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Process Biochemistry 42 (2007) 210–214

Changes of lipid content and fatty acid composition of Schizochytrium

limacinum in response to different temperatures and salinities

Luying Zhu a,b, Xuecheng Zhang a,*, Lei Ji a,c, Xiaojin Song a, Chenghong Kuang a

a College of Marine Life Sciences, Ocean University of China, No. 5 Yushan Road, Qingdao, Shandong 266003, PR Chinab College of Life Sciences, Ludong University, Yantai, Shandong 264025, PR China

c Chemicals and Mineral Laboratory, China Entry/Exit Inspection and Quarantine Bureau, Qingdao, Shandong 266002, PR China

Received 19 March 2006; received in revised form 9 July 2006; accepted 6 August 2006

Abstract

The growth, lipid content and fatty acid composition of Schizochytrium limacinum OUC88 at different temperatures (16, 23, 30 and 37 8C) and

salinities (0, 0.9, 1.8, 2.7 and 3.6%, w/v) were analyzed. The strain grew better and lipid contents were higher at 16–30 8C and salinity at 0.9–3.6%

(w/v). The adaptive responses of this microbe to temperature and salinity were mainly to regulate the degree of fatty acid unstauration to maintain

the normal membrane lipid physical state. However, at 37 8C and 0 salinity, the growth of the strain was inhibited obviously and the lipid content

reduced significantly and, some important changes occurred in fatty acid composition, especially the odd-numbered fatty acids 15:0 and 17:0

which amounts increased greatly. In addition, the ratio of DHA to DPA changed at different temperatures and salinities.

# 2006 Published by Elsevier Ltd.

Keywords: Fatty acid compositions; Lipid content; Salinity; Temperature; Schizochytrium limacinum

1. Introduction

Lipids in microbial cells play various biological roles and,

consequently, lots of research has been done on lipids and their

role in cell physiology. The lipid composition of microorgan-

isms can exhibit considerable variations with a changing

environment [1–3]. These changes in microbial lipid composi-

tion may result in alteration in membranes physical character-

istics which enable microbes to maintain membrane fluidity,

integrity and functionality in the face of environmental

fluctuations [4]. Temperature is one of the most important

environmental factors that affect all aspects of the growth and

development of living organisms, and affect significantly the

fatty acid composition of most microorganisms [2,3,5].

Additionally salinity also affects the fatty acid compositions

of many microorganisms [2,3].

Schizochytrium sp., a traustrochytrid, is a heterotrophic

marine fungal. This microbe contains large amounts of DHA

(docosahexaenoic acid, C22:6 n � 3) which can help improve

human health [6,7] and, it has attracted increasing interest from

* Corresponding author. Tel.: +86 532 82032789; fax: +86 532 82032017.

E-mail address: xczhang@ouc.edu.cn (X. Zhang).

1359-5113/$ – see front matter # 2006 Published by Elsevier Ltd.

doi:10.1016/j.procbio.2006.08.002

researchers [8–12]. To further understand the physiological and

biochemical characteristics of this marine fungal, it is necessary

to analyze cellular fatty acids’ behaviors under different

cultivating conditions. Although the fatty acid compositions of

Schizochytrium at different developmental stages and nutrient

levels have been reported recently [11,13], the fatty acid profile

at different temperatures and salinities has not been studied. In

this work, we investigated the effects of cultivating tempera-

tures and salinities on the growth, lipid content and the fatty

acid composition of Schizochytrium limacinum OUC88. The

goal is to analyze how different environmental factors will

affect the organism and the resulting changes in fatty acid

composition.

2. Materials and methods

2.1. Cultures and culture conditions

The S. limacinum OUC88 used in this study was UV-induced mutant of S.

limacinum SR21 provided by the Institute for Fermentation Osaka (Japan) (IFO

number is 32693). The culture was maintained on GSA slant (20 g/l glucose,

10 g/l soybean cake hydrolysate [14], 50% (v/v) natural seawater (the normal

salt concentration of nature seawater in this region is 3.6% (w/v)) and 2.0% (w/

v) agar) at 12 8C and inoculated monthly. GS (60 g/l glucose, 40 g/l soybean

cake hydrolysate, 50% (v/v) nature seawater and pH 7.0) medium was used as

L. Zhu et al. / Process Biochemistry 42 (2007) 210–214 211

Fig. 1. Effect of temperature on the growth and lipids content of Schizochytrium

limacinum OUC88. (Data were means � S.D. of three replicates.)

the basal medium. Seed cultures were grown in flasks containing GS medium at

23 8C with shaking rotationally (200 rpm) for 2 days.

2.2. Effect of temperature

Cultures were grown in flask containing 50 ml GS medium at 16, 23, 30 and

37 8C, respectively, shaking rotationally (200 rpm) for 5 days. After the cultures

were harvested, biomass, total lipid and fatty acid composition were analyzed.

Each treatment was repeated three times.

