Effect of carbon source and high C/N ratio in the cellulosic hydrolysate- based media
on the crude lipid contents and unsaturated fatty acid compositions of yeasts
Yi-Huang Changa, Ku-Shang Changa, Hung-Der Janga, *, Chuan-Liang Hsub, **
a, *
Department of Food Science, Yuanpei University, Hsinchu 300, Taiwan (E-mail:
hungder@mail.ypu.edu.tw)
b,**
Department of Food Science, Tunghai University, Taichung 407, Taiwan (E-mail: clh@thu.edu.tw)
ABSTRACT
Microbial lipid production by the oleaginous yeast Cryptococcus sp. was studied using glucose and cellulosic
hydrolysate as carbon source, to realize the feasiblility of biodiesel production from high-lipid cell culture.
The crude lipid contents of Debaryomyces nepalensis and Cryptococcus sp. cultured in YM Agar reached as
high as 32-35 % on dry weight basis, which were higher than those of Pseudozyma spp and Moesziomyces
eriocauli. Accumulation of lipids strongly depended on the C/N ratio and nitrogen concentration. The highest
content of lipids was measured at a C/N ratio of 60-90 and a nitrogen concentration of 0.2% with 60-57%
lipids of the dry biomass. The crude lipid contents of biomass could be increased with the addition of carbon
source and adjusting the C/N ratio of the cellulosic hydrolysate-based media. Batch cultures using cellulosic
hydrolysate demonstrated that there was little inhibitory effect with a reducing sugar concentration of up to
60 g/l. Batch cultures were run for 4 days and reached a dry biomass, lipid concentration, and lipid content of
12.6, 6.3 g/l, and 50.2% (w/w), respectively. The lipids from Cryptococcus sp. contained mainly long-chain
saturated and unsaturated fatty acids with 16 and 18 carbon atoms. The four major constituent fatty acids
were oleic acid, palmitic acid, stearic acid and linoleic acid. The contents of oleic acid methyl ester and
linoleic acid methyl ester were 58.9 % and 7.0 %, respectively. Based on the results, microbial lipids from
Cryptococcus sp. are a potential alternative oil resource for biodiesel production.
Keywords: Cryptococcus spp.; Cellulosic hydrolysates; Microbial lipid; Batch fermentation; Biodiesel
INTRODUCTION
Certain yeasts have been identified rich in crude lipid contents and have the potential for use in biodiesel
production. Lipids serve as storage materials in some lipid accumulating yeasts, such as Rhodosporidium sp.,
Rhodotorula sp. and Lipomyces sp., which can accumulate intracellular lipids as high as 70% of their
biomass dry weight [1]. Several different yeasts were studied, including Candida curvata, Trichosporon
cutaneum, Rhodosporidium toruloides, and L. starkeyi, where L. starkeyi was reported to store the largest
quantities of lipids [2]. In addition, various microorganisms are capable of accumulating huge quantities of
lipid when cultured on nitrogen-limited sugar-based media [3, 4]. It was observed that under nitrogen
limiting conditions and the presence of a carbon-source in excess organisms started to store lipids. Therefore
a high carbon to nitrogen (C/N) ratio, around 90, is a basic requirement for the accumulation of lipids.
However, the effects of carbon source and C/N ratio on the crude lipid content need to be studied. The basic
physiology of lipid accumulation in microorganisms has been well studied. It is known that lipid production
requires medium with an excess of sugars or similar components (e.g., glycerol, polysaccharides, etc.) and
other limited nutrients, such as nitrogen. Thus, oleaginous potential is critically affected by the C/N ratio of
the culture and other factors like aeration, inorganic salt presence, etc. [5]. The aim of the study was to obtain
the biomass with high crude lipid contents from the yeast cultures through the addition of organic carbon
source to the cellulosic hydrolysate-based medium. Special interest was on the composition of fatty acids,
which related to the quality of biodiesel fuels.
MATERIALS & METHODS
Microorganisms and cultivation
Yeast strains were collected and isolated from peka, leaf and forest soil in Taiwan. To isolate yeasts from the
soil, 1.0 g of each sample soil was diluted with 9 ml of sterilized water and vortex-mixed. Five potential
strains for lipid production were identified as Cryptococcus sp. SM5S05, Moesziomyces eriocauli SJ3L01
and SJ4L09, and Pseudozyma spp. FN20L03 and SJ4L03. One tenth of a milliliter of successive decimal
dilutions was spread on acidified yeast malt agar (YMA) (1% glucose, 0.5% peptone, 0.3% yeast extract,
0.3% malt extract, 1.5% agar, pH3.5). The plates were then incubated at 24°C for 3 days. Representative
colonies were picked and preserved on YMA slant at 4℃.
