Bioresource Technology 135 (2013) 304–308

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Cultivation and lipid production of yeast Cryptococcus curvatus

using pretreated waste active sludge supernatant
Yeong hwan Seo, Il gyu Lee, Jong in Han ⇑
Department of Civil and Environmental Engineering, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Oleaginous yeast Cryptococcus curvatus has become some of the most promising feedstock for biodiesel
Available online 17 October 2012 production due to their high production efficiency. However, high cost of cultivation, especially substrate
cost, hinders rapid commercialization of yeast-based biodiesel. In this study, waste activated sludge
Keywords: (WAS), which is rich in nutrients and organic matters, was examined as an economic substitute for
Biodiesel organic substances. To be efficiently bioavailable, WAS must be pretreated. Hydrodynamic cavitation
Cryptococcus curvatus reaction time and pH were identified as experimental factors, whereas growth rate was selected as
Sludge
response parameter. The experimental factors were optimized employing Response Surface Methodology
Hydrodynamic cavitation
Pretreatment
(RSM). Growth rate of 1.13/h was optimized at reaction time 12.5 min and pH 8.65. When sludge pre-
treated under these optimal conditions was used as a substitute to yeast extract, 9.84 g/L of biomass
was obtained in a day and lipid was accumulated up to 23% of dry weight.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction incomparably high productivity and increased final cell density

Biodiesel has drawn increasing attention as a renewable, biode-
gradable and environment friendly fuel. However, sources used for
the production of commercial biodiesel are mainly food based such
as soybean oil, rapeseed oil, palm oil, and corn oil. These food-
based biofuels have raised intense public concerns. For example,
if grain needed to fill a 25 gallon oil tank of sport utility vehicle
was instead consumed for food, it would provide one person with
enough calories for a year (Courtney, 2008).
Oil originated from microbes is considered as an ideal alterna-
tive for biodiesel production due to its high productivity (Chisti,
2007; Miao and Wu, 2006; Xu et al., 2006; Li et al., 2007). Ethical
controversies are not of an issue here, since microbes are not nor-
mally used for food. These oil-rich microbes, collectively called ole-
aginous microbes, include microalgae, yeasts, fungi, and even
bacteria. Among these microbes, oleaginous yeast Cryptococcus
curvatus has drawn noticeable attention, as it accumulates oil up
to 60% of cell dry weight (Ratledge, 1991). The composition of fatty
acid in this microbe is quite similar to that of plant seed oils such
as palm oil (Davies, 1988).
Oleaginous yeast utilizes organic carbon substances for their
growth, unlike photoautotrophic microalgae. However, the cost
of substrates accounts for up to 50% of the biodiesel production
(Li et al., 2007). Even with this cons, oleaginous yeast presents
distinct pros such as easy maintenance of growth conditions,
⇑ Corresponding author. Tel.: +82 42 350 3629; fax: +82 42 350 3610.
E-mail address: hanj2@kaist.ac.kr (J.in Han).
(Chen, 1999; Yee and Blanch, 1993; Shay et al., 1987; Chang
et al., 1993). Therefore, if the cost of substrates can be reduced to
economically sufficient level, the oleaginous yeast would become
a competitive route for the biofuel production.
Waste activated sludge (WAS) is a promising feedstock due to
high content of organic carbon and nutrients. However, WAS is
limited because of its low biodegradability, and hence limited bio-
availability. Nutrients in WAS are insoluble and are trapped in sus-
pended solids such as aggregates of organic matter, extracellular
polymers, cells and cellular debris or absorbed on their surfaces
(Pham et al., 2007). The proper disintegration of WAS is required
before its use as a substrate for the yeast growth.
Alkali treatment has been known to be relatively effective in
sludge solubilization, in the efficiency order of NaOH > KOH >
Mg(OH)2 and Ca(OH)2 (Kim et al., 2003). Physical disintegration
with ultrasonic waves can synergistically boost up the efficiency
of the alkaline pretreatment. However, issues of high energy con-
sumption and scaling-up make impractical for the purpose of com-
mercialization (Chakinala et al., 2008). Hydrodynamic cavitation
(HC), which employs similar working principles to the sonication,
is an excellent alternative, as it is much more energy-efficient
and can be easily scaled-up (Wang et al., 2009; Braeutigamm
et al., 2009). HC can simply generated by a constriction using an
orifice plate. Fluid passing through an orifice will experience a drop
in pressure. If a pressure falls below the threshold level for
cavitation, micro bubbles are generated. Subsequently, as a liquid
jet expands, pressure recovers and bubbles are collapsed. At the

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.10.024
 Y.hwan Seo et al. / Bioresource Technology 135 (2013) 304–308 305

point of collapse, temperature within micro bubble may be several

thousand kelvin and pressure is several atmospheres.
This paper focusses on finding the optimal condition for the
mass cultivation of C. curvatus using WAS as a cheap substrate.
To this end, sludge pretreatment was conducted, taking advantage
of HC as a physical means and NaOH as a chemical catalyst. Lipids
in C. curvatus can then be used for biodiesel production.

