methanol
and 1 mL benzene in a sealed tube and heating in a hot water bath at 90°C
for 90 min. The tube was allowed to cool, then 4 mL of distilled water and 2.5
mL petroleum ether were added and shaked well. The ether layer (upper layer)
was removed in a small vial and evaporated and then 50 mL of n-hexane was added.
Gas Chromatographic Conditions
Sample was injected into a Shimadzu Model GC-8A gas Chromatograph equipped
with a Flame Ionization Detector (FID) and a 2.5 M x 3 mm glass column under
the following conditions, 5% DEGS coated on 80-100 mesh with chromosorb WHP).
The flame ionization detector temperature was 270°C and the column set was
temperature programmed from 150 to 180°C at 2°C min-1 The
carrier gas was nitrogen, with a flow rate of 20 mL min-1; hydrogen
flow rate was 75 mL min-1 and chart speed 2.5 mm min-1
The hydrogen and air flow-rates were 75 and 0.5 mL min-1, respectively.
The FID sensitivity was16x102. Peaks of fatty acids were identified
by cochromatography with the standards and relative retention times (Radwan,
1978).
RESULTS AND DISCUSSION
Carbon Source
Four of agro-industrial by-products used as carbon sources were individually
examined for single cell oil production by the tested yeast Cryptococcus
curvatus NRRLY-1511 namely, sugar beet molasses, beet cane molasses, potato
peels and tomato peels. Each of these sources was replaced with glucose in the
cultivation medium as control in a concentration of 100 g L-1 at
initial pH of 6.0. Different parameters were determined and obtained results
are listed in Table 1. As shown, great change in pH value
was noticed up to the lowest value to be 4.6 which recorded with potato peels
as carbon source.
| Table 1: | Effect of carbon source on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
| dx:Δ biomass, dt: Δ time, dp: Δ product | |
The highest pH value was observed with beet molasses being 7.2. For sugar consumed (g L-1), the highest value found when glucose was used as carbon source to be 36.2 g L-1 while the lowest sugar consumed value was observed in case of suger beet molasses using as carbon source to be 16.6 g L-1. Results obtained by Syed et al. (2006) showed that glucose was the best carbon source between the five tested sources. Their results exhibited dry biomass production about 34.6 g L-1 and 5.8% of γ-linolenic acid from different strains belonging to Mucorales.
For the dry cell weight (g L-1) obtained by the used yeast Cryptococcus curvatus NRRLY-1511, sugar beat molasses recorded the highest dry cell weight being 3.2 g L-1 as shown in Table 1. On the other hand, tomato peels showed the lowest value of obtained dry biomass to be 1.8 g L-1 compared to that value obtained with control being 3.6 g L-1. The cell weight correlated with oil weight produced (g L-1) since suger beet molasses gave the highest produced single cell oil to be 1.8 g L-1. This means that the increase fold equal to 0.89 and 1.5 for yeast cell weight and single cell oil weight compared to control, respectively. So, one can detect that sugar beet molasses showed to be the best agro-industrial by-product can be used in single cell oil production by Cryptococcus curvatus NRRLY-1511. Results of Syed et al. (2006) proved that tapioca starch was the best source for lipid production among four different carbon source namely sucrose, lactose, soluble starch and tapioca starch. Similar results have also been reported earlier (Somashekar et al., 2002).
Certik et al. (1997) illustrated that carbohydrates are usually metabolized via the Embden-Myerhof Pathway to generate pyruvate or acetyl-CoA, which are then used for proteosynthesis, respiration and synthesis of other compounds including membrane and storage lipids. Furthermore, the efficiency of biomass and SCO are illustrated in Fig. 1.
| Fig. 1: | Effect of carbon source on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
Carbon Source Concentration
In order to select the optimum concentration of the sugar beet molasses
as carbon source, five concentrations were used as shown in Table
2. Tabulated data showed that the maximum sugar consumed was 36.2 g L-1
recorded in case of 175 g L-1 of sugar beet molasses. This result
was not related to the dry cell weight since the growth of Cryptococcus curvatus
NRRLY-1511 was 2.3 g L-1 while the highest cell weight obtained was
4.6 g L-1 that obtained with 100 g L-1 of sugar beet molasses
. The concentration of 125 g L-1 of carbon source was the optimum
concentration required for single cell oil production being 1.2 g L-1
and oil percentage of 29.27%. This result was confirmed by that result obtained
by Syed et al. (2006) who found that the increase in initial glucose
leads to dry biomass decrease. This might be due to intolerance of the yeast
cells to high concentration of glucose which increase the osmotic potential
of the medium. In addition, the values of single cell protein (SCP) and SCO
efficiencies are shown in Fig. 2.