Cellulosic material pretreatment and hydrolysis with enzyme mixtures
The corncob materials, purchased from a local market, were oven-dried (≥24 h at 50°C) and grounded into
particles (diameter 2-10 mm) and stored in pill vials at 25°C. The corncob materials consisted mainly of 42%
cellulose and 28% hemicellulose, which could be hydrolyzed to reducing sugar. The solid substrate was
mixed with distilled water at a solid-to-liquid ratio of 1:10. The mixture was filtered and then the filtrate was
further hydrolyzed by autoclaving and acid as our previous methods [6].
The mixture of prehydrolysate obtained from the acid and autoclaving pretreatments was collected and
filtered with Whatman No. 4 filter paper. Enzymatic hydrolysis was performed with a 100 ml prehydrolysate
and commercial cellulase solution. The protocols of enzymatic hydrolysis of prehydrolysate to produce
reducing sugar were also according to our previous report [6]. The mixtures were incubated at 50°C in an
orbital shaker with a speed of 160 rpm for 72 h. Samples were analyzed for levels of total reducing sugar and
used for batch culture of yeast cell.
Lipid production and batch culture conditions
The yeast strains were grown on YMA at 25°C for 3 days. One inoculum loop of yeast cells were transferred
into base medium (2% glucose, 0.5% yeast extract, 0.5% peptone, 0.15% MgSO4·7H2O, pH 6.0) and
incubated in a rotary shaker at 180 rpm for 48 h at 25°C. Then, 5% (v/v) of each yeast inocula (5×1065×107/ml) was transferred into the base medium and incubated in a rotary shaker at 180 rpm for 4 days at
25°C. The base medium for lipid production contained 4% or 8% carbon source (glucose or sucrose), 0.25%
yeast extract, 0.25% peptone and 0.15% MgSO4·7H2O at pH 6.0. After incubation, yeast cells were
centrifuged at 3,000 rpm for 15 min and collected for lipid extraction.
To determine oleaginous potential for future application, the strain Cryptococcus sp. SM5S05 was
inoculated and cultured. The base medium contained different levels of glucose (from 2-10%) or corncob
hydrolysate (4 and 6%), yeast extract (from 0.05 to 0.5%), peptone (from 0.05 to 0.5%) and 0.15%
MgSO4·7H2O at pH 6.0. After incubation, the yeast cells were centrifuged and collected for lipid extraction.
Yeast biomass was determined by measuring cell optical density at 600 nm with a spectrophotometer.
Lipid extraction and fatty acid analysis
Total yeast lipids were extracted according to the procedures described by Folch et al. [7]. After incubation,
the yeast cells were harvested by centrifugation under 3,000 rpm for 15 min. The yeast cells were collected
and washed once with distilled H2O, and ready for lipid extraction. Briefly, approximate 2 grams (wet weight)
of the rinsed cells was extracted with 40 ml of chloroform/methanol (2:1, v/v) at room temperature for 1 hour.
The extracted lipids in chloroform phase were separated from the aqueous phase by adding 8 ml of 0.9%
NaCl. The lipid extract in chloroform was collected into a weight-measured test tube, and solvents were
evaporated by nitrogen gas. After the dryness, the amount of lipid extracted from individual yeast could be
obtained by subtracting the weight of empty tube from that of tube with the sample.
According to the level of lipid content, the extracted lipids were then re-constituted with appropriate
amount of chloroform. Triheptadecanoin was added to the re-constitute lipid extract as the internal standard.
The extracted lipids with internal standard were treated with BF3 for 20 min at 95˚C to prepare fatty acid
methyl esters (FAMEs). The FAMEs were extracted by hexane and then analyzed by an Agilent 6890 GC
with a flame-ionization detector and a fused-silica capillary column (Omegawax; 30 m × 0.32 mm, i.d., film
thickness 0.25 μm , Supelco, PA, USA). Helium was used as the carrier gas. Temperature of injector was set
at 205˚C, and that of detector set at 240˚C. The temperature of oven was initially at 140˚C, raised to 205˚C at
5˚C/min and held for 10 min, then raised to 235˚C at 20˚C/min and held for 5 min. The peaks of fatty acids
were identified by comparing their retention times to those of known standard materials. The quantification
was determined by the technique of internal standardization.