2. Methods

2.1. Yeast strain, media, and cultivation

C. curvatus was purchased from the Korean Collection for Type

Culture (KCTC) with depository number KCTC 17162. YM Agar Med-
ium (KCTC) was used for pre-culture and solid culture, and it con-
tained the following ingredients in 1 L of deionized water: 3 g of
yeast extract, 3 g of malt extract, 5 g of peptone, 10 g of dextrose,
in 20 g/L of agar. For the cell growth, Cryptococcus Growth
Medium (CGM) was mainly used, containing 40 g of glucose, 0.5 g
of NH4Cl, 2.7 g of KH2PO4, 0.95 g of Na2HPO4, 1 g of MgSO47H2O,
trace elements solution (DSMZ medium 320) 10 mL in 1 L of deion-
ized water. To confirm its known effect on boosting-up the micro-
bial growth, yeast extract was supplemented at different levels: 0,
0.1, 0.3, 0.5, 0.7, 1.0, and 1.2 g. Both YM and CGM media were auto-
claved. CGM media was cultivated in a shaking incubator (200 rpm). Fig. 1. Schematic diagram of hydrodynamic cavitation system.

2.2. Sludge pretreatment 2.4. Optimization of sludge disintegration

Sewage samples were obtained from the Sewage Treatment
Plant in Daejeon, Republic of Korea. The properties of the activated
sludge, whose approximate concentration was 1% (w/v), are listed in
Table 1.
Hydrodynamic cavitation (HC) system was employed to
mechanically disrupt cell structure and any extra materials limiting
bioavailability. A reactor for HC reactor was made of stainless steel
as Fig. 1. The reactor was connected to a water pump (TPH2TK6KS;
Walrus Pump (Taiwan) Ltd.). The maximum capacity of the system
was 1.5 L. The solution pH was adjusted between 8–10 using so-
dium hydroxide (5 M). After the HC-treated sludge was centrifuged
at 12000 rpm for 10 min, the supernatant was neutralized to pH 5.5,
filtered with 0.45 lm PES filter (Whatman), and thereafter used.
2.3. Sludge as substrates
Pretreated activated sludge supernatant was used for the
growth of C. curvatus. Two different experiments were performed
to see how the pretreated sludge affected the growth, i.e., either
as a substitute for yeast extract or as a nearly complete nutrient
source (i.e., carbon source). As a yeast extract substitute, the sludge
supernatant was mixed with CGM which contains glucose but not
yeast extract (1:4, v/v). As the complete nutrient source, the sludge
supernatant was mixed with CGM without glucose and yeast ex-
tract (1:1, v/v). Cultivation was done at 200 rpm and 30 °C.
Response surface methodology (RSM) was employed to obtain
the optimal pretreatment conditions that exhibited the shortest
growth rate (Y). Two factors were used: pH (A), and hydrodynamic
cavitation operating (HC) time (B). The pH level was varied be-
tween 8 and 10, whereas HC time was varied between 5 and
20 min. Total 13 experimental runs including five replicates of a
center point were executed. Data analysis was carried out via De-
sign Expert.
2.5. Measurement of disintegration degree of sludge and microbial
growth rate
The pretreated sludge supernatant was analyzed for the compo-
sition of soluble substances such as chemical oxygen demand
(COD), nitrogen (N), phosphorus (P), and protein. Protein concen-
tration was measured according to Lowry et al. (1951). Concentra-
tions of N, P, and COD were measured using a Hach DR 5000 UV–Vis
Laboratory Spectrophotometer (Hach, USA).
The growth of yeast was monitored by measuring absorbance at
600 nm. A calibration curve was plotted with the absorbance ver-
sus cell dry density. Dry cell was obtained using the following pro-
cess: (1) five milliliters of culture was centrifuged at 4000 rpm for
10 min. (2) The cells were washed twice with 5 mL of deionized
water. (3) And then dried to constant weight at 60–80 °C.