| Table 2: | Effect of carbon source concentration on biomass and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
| dx: Δ Biomass, dt: Δ Time, dp: Δ Product | |
| Fig. 2: | Effect of carbon source concentration on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
Nitrogen Source
Four different of agro-industrial by-products were individually used as
nitrogen sources with replaced each of them by NaNO3, the nitrogen
source in the cultivation medium as control. As shown in Table
3, little change in pH value of the cultivation medium was found up to the
lowest value of 4.2 that recorded with rice bran as nitrogen source. For sugar
consumptions, the highest value of sugar consumed was 65.37 g L-1
that recorded when corn gluten meal used as nitrogen source. The highest yield
of dry biomass to be 2.8 g L-1 was also recorded with corn gluten
meal that correlated with single cell oil weight produced being 1.6 g L-1
and oil percentage of 64%. These results proved that corn gluten meal considered
to be the most favourable nitrogen source required for single cell oil production
by Cryptococcus curvatus NRRLY-1511. Furthermore, the efficiency of either
SCP or SCO is illustrated in Fig. 3.
| Table 3: | Effect of nitrogen source on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
| dx: Δ Biomass, dt: Δ Time, dp: Δ Product | |
| Fig. 3: | Effect of nitrogen source on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
Nitrogen Source Concentration
Five concentrations of the nitrogen source were individually replaced in
the cultivation medium to select the optimum concentration required for single
cell oil production. Little change in final pH values was observed as shown
in Table 4. Cryptococcus curvatus NRRLY-1511 consumed
36.6 g L-1 sugars when using N-concentration in the cultivation medium
0.130 g L-1 that produced 3.2 g L-1 of dry biomass. The
obtained SCO weight was equal to 1.5 g L-1 and oil percentage was
46.88%. Results obtained by Syed et al. (2006) showed that the total
lipid content produced from the medium containing yeast extract was higher than
that medium containing peptone. They reported that yeast extract was the best
nitrogen source for obtaining biomass and lipid. In addition, the efficiency
of either SCP or SCO is shown in Fig. 4.
| Table 4: | Effect of nitrogen source concentration on biomass and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
dx: Δ Biomass, dt: Δ Time, dp: Δ Product | |
| Fig. 4: | Effect of nitrogen source concentration on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
Cultivation Medium pH
In order to examine the effect of initial pH value of the cultivation medium,
seven levels of different pH were performed. Appreciate differences between
the initial and final pH values were noticed as shown in Table
5. Little change in the final pH value was found between the different treatments.
At pH 5.5 treatment, the dry cell weight of Cryptococcus curvatus NRRLY-1511
reached to 2.4 g L-1 with SCO weight of 1.7 g L-1. On
the other hand, pH 6.5 recorded the highest sugar consumption to be 56.2 g L-1.
It was found that microbial oil production was maximum when the mould was cultivated
at pH 6.5 (Syed et al., 2006). They also found that total lipid drastically
decreased at pH 8.0 and at pH 4.0. They also reported that there was an increase
in total lipid concentration in the pH range of 3.0 to 6.0. The efficiency of
either SCP or SCO is illustrated in Fig. 5.
| Table 5: | Effect of initial pH value of the cultivation medium on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
| dx: Δ Biomass, dt: Δ Time, dp: Δ Product | |
| Fig. 5: | Effect of initial pH value of the cultivation medium on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
Incubation Period
The behavior of Cryptococcus curvatus NRRLY-1511 exhibited different
activities with different incubation period as shown in Table
6. Very little change was found in final pH value of the cultivation medium.
The highest value of sugar consumption was observed at 48 h to be 44.3 g L-1.
After 72 h of incubation, the dry biomass weight was 3.7 g L-1. For
microbial oil (SCO) weight production, data showed that 2.2 g L-1
of microbial oil with 59.46% oil percentage was produced after 72 h of incubation.