2.3. Effect of salinity

Cultures were grown in 50 ml GS medium contained different salt con-

centrations (0, 0.9, 1.8, 2.7 and 3.6% (w/v)) by adjusting the concentration of

seawater in culture medium to 0, 25, 50, 75 and 100% (v/v). Cultures were

harvested after growing at 23 8C with shaking rotationally (200 rpm) for 5 days

and analyzed for biomass, total lipid and fatty acid composition. Each treatment

was repeated three times.

2.4. Biomass determination and lipid extraction

The cell biomass was determined by centrifuging the cell suspension,

washing twice with distilled water and freeze-drying. Total lipid was extracted

with chloroform: methanol (2:1) for 1 h. The extracted lipid was centrifuged to

obtain a clear supernatant and anhydrous sodium sulphate was added to remove

any residual moisture. The solvent was removed by flushing with nitrogen and

the total lipid estimated by a gravimetric method [15].

2.5. Fatty acid analyses

The dried cells were suspended in 0.4 M methanolic KOH at 60 8C for 1 h,

and fatty acids were esterified at 60 8C for 1 h in BF3/methanol (14%, w/w)

reagent. The esterified fatty acids were extracted with n-hexane and then

analyzed by Agilent 6890 GC equipped with a FID and a DB-23 capillary

column (30 m � 0.25 mm). Nitrogen was used as carrier gas. Initial column

temperature was set at 170 8C for 1 min, then raised to 230 8C at 15 8C/min and

Table 1

Fatty acid composition (% total fatty acids) of S. limacinum OUC88 cultured at d

Fatty acid Temperature (8C)

16 23

12:0 0.20 � 0.02 0.2

14:0 8.95 � 0.16 8.4

15:0 2.19 � 0.05 2.2

16:0 38.01 � 1.22 39.1

17:0 0.85 � 0.03 0.8

18:0 1.47 � 0.04 1.6

18:2 n � 6 0.23 � 0.01 0.2

18:3 n � 3 0.59 � 0.02 0.6

18:3 n � 6 0.23 � 0.02 0.2

20:0 0.52 � 0.02 0.5

21:0 0.33 � 0.03 0.3

20:3 n � 6 0.33 � 0.02 0.3

20:4 n � 6 0.49 � 0.03 0.4

22:0 0.39 � 0.02 0.4

20:5 n � 3 0.72 � 0.04 0.7

22:5 n � 6 6.74 � 0.22 7.5

22:6 n � 3 37.63 � 1.25 36.1

Saturated 52.91 � 1.17 53.8

Unsaturated 46.96 � 0.78 46.3

Odd 3.37 � 0.25 3.4

Even 96.50 � 1.54 96.7

22:6 n � 3/22:5 n � 6 5.58 � 0.07 4.8

Data were means � S.D. of three replicates.

maintained 23 min. The injector was kept at 250 8C with an injection volume of

1 ml under splitless mode. The FID detector temperature was set at 260 8C. Fatty

acid methyl esters (FAMEs) were identified by comparison with the retention

time of authentic standards (Sigma Co., USA). The quantities of individual

FAMEs were estimated from the peak areas on the chromatogram using

nonadecanoic acid (19:0) (Sigma Co., USA) as an internal standard.

3. Results

3.1. Effects of the temperature

Results obtained for S. limacinum OUC88 growth and lipids

content at different temperatures are shown in Fig. 1. When

growing at 16–30 8C cultures grew better, lipid contents were

higher and there were only slight fluctuations in biomass and

lipids content with the changing temperature. However, when

the temperature was raised to 37 8C, cells hardly grew, and

ifferent temperature

30 37

2 � 0.02 0.26 � 0.02 0.42 � 0.03

1 � 0.23 8.35 � 0.12 6.76 � 0.25

1 � 0.13 2.23 � 0.07 10.30 � 0.27

4 � 1.74 41.58 � 0.86 32.63 � 0.55

8 � 0.03 0.93 � 0.05 4.48 � 0.22

8 � 0.10 1.93 � 0.07 2.06 � 0.18

6 � 0.02 0.30 � 0.02 0.55 � 0.04

4 � 0.03 0.78 � 0.03 1.76 � 0.07

3 � 0.02 0.26 � 0.03 0.42 � 0.02

5 � 0.03 0.63 � 0.05 0.92 � 0.03

4 � 0.02 0.36 � 0.02 0.61 � 0.03

5 � 0.02 0.41 � 0.04 0.73 � 0.04

8 � 0.02 0.49 � 0.04 0.85 � 0.03

2 � 0.03 0.48 � 0.03 0.73 � 0.03

2 � 0.04 0.87 � 0.03 1.24 � 0.06

1 � 0.40 7.76 � 0.18 10.32 � 0.77

6 � 0.87 32.12 � 1.64 24.83 � 0.80

5 � 0.76 56.75 � 0.89 58.91 � 0.75

5 � 0.58 42.99 � 0.47 40.68 � 1.19

3 � 0.16 3.52 � 0.21 15.39 � 0.84

7 � 1.17 96.22 � 0.98 84.20 � 1.24

1 � 0.23 4.14 � 0.10 2.41 � 0.05

L. Zhu et al. / Process Biochemistry 42 (2007) 210–214212

Fig. 2. Effect of salinity on the growth and lipids content of S. limacinum

OUC88. (Data were means � S.D. of three replicates.)

biomass decreased from 22.15 g/l at 30 8C to 8.53 g/l, and

lipids contents also lowered significantly from 39.20% at 30 8Cto 24.08% at 37 8C.