Table 1
Compositional change in activated sludge before and after being pretreated at pH 8.65 2.6. Lipid extraction and analysis
for 12.5 min using hydrodynamic cavitation.

Values (mg/L) Lipid contents and compositions were analyzed according to the
procedure described by Liang et al. (2010) with slight modification.
Before pretreatment After pretreatment
The cells were harvested by centrifugation (12000 rpm, 10 min),
T-COD 10110 2143 washed twice with deionized water, oven-dried at 50 °C for
S-COD 15 2143
T-Protein 93 21
1 day, and then disrupted in 15 mL falcon tubes with a bead-beater
S-Protein 11 21 (BioSpec Products). Bead-beating was conducted for 2 min with
T-N 650 135 1 mm glass beads to approximately 5 mL and 2 mL of methanol.
S-N 5 135 The entire content was then transferred to a 50 mL centrifuge tube
T-P 287 61
and chloroform added to make a 2:1 (v/v) chloroform/methanol ra-
S-P 6 61
tio. The tube was vortexed for 5 min and was allowed to stand for
306 Y.hwan Seo et al. / Bioresource Technology 135 (2013) 304–308

24 h. Afterwards, the tube was centrifuged (12,000 rpm, 15 min) to
remove yeast residues. The supernatant was collected, and the sol-
vent was vaporized.
Fatty acid methyl esters (FAMEs) were produced from lipid
through transmethylation overnight at 50 °C as described by
Christie (1982). After the reaction, 1 mL of deionized water was
added, and organic phase was separated from water phase by
centrifugation at 4000 rpm for 10 min. FAMEs in organic phase
were analyzed by gas chromatography (HP5890, Agilent, USA) with
a flame ionized detector (FID) and INNOWAX capillary column
(Agilent, USA, 30 m  0.32 mm  0.5 lm).
3. Results and discussion
3.1. Optimization of growth condition of C. curvatus
The growth characteristics of C. curvatus were examined with
glucose as a sole carbon and energy source under several temper-
ature conditions. The culture temperature had major effect on
growth rate. There is an optimal temperature and the optimal
growth rate (0.33/h) was found at 30 °C.
To enhance the growth rate, the effect of yeast extract as a cell
growth promoter was investigated. As expected, yeast extract
substantially accelerated the growth with a reduced tendency of
growth-boosting effect at high concentration. 1 g/L of yeast
extract turned out to be optimal, supporting only growth rate of
0.693/h.
3.2. Sludge as a substitute of yeast extract
The optimization of sludge pretreatment parameters, such as
pH and HC operating time, was made during the cultivation of C.
curvatus using WAS as a substitute for yeast extract. This was sys-
tematically done with Central Composite Design (CCD) using Re-
sponse Surface Methodology (RSM). CCD based on the prediction
of growth rate with different combinations of two independent
variables and their experimental data is summarized in Table 2.
The growth rate of 0.982 ± 0.09/h was obtained at the center point
condition of pH 9 at 12.5 min. The optimization of the pretreat-
ment conditions was obtained via CCD and the second-order poly-
nomial equation that describes the growth rate in terms of actual
parameters is shown in Eq. (1)
Growth rate ¼ 16:98 þ 3:747  pH þ 0:300  Time
 0:027  pH  Time  0:197  pH2  2:648
2
 103  Time
This result clearly indicates that the experimental values were
distributed near to a straight line and showed high correlation
(R2 > 0.90) with the predicted values.
To examine the adequacy of the developed model, Analysis Of
Variance (ANOVA) for the quadratic model was carried out and re-
sults are presented in Table 3. The model is regarded significant if
p-value (also known as the Prob > F; value) is lower than 0.05, indi-
cating that a ‘Model F-value’ could occur because of noise with only
a 0.07% chance (Tan et al., 2011). The model F-value of 17.87 with a
low p-value (p < 0.05) implies that this model was statistically sig-
nificant at 95% confidence level. In addition, the model terms A, B,
AB, A2 and B2 had significant effect on the growth rate, since each
p-value was less than 0.05.
The experimental results were visualized in three dimensional
response surface plots indicating the correlations. As this response
surface plot was found to have a peak, the highest growth rate
could be obtained near at the center point. The interactions of alka-
li concentration and time for growth rate are depicted in Fig. 2. An
optimal pretreatment condition, which supported the highest
growth rate, was at pH 8.65 and for 12.5 min, which resulted in a
predicted growth rate of 1.11/h. To confirm the result of the pre-
dicted optimal value, experiments were conducted at the optimal
conditions, displaying growth rate of 1.13/h. From the ANOVA test,
pH was more significant (0.0028 < 0.0193) than reaction time, indi-
cating that alkali concentration had a strong influence on the
growth rate. Growth rate increased with increase in pH and time,
but it decreased with further increase.
After being pretreated at the optimal condition (i.e., pH 8.65 and
reaction time of 12.5 min), the composition of the sludge superna-
tant was analyzed. Around 20% insoluble components were found
to become solubilized (Table 1).
3.3. Cell biomass and lipid accumulation by C. curvatus cultivation on
pretreated sludge
Even though the pretreated sludge supernatant could be used as
a complete nutrient source, i.e., a carbon source, the growth was
suboptimal: final optical densities at 600 nm reached only 0.3–
0.5 and maximum lipid contents were 10%. One obvious reason
for such low final cell concentration was that the absolute amount
of sludge in the medium was too low. This limitation was resolved
by the subsequent addition of sludge (Fig. 3.). The sludge
pretreated at the optimal condition of pH 8.65 and treatment time
of 12.5 min was used for microbial growth. A possible reason for
low lipid content would be an inherently low initial C/N ratio of
the sludge, which plays a key role in lipid accumulation. The effect
ð1Þ of C/N ratio on lipid accumulation is well-known and has already
been documented in the context of metabolic pathway (Ratledge,

