الفهرس 
Biodiesel productionOnly 14 pages are availabe for public view
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Abstract
Biodiesel is a renewable resource of energy. It is regarded to face
high energy prices and potential depletion of fossil oils reservoirs. It
contributes to the reduction of carbon dioxide and sulfur emissions to the
atmosphere and has potential of being an environmentally solution for
global warming, the energy crisis and depleted fossil fuel supplies. It is a
mixture of fatty acids (FAs) methyl esters derived from triacylglycerols
(TAGs). The biomass of oleaginous yeast is an optimal and abundant
source of TAGs which exhibit similar FAs composition to plant oils.
The present work aimed to study lipid-producing yeasts and
promising for biodiesel production. The study included isolation of yeasts
capable to produce lipid, screening for the most efficient isolates,
estimation of biomass, lipid weight and lipid content of this isolate,
analysis the fatty acid profiles and determination of the optimum
nutritional and environmental conditions for lipid production.
Results could be summarized as follow:-
1. Seventy six isolates of yeast were isolated from different sources
included 23 isolates from soil samples and 2 isolates from organic
manure, as well 22 yeast isolates from rotten fruits, 1 isolate from
sugarcane juice, and 28 isolates from pickles, based on the typical
morphological character of yeast using light microscope.
2. The lipid accumulation capacity of the isolated yeast was revealed by
Sudan Black B staining of the cells grown in nitrogen-limited
medium. The preliminary screening of all 76 yeast isolates showed
that 33 isolates of yeast had the ability to accumulate lipids.
3. The quantitative assessment of selected 33 yeast isolates after
growing on N-limiting medium was determined by extraction and
estimation of cell dry weight, lipid weight and lipid content. It was
observed that there were wide variations in cell dry weight, lipid
weight and lipid content, between the different examined isolates.Some isolates exhibited high values of lipid content as clearly seen in
isolates number S5, D5, J3 and C9 being (0.59, 1.24, 1.16 and 0.69
gl-1 lipids), respectively. The corresponding of lipid content (%) were
22.18, 23.48, 18.77 and 24.47% Whereas some other isolates
appeared less values as in S6, F1, F3, S12, L3 and S11. The choice of
high lipid content yeast was done due to their potentiality in
accumulation of the intracellular lipids which could be calculated by
dividing lipid weight on cell dry weight (gl-1) as percentage (%).
4. Time course of biomass production and lipid accumulation was
carried out on the high efficient isolates namely S5, D5, J3 and C9
grown in N- limited medium no. 7 under shaking condition at 28-30
ºC. It was observed that lipid production was started during the
exponential phase of growth then sharply increased to achieve the
maximum values of lipid weight (gl-1) and lipid content (%) during
stationary phase after 80, 120, 96 and 104 hrs of fermentation time
for S5, D5, J3 and C9 isolates respectively. The corresponding values
of lipid content were 35.06, 30.06, 37.90 and 37.50%, respectively.
The corresponding figures of sugar utilization efficiency (SUE %)
and conversion coefficient (C.C%) were 99.09, 4.94% for S5, 99.86,
3.75% for D5, 94.70, 4.67% for J3 and 97.15, 5.25 % for C9,
respectively.
5. The fatty acids profile analysis demonstrated that the lipid extracted
from the four promising yeast isolates mainly contained the principal
fatty acids triacylglyceols, (TAGs) as palmitic acid (C16:0), stearic
acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2) which is
similar to that of vegetable oils. The isolates (S5, D5, C9 and J3)
mainly their content of triacylglycerols (TAG) as palmitic acid
(C16:0), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid
(C18:2) which is similar to that of vegetable oils. The highest
dominant fatty acid among the fatty acids profile of the 4 isolates was
the oleic acid (C18:1) with content ranging between 29.14% and
55.69%, followed by palmitic acid (C16:0) with content ranging between 16.08% and 31.52% among the 4 isolates. In addition,
palmitoleic acid (C16:1) content was 13.75%, 15.74% and 8.08%, as
one of the main products with S5, D5 and C9 whereas isolate J3
produced negligible amounts of this fatty acid.
6. The most potent isolates S5 and D5 were identified using a molecular
taxonomic approach. BLAST analysis of the 26S rRNA gene
sequence of the yeast isolates S5 & D5 were revealed to be a perfect
match with that of Candida tropicalis and Issatchenkia orientalis
type strains, respectively.
7. Optimization of culture conditions for lipid production:
- Effect of different media and extraction methods on lipid
production :
Candida tropicalis (S5) gave the highest cell dry weight with Nlimited
medium no. 5 (15.47 gl-1) whereas the maximum figure of
lipid weight was obtained with N- limited medium no. 7 being
0.307 gl-1 & 0.46 gl-1 by extraction method 1 (Folch et al. 1957)
and method 2 (Bligh & Dyer (1959) modified by Manirakiza et al.