RESULTS & DISCUSSION
Lipid production by oleaginous yeasts
M. eriocauli SJ3L01 and SJ4L09, Pseudozyma spp. FN20L03 and SJ4L03, and Cryptococcus sp. SM5S05
were selected from our lipid accumulating yeast pool to further determine whether these candidates could be
applied for future microbial oil production. Among strains tested, Cryptococcus sp. SM5S05 accumulated
highest levels of intracellular lipids no matter which modified yeast extract-peptone dextrose or sucrose
culture medium was utilized (Table 1). When lipid accumulation trends in Cryptococcus sp. SM5S05 treated
with different carbon sources were compared, the amounts of total lipids dropped 42% (from 258.8 to 150.8
mg) or 44% (223.5 to 124.6 mg) as levels of glucose or sucrose in the culture medium increased from 4% to
8%, respectively. Similar trends were also observed on M. eriocauli SJ3L01, indicating that both yeast strains
(SM5S05 and SJ3L01) were capable of assimilating and metabolizing dextrose and sucrose to synthesize
intracellular lipids. In contrast, Pseudozyma sp. FN20L03 only accumulated relatively high levels of lipids
(201.5 mg and 143.2 mg) when glucose appeared in the medium. The addition of sucrose significantly
reduced its oleaginous activity. The other two strains, Pseudozyma sp. SJ4L03 and M. eriocauli SJ4L09,
produced high amounts of lipids, but high level of sucrose (8%) in the culture medium might restrict lipid
accumulation. Since lipid production in Cryptococcus sp. SM5S05 was higher, this yeast strain was selected
to perform the following experiments.
Table 1. The effect of glucose or sucrose concentration on lipid content of various yeast strains
Concentration
Lipid content (mg/g dried biomass)
Carbon
source
(%)
FN20L03
SM5S05
SJ4L03
SJ4L09
Glucose
4
201.5± 6.3
258.8± 12.0
154.2± 3.0
142.7± 5.5
8
143.2± 5.5
150.8± 4.4
136.1± 2.9
100.8± 4.4
Sucrose
4
90.1± 4.0
223.5± 3.5
128.9± 2.2
124.5± 2.2
8
44.5± 0.9
124.6± 2.3
78.8± 3.0
48.6± 1.4
SJ3L01
117.3± 3.5
94.3± 3.1
100.5± 3.0
77.4± 1.8
The concentrations of lipid content are expressed as mean ± standard deviation of triplicate experiments.
Effect of glucose concentration and C/N ratio
In general, lipid production by yeast requires environment with excess quantities of carbon source (sugars or
other sugar-related compounds) and limited nitrogen source [8]. It is well known and studied that oleaginous
potential is critically affected by the C/N ratio of the culture and other factors like aeration, inorganic salt
presence, etc. [5]. Results in Table 2 also indicated that lipid accumulation in each strain decreased
noticeably as the carbon source increased. It suggested that 0.5% nitrogen source (yeast extract and peptone)
in the culture medium might be excessive for optimal lipid production by oleaginous yeast. Therefore, to
enhance higher lipid accumulation were to change C/N ratio; either increasing concentration of glucose or
reducing levels of nitrogen source. The results showed that higher concentrations (0.5% and 1.0%) of
nitrogen source in culture medium limited cell growth in comparison with the other two (0.1% and 0.2%),
and significantly reduced lipid production. Conversely, low nitrogen source (0.1%) in medium might not
significantly affect cell mass, but it restricted lipid accumulation. This observation did not dramatically
improved even by raising levels of glucose up to 10%, suggesting 0.1% nitrogen source could only maintain
yeast cellular metabolism. Furthermore, when the initial glucose concentration increased from 2% to 4% and
at a nitrogen concentration of 0.2%, the biomass and lipid content of dry weight increased from 7.9 and 133.4
mg/g to 10.1 and 600.4 mg/g, respectively. However, the biomass and lipid accumulation slightly dropped to
9.2 and 568.8 mg/g for cultures with an initial glucose concentration of 6%. These data were further reduced
to 8.2 and 549.7 mg/g, respectively for cultures with 8% glucose. When the substrate concentration reached
10%, biomass and lipid production were greatly decreased to 7.9 and 412.2 mg/g, respectively, suggesting
that a considerable inhibitory effect had occurred. High amount of lipids (around 60% of the dry biomass)
can be accumulated in the cultures of Cryptococcus sp. SM5S05. When this yeast strain was incubated with
the medium containing 0.2 % nitrogen source, amounts of cell biomass and accumulated lipids were the
highest as compared to the media with other proportions of nitrogen at the same level of glucose. Except for
the medium with 2% glucose in the medium, this formula might not meet the minimal requirement of carbon
source for yeast. Based on results in Table 2, we suggest a culture medium for mass production of lipids or
cell mass would contain 0.2% nitrogen source and 4% to 6% glucose.