Table 2
The Central-Composite Design data and corresponding Growth rate with different combinations of two independent variables when pretreated sludge was used for substitute of
yeast extract.

Run Coded variable level Experimental factors Growth rate (/h)

A B pH Time (min) Experimental Theoritical
1 0 0 9 12.5 0.939 1.084
2 1 1 8 5 0.653 0.741
3 1.414 0 7.585 12.5 0.770 0.883
4 0 0 9 12.5 1.034 1.084
5 1 1 10 20 0.226 0.330
6 1 1 10 5 0.729 0.873
7 0 0 9 12.5 0.884 1.084
8 -1 1 8 20 0.977 1.008
9 0 0 9 12.5 1.117 1.084
10 0 0 9 12.5 0.936 1.084
11 1.414 0 10.41 12.5 0.382 0.500
12 0 1.414 9 23.1 0.447 0.689
13 0 0.1414 9 1.893 0.900 0.884
 Y.hwan Seo et al. / Bioresource Technology 135 (2013) 304–308 307

Table 3
ANOVA for the quadratic model when pretreated sludge was used for substitute of yeast extract.

Source Sum of squares df Mean squares F value p-value Prob > F

Model 0.82 5 0.16 17.87 0.0007 Significant
A-pH 0.19 1 0.19 20.35 0.0028
B-Time 0.084 1 0.084 9.15 0.0193
AB 0.17 1 0.17 18.61 0.0035
A2 0.27 1 0.27 29.55 0.0010
B2 0.15 1 0.15 16.80 0.0046
Residual 0.064 7 9.186  103
Lack of fit 0.030 3 9.862  103 1.14 0.4351 Not significant
Pure error 0.035 4 8.679  103
Cor total 0.89 12

Fig. 2. Contour plot (a) and Response surface plot (b) of the combined effects of pH and hydrodynamic cavitation operating time on growth rate when pretreated sludge
supernatant was used for substitute of yeast extract.

10
cycle is then excreted and involves the lipid accumulation. Gener-
ally, oleaginous yeast cannot accumulate a high content of lipid
with a C/N ratio less than 20/1, whereas a C/N ratio from 40/1 to
80/1 appears to be well suited for lipid production in most oleagi-
8
nous microbes (Moreton, 1988).
When sludge supernatant was used as yeast extract substitute,
Dry cell weight (g/L)