2001) of lipids, respectively. Issatchenkia orientalis (D5) showed
the highest value of cell dry weight with N- limited medium no. 8
(4.56 gl-1) and the highest lipid weight being 0.29 gl-1 with
extraction method 1 and 0.56 gl-1 with extraction method 2 on Nlimited
medium no.6. It could be concluded the nitrogen limited
medium no. 7 & 6 were the preferable media for lipid production
by Candida tropicalis (S5) and Issatchenkia orientalis (D5),
respectively using extraction method 2.
- Effect of carbon sources :
The values of lipid yield, conversion coefficient and lipid
productivity on glucose were higher than other carbon sources
used by Candida tropicalis (S5) being 3.30%, 4.10%, 0.26 gl-1/day
and the accumulated lipid which presented by lipid weight and
lipid content were 1.32 gl-1 and 23.04 %, respectively with 32.16
gl-1 of consumed sugar. Issatchenkia orientalis (D5) exhibited the same trend for lipid content, it was reached to 26.57 % while lipid
weight, consumed sugar and SUE were 1.73 gl-1, 42.16 gl-1 and
84.32%, respectively.
- Effect of nitrogen sources:
Yeast extract was the most effective N- source on biomass value
(cell dry weight) of Candida tropicalis (S5) being 8.62 gl-1 and the
optimize value of lipid weight, consumed sugar, lipid yield, lipid
productivity and SUE were with the mixture of yeast extract and
(NH4)2SO4. Peptone gave very high result with cell dry weight
with Issatchenkia orientalis (D5) and the best value of lipid
weight on urea (2.75 gl-1) than the other nitrogen sources.
- Effect of C/N ratio:
The data of Candida tropicalis (S5) showed that biomass
decreased gradually with increase of C/N ratio and the maximum
biomass (7.90 gl-1) at 54 of C/N ratio. Whereas cellular lipid
accumulation gave the lowest value of lipid weight at 40 of C/N
ratio , then showed sharp increase when C/N ratio increased to 115
to record the maximum of lipid content (27.77%) with sugar
utilization efficiency, conversion coefficient and lipid productivity
being 99.18%, 1.26%, and 0.20 gl-1/day, respectively. Issatchenkia
orientalis D5 exhibited that cell dry weight, lipid weight, SUE,
C.C and lipid productivity reached to the maximum value at 100
of C/N ratio.
- Effect of incubation temperature :
The maximum value of lipid content was recorded at 30 ºC by
Candida tropicalis (S5) being 28.90% of dry biomass then sharply
decrease by increasing the temperature to reach 45 ºC giving the
lowest value (11.53%) . At 30 ºC, consumed sugar, SUE, yield
factor, lipid yield, C.C and lipid productivity recorded the
maximum values. Issatchenkia orientalis (D5) exhibited the
highest values at 30 ºC. Cell dry weight, lipid weight and lipid
content were 1.90, 0.68 gl-1 and 35.79%, respectively when consumed sugar, yield factor and conversion coefficient were
153.49 gl-1, 1.24 % and 0.44 %.
8. Biological activity of lipid producing yeast strains
The two strains Issatchenkia orientalis (D5) and Candida
tropicalis (S5) grown in 5 l Erlenmeyer flask containing 2 l of optimal
fermentation medium (N- limited media) under shaking conditions and
the optimum conditions during 9 days with sampling time of 24 h. It was
observed that cell biomass (gl-1) was gradually increased along with the
time sample (9 days) in both strains and associated with consumed sugar
(gl-1) to reach maximum values of cell dry weight (11.22 & 13.30 gl-1) at 8 and
9 day with consumed sugar equal 79.52 and 156.29 gl-1 for Candida tropicalis
(S5) and Issatchenkia orientalis (D5), respectively. With respect to lipid
production , it was found that Candida tropicalis (S5) gave the highest
values of lipid weight (gl-1), lipid content (%), lipid yield (%), conversion
coefficient (%) and lipid productivity (gl-1/day) after 6 days being 2.16
gl-1, 34.17 %, 2.70 %, 2.73% and 0.36 gl-1/day, respectively. Issatchenkia
orientalis (D5) recorded the highest lipid concenteration (gl-1) and lipid
yield (%) after 4 days of the fermentation period being 3.24 gl-1 and
2.06%. The data showed that the specific growth rate, doubling time,
multiplication rate and number of generation were 0.317 day-1, 2.18 days,
0.458 day-1 and 1.37 for Candida tropicalis (S5) and 0.275 day-1, 2.52
days, 0.396 day-1 and 0.793 for Issatchenkia orientalis (D5). There are
high positive correlation between cell dry weight and lipid weight (gl-1)
with incubation time. The regression analysis showed that the specific
production rate of lipid production were 0.358 day-1 for Candida
tropicalis (S5) and 0.385 day-1 for Issatchenkia orientalis (D5), the first
strain was lower than that observed by other strain. The doubling time of lipid contents were 1.94 and 1.80 days, respectively .