Accumulation of lipids strongly depended on the C/N ratio and nitrogen concentration. The highest lipid
content was obtained at a C/N ratio of 60-90 and a nitrogen concentration of 0.2% (60-57% lipids of the dry
biomass), while at a higher C/N ratio of 150 only 41% were accumulated. However, there was a limit from
the critical effect of nitrogen concentrations on the lipid content of the dry biomass. No matter how the C/N
ratio was, the content of lipids of the dry weight significant decreased to less than 28% when the nitrogen
concentrations were higher or lower than 0.2%. Changing C/N ratio in the culture medium has been known to
affect lipid accumulation by oleaginous yeasts [9]. Usually, when cells in a high C/N ratio (> 50)
environment would deplete nitrogen source quickly, and excess nutrients continue to be taken up by the cells
and converted into triacylglycerols for storage. Upon nitrogen depletion, however, cell propagation could be
significantly depressed and resulted in the decrease in cell density. Thus, many attempts including various
substrates and culture modules have been taken to achieve a high-cell-density cultivation for lipid
accumulation [9, 10]. In the current study, we found that the culture of Cryptococcus sp. SM5S05 in a series
of modified nitrogen-limited medium could maintain both high cell biomass and lipid accumulation. Further
investigation is required to determine oleaginous potential of Cryptococcus sp. SM5S05 by modulating
different substrates and incubation parameters. Accumulation of lipids strongly depended on the C/N ratio
and nitrogen concentration. The highest content of lipids was measured at a C/N ratio of 60-90 and a nitrogen
concentration of 0.2% with 60-57% lipids of the dry biomass.
Table 2. Effect of concentration of glucose and C/N ratio on crude lipid production of Cryptococcus sp.
Concentration of
C/ N
Dried biomass (g/L)
Lipid content
Concentration of
carbon source (%)
nitrogen source*(%)
ratio
(mg/g dried biomass)
0.1
60
8.3 ± 0.3**
170.3 ± 10.1
0.2
30
7.9 ± 0.1
133.4 ± 14.2
2
0. 5
12
6.1 ± 0.6
121.5 ± 10.3
1.0
6
5.6 ± 0.1
113.5 ± 8.4
0.1
120
9.4 ± 0.3
280.9 ± 14.3
0.2
60
10.1 ± 0.2
600.4 ± 45.3
4
0. 5
24
7.8 ± 0.5
231.5 ± 12.1
1.0
12
7.5 ± 0.4
114.8 ± 10.3
0.1
180
8.4 ± 0.5
229.9 ± 20.4
0.2
90
9.2 ± 1.0
568.8 ± 33.5
6
0. 5
36
7.8 ± 0.2
313.1 ± 27.4
1.0
18
7.3 ± 0.1
231.5 ± 15.9
0.1
240
6.8 ± 0.3
281.9 ± 18.3
0.2
120
8.2 ± 0.2
549.7 ± 40.7
8
0. 5
48
5.2 ± 0.2
154.3 ± 6.3
1.0
24
5.3 ± 0.2
58.8 ± 8.6
0.1
300
6.7 ± 0.4
184.4 ± 16.8
0.2
150
7.9 ± 0.1
412.3 ± 19.8
10
0. 5
60
5.2 ± 0.1
116.0 ± 6.3
1.0
30
5.1 ± 0.1
38.1 ± 7.3
* The nitrogen source is supplied with yeast extract and peptone at the ratio of 1:1 into media.