cell concentration was approximately 9.84 g/L and lipid content

6
roughly 23% after 24 h of cultivation (Fig. 3.). A variety of sub-
strates have been used in the cultivation of C. curvatus (Table 4).
Evans and Ratledge (1983) studied the growth of Crytococcus on
4
glucose, sucrose, lactose, ethanol, and xylose, and obtained bio-
Sludge mass of around 3 g/L from all substrates. A higher biomass produc-
tion rate of 6.57 g/L/day was reported by Zhang et al. (2011). They
2 Sludge performed screening experiments among Rhodotorula glutinis,
Rhodococcus opacus and C. curvatus using N-acetylglucosamine
(GlCNAc) as a sole carbon source. Meesters et al. (1996) obtained
0
cell densities of up to 21 g/L and lipid production of 2.3 g/L on
0 5 10 15 20 25
the first day using glycerol and fed-batch cultivation. In this study
Time (h)
using the sludge, the absolute value of biomass productivity of
Fig. 3. Growth curve of C. curvatus when pretreated sludge supernatant was 9.84 g/L/day was not as high as Meesters et al.’s results, probably
substituted for organic matters. Sludge pretreated on pH 8.65, operating time because of the different cultivation approach, but higher than most
12.5min (Optimal condition) was used. -- : Sludge was used as yeast extract others’. Fed batch system has some distinct advantages such as
substitute, -s-: Sludge was used as glucose substitute, -4-: CGM without yeast
constant substrate concentration (Rao, 2010).
extract, -.-: CGM with 1g/L of yeast extract.
As shown in Table 5, the composition of fatty acid in Cryptococcus
oil is similar to oil from Jatropha which is currently used as a
2002; Rossi et al., 2011). In oleaginous yeasts, nitrogen limitation commercial feedstock in biodiesel production. The yeast oil is actu-
activates AMP-deaminase (Ratledge and Wynn, 2002), which ally better in terms of saturation degrees of fatty acids (FAs): 25% of
serves to supply ammonium to nitrogen deficient cells. This reac- palmitic acid versus 14.66% in Jatropha, and 11.21% of stearic acid
tion causes to decrease mitochondrial AMP concentration, blocking versus 6.86% in Jatropha (Thiru et al., 2011). Compared to microalgal
the TCA cycle. Excess citrate accumulated in the interrupted TCA lipids in which only 22% is saturated fatty acids, Cryptococcus oil
308 Y.hwan Seo et al. / Bioresource Technology 135 (2013) 304–308

Table 4
Lipid production rates of Cryptococcus curvatus on various substrates.

Substrate Biomass productivity (g/L/day) Lipid content (%) Lipid productivity (g/L/day) Reference
Glucose 2.72 33.2 0.90 Evans and Ratledge (1983)
Sucrose 2.98 37.4 1.11
Lactose 3.33 39.2 1.30
Xylose 2.64 48.6 1.28
Ethanol 2.26 30.1 0.68
N-acetylglucosamine 6.57 17.3 1.14 Zhang et al. (2011)
Glycerol 21 10.9 2.3 Meesters et al. (1996)
Sludge 9.84 23.0 2.26 This study

Table 5
Comparison of fatty acid profile of oil from Cryptococcus with Jatropha oil when the
pretreated sludge supernatant was used as a yeast extract substitute.
Type of fatty acid Relative amount of total fatty acids (% w/w)
Jatropha oil Cryptococcus oil
C 16:0 (Palmitic acid) 14.66 25.81
C 16:1 (Palmitoleic acid) 0.94 1.32
C 18:0 (Stearic acid) 6.86 11.21
C 18:1 (Oleic acid) 39.08 49.21
contains about 37% of saturated FAs (Chinnasamy et al., 2010). High
saturation indicates an excellent cetane number and oxidative sta-
bility of biodiesel. Biodiesel quality is directly associated with a ce-
tane number which is a measure of ignition delay in engine (Van
Gerpen, 2009). However, highly saturated fatty acid can suffer from
poor cold properties (Joshi et al., 2011). Therefore, mono-unsatu-
rated fatty acids are known as the best balance between cold flow
properties and oxidative stability to be a better biodiesel (Fallen,
2009). From this point of view, 50% of mono-unsaturated fatty acids
found in the C. curvatus’ oil are nearly ideal source for biodiesel.
4. Conclusion
C. curvatus can grow on pretreated sludge, at the level compara-
ble to yeast extract. The combination of hydrodynamic cavitation
and alkaline treatment was found to be an effective pretreatment
method for sludge. Lipid from Cryptococcus is similar to Jatropha
oil which has been established for biodiesel production. Compared
to Jatropha and algal oil, yeast oil shows more saturation number
which is related to biodiesel quality. Yeast, which can grow rapidly
without any environmental or ethical issues, can be an excellent
alternative source for the biodiesel production, provided that var-
ious cheap feedstock such as activated sludge is used.
Acknowledgements
This work was financially supported by the Advanced Biomass
R&D Center (ABC) of Korea Grant (2011-0031348) funded by the
Ministry of Science, Korea Minister of Ministry of Land, Transport
and Maritime Affairs (MLTM) as ‘‘U-city Master and Doctor Course
Grant Program’’ and Technology of Korean government of the
Republic of Korea.
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