This result was proved by plotting the efficiency of either SCP or SCO that
illustrated in Fig. 6.
| Table 6: | Effect of incubation period on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
dx: Δ Biomass, dt: Δ Time, dp: Δ Product | |
| Fig. 6: | Effect of incubation period on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
Growth Temperature
Six growth temperatures were used to examine their effect on the single
cell oil production by Cryptococcus curvatus NRRLY-1511.As shown in Table
7, the treatment of 28°C exhibited change in final pH value of the cultural
medium. In addition, the value of sugar consumed by the tested yeast strain
that observed with the same treatment being 45.6 g L-1. The highest
production of either dry cell weight or SCO weight to be 5.0 g L-1or
2.4 g L-1, respectively. The growth of the tested yeast was low at
temperature below 24°C and above 30°C as illustrated in Table
7. Carvalho et al. (1999) found that 28°C was the optimum temperature
for dry biomass production. They obtained 2.47, 5.83 and 4.29 g L-1
dry biomass of Mucor sp. LB-54 at 12, 28 and 38°C, respectively.
At the same temperature, the production of lipid content was 0.39, 1.21 and
0.49 g L-1, respectively. Furthermore, the efficiency of either SCP
or SCO is clearly shown in Fig. 7.
| Table 7: | Effect of growth temperature on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
| dx: Δ Biomass, dt: Δ Time, dp: Δ Product | |
| Fig. 7: | Effect of growth temperature on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
Agitation Speed
The effect of agitation speed on the SCO production was investigated. Five
agitation levels were performed and obtained results are listed in Table
8. From tabulated data one can see little change in the values of final
pH of the cultural medium. Concerning the sugar consumption, it was very clear
that Cryptococcus curvatus NRRLY-1511 used sugar in increase ratio with
the increase agitation speed used. The increase of sugar consumption reached
to the highest value to be 58.6 g L-1 in case of 200 rpm. The same
trend was observed with data of dry cell weight (SCP) since the values gradually
increased to reach the highest value of 4.6 g L-1 that recorded in
case of 200 rpm. Choi et al. (1982) reported that both biomass and its
lipid content increased with increase in dissolved oxygen concentration. They
added that Rhodotorula glutinis as obligate aerobic yeast, is dependent
on oxygen for its energy metabolism and synthesis of cellular components. So,
it is not surprising therefore, that increasing oxygen concentration showed
a positive effect on the lipid content of these aerophilic yeast cells, even
though lipid were more reduced than the other major components of living cells.
Data of the efficiency of either SCP or SCO produced by the tested yeast strain
is illustrated in Fig. 8.
| Table 8: | Effect of agitation speed of cultivation on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
| dx: Δ Biomass, dt: Δ Time, dp: Δ Product | |
| Fig. 8: | Effect of agitation speed of cultivation on single cell protein and single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
Quantitative Values of Fatty Acids
Table 9 showing the profile of fatty acid composition
of microbial oil (SCO) obtained by Cryptococcus curvatus NRRLY-1511 at
200 rpm agitation speed. This Table showing 15 appeared compounds for the tested
sample and only nine detected for the control sample (stagnant culture). Of
appeared compounds, 9 saturated fatty acids (n = 9) of 41.96%. The most incidence
one was palmitic acid C16H32O2 (C16:0) since
it gave 16.74% of the tested sample. Of the total fatty acids, 26.91% was monosaturated
fatty acids (n = 4). Oleic acid C18H34O2 (C18:1
ω9) showed to be the highest one by 22.66% of the tested sample. The percent
of polyunsaturated fatty acids (C18:2) was 31.13%. Of these, linoleic acid C18H30O2
(C18:2 ω6) exhibited 30.68% of the tested sample.
| Table 9: | Quantitative values of fatty acids content in single cell oil production by an oleaginous yeast strain, Cryptococcus curvatus NRRL-Y-1511 |
| aSCO obtained from stagnant cultivation. bAfter Abou El-Hawa et al. (2004) | |
The importance of obtained microbial oil that is near close to canola oil in its composition. So, comparing the obtained microbial oil with canola oil it can be found, more or less, the same fatty acids with different concentration; Palmitic acid (16:0), Oleic (18:1), Linoleic (18:2), Linolenic (18:3), and Myristic (14:0) as reported by Abou El-Hawa et al. (2004).
CONCLUSIONS
With this strategy, it is able to stimulate single cell oil (SCO) production by Cryptococcus curvatus NRRLY-1511 from the cultivation medium containing some agro-industrial by products as carbon and nitrogen sources. The data presented in this investigation showed the significant influence of some nutritional and environmental factors. So these results suggest that Cryptococcus curvatus NRRLY-1511 may have potential for commercial development for the production of single cell oil (SCO) by fermentation technique.