** Data were expressed as the average of triplicates ± standard deviation.
Batch culture of cellulosic hydrolysate for the production of yeast lipids
Fig. 1 shows the cell biomass, pH values and glucose concentrations against fermentation time in the
cellulosic hydrolysate medium containing 4% of reducing sugar. The pH decreased rapidly from 6.0 to 3.2
after 6 d of cultivation. The cell biomass increased rapidly after 1 d from 0.8 g/L to 8.1 g/L after 7 d of
incubation in the corncob hydrolysate medium. In general, the cell biomass reached the maximal values and
showed no significant difference after 4 d. The reducing sugar was rapidly used by yeast and
decreased from 40 to 26 g/L within 2 d, where the cell biomass increased rapidly. However, 17 g/L
of reducing sugar were detected and could not be utilized by the yeast cells after 7 d of incubation.
The maximal concentrations of cell biomass, lipid and lipid content were 8.2 g/L, 4.7 g/L and 59.1%
(w/w), respectively (Table 3). Thus, the overall lipid yield and productivity were estimated to be 0.57 g/g and
0.67 g/L/d. In conclusion, these batch experiments demonstrated that Cryptococcus sp. SM5S05 had the
robust ability to grow in the media and reach very high cell density. The lipids from Cryptococcus sp.
contained mainly long-chain saturated and unsaturated fatty acids with 16 and 18 carbon atoms. The four
major constituent fatty acids were C18:1 (oleic acid), C16:0 (palmitic acid), C18:0 (stearic acid) and C18:2
(linoleic acid). The contents of oleic acid methyl ester and linoleic acid methyl ester were 58.9 % and 7.0 %,
respectively.
Fig. 1. Time course of biomass, pH and glucose changes of batch fermentation using the cellulosic hydrolysate. The
initial concentration of reducing sugar is 4%.
Table 3. Biomass, lipid and fatty acid composition of Cryptococcus sp. SM5S05 cells in glucose-based and corncob
hydrolysate-based media
Relative amount of total fatty acids (%, w/w)
Reducing
Sugar
(%, w/v)
4
6
Biomass
(g/L)
Lipid
(g/L)
Lipid
content
(%, w/w)
8.22
4.66
59.06
±0.11
±1.05
±12.90
12.57
±0.25
6.31
±0.23
50.20
±0.81
C14:0
C16:0
C18:0
C18:1
C18:2
C18:3
C20:0
C22:0
0.4
21.1
7.1
58.9
7.0
0.7
1.6
0.7
0.5
21.0
7.5
57.5
7.2
0.8
2.0
0.9
Some other fatty acids were also detected in trace amounts (less than 1%) and were not included in this table.
The results of cell biomass, pH values and glucose concentrations against fermentation time in the
cellulosic hydrolysate medium containing 6% of reducing sugar were showed in Fig. 2. The cell biomass
increased rapidly after 1 d from 0.9 g/L to 12.6 g/L after 7 d of incubation. The cell biomass reached the
maximal values and did not have significant difference after 4 d. Approximately, 26 g/L of reducing
sugar were detected and could not be utilized by the yeast cells after 7 d of incubation. The
maximal concentrations of cell biomass, lipid and lipid content were 12.6 g/L, 6.3 g/L and 50.2% (w/w),
respectively (Table 3). Thus, the overall lipid yield and productivity were estimated to be 0.50 g/g and 0.9
g/L/d. These data also show that the distribution of the major fatty acids, were almost constant in the yeast
lipid, no matter how the content of reducing sugar in the cellulosic hydrolysate media changed. In addition, it
is known that fatty acid distribution impacts on the cetane number (CN) of the biodiesel product. According
to an empirical equation [11], with the percentage distribution shown in Table 3, all the lipid samples could
produce biodiesel with CN values of 51.0. Minimal CN values have been set at 47, 49 and 51, by biodiesel
standards ASTMD 6751 (USA), DIN 51606 (Germany) and EN 14214 (European Organization), respectively.
Therefore, the FAMEs produced from the microbial lipids of Cryptococcus sp. SM5S05 met these standards
and could be manufactured into high quality biodiesel for practical uses.
Fig. 2. Time course of biomass, pH and glucose changes of batch fermentation using the cellulosic hydrolysate. The
initial concentration of reducing sugar is 6%.
CONCLUSION
The crude lipid contents of biomass could be increased with the addition of carbon source and adjusting the
C/N ratio of the culture media. The abundant unsaturated fatty acid contents in the crude lipid ensured the
high quality of biodiesel. The results obtained in this study were significant for industrial production of
biodiesel from yeast biomass.
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