The fatty acid composition of the cultures showed some

variations when the temperature changed (Table 1). The amount

of 16:0, the major saturated fatty acid (SFA), increased from

38.01 to 41.58% of the total fatty acids when the temperature

increased from 16 to 30 8C, and fell to 32.63% at 37 8C. 14:0

showed decreased contents while 18:0 showed increased

contents with the temperature increasing from 16 to 37 8C. A

similar changing pattern was observed in the two odd-

numbered fatty acids 15:0 and 17:0 at different temperatures,

the contents of which did not change significantly as

temperature raised from 16 to 30 8C, however, when

temperature raised to 37 8C, they increased up to 4.62- and

4.82-times respectively of those at 30 8C. In addition, the traced

Table 2

Fatty acid composition (% total fatty acids) of S. limacinum OUC88 cultured at d

Fatty acid Salinity (% w/v)

0 0.9

12:0 0.30 � 0.01 0.22 � 0.01

14:0 3.86 � 0.08 7.62 � 0.12

15:0 10.16 � 0.21 2.86 � 0.13

16:0 32.62 � 1.16 38.09 � 0.88

17:0 4.59 � 0.52 1.17 � 0.22

18:0 2.12 � 0.27 1.90 � 0.16

18:2 n � 6 0.41 � 0.04 0.25 � 0.01

18:3 n � 3 0.98 � 0.03 0.65 � 0.02

18:3 n � 6 0.34 � 0.02 0.25 � 0.02

20:0 0.85 � 0.07 0.52 � 0.06

21:0 0.47 � 0.03 0.31 � 0.02

20:3 n � 6 0.47 � 0.04 0.34 � 0.03

20:4 n � 6 0.55 � 0.03 0.40 � 0.02

22:0 0.60 � 0.03 0.42 � 0.03

20:5 n � 3 0.92 � 0.12 0.71 � 0.05

22:5 n � 6 8.27 � 0.18 7.63 � 0.27

22:6 n � 3 31.97 � 1.28 36.65 � 1.06

Saturated 55.57 � 1.53 53.11 � 1.14

Unsaturated 43.91 � 0.88 46.88 � 0.56

Odd 15.22 � 0.25 4.34 � 0.08

Even 84.26 � 1.27 95.65 � 1.11

22:6 n � 3/22:5 n � 6 3.87 � 0.11 4.80 � 0.14

Data were means � S.D. of three replicates.

SFAs including 12:0, 20:0, 21:0 and 22:0 all increased slightly

when the temperature increased from 16 to 30 8C, and then at

37 8C they increased to 1.62-, 1.46, 1.69- and 1.52-times of

their quantities at 30 8C.

The change of temperature also affected unsaturated fatty

acids (UFAs) in the cell. The amount of 22:6 n � 3, the main

UFA, decreased from 37.63 to 24.83% and the amount of 22:5

n � 6 increased from 6.74 to 10.32% when the temperature

changed from 16 to 37 8C. These changes resulted in a decrease

of the 22:6 n � 3 to 22:5 n � 6 ratio from 5.58 at 16 8C to 2.41

at 37 8C. Additionally, the other UFAs exhibited activities

similar to those observed in the traced SFAs with the changing

temperature.

Those changes as described above resulted in an increase of

total SFA and a decrease of total UFA when the temperature

increased from 16 to 37 8C. Therefore, the ratio of SFA/UFA

increased from 1.13 at 16 8C to 1.45 at 37 8C. In addition, a

4.37-times increase was observed for the odd-numbered fatty

acids caused by the significant increases of 15:0 and 17:0 when

temperature changed from 30 to 37 8C.

3.2. Effect of salinity

The growing process and the lipids content of the cultures

also showed some differences at different salt concentrations

(Fig. 2). The strain grew better and biomass remained steady

(average 24.51 g/l) with salinity at 1.8–3.6% (w/v). When

salinity decreased from 0.9% (w/v) to 0, the growth of the cell

was inhibited obviously and the biomass lowered significantly

from 18.85 to 7.86 g/l. Lipids content increased from 41.34 to

48.97% with the decreased salt concentration from 3.6 to 0.9%

ifferent salinity

1.8 2.8 3.6

0.23 � 0.02 0.24 � 0.02 0.27 � 0.02

8.34 � 0.15 8.95 � 0.09 9.66 � 0.13

2.10 � 0.08 2.20 � 0.04 2.17 � 0.07

39.79 � 0.53 41.53 � 1.04 43.38 � 0.75

0.90 � 0.18 0.95 � 0.09 0.99 � 0.05

1.91 � 0.25 1.93 � 0.08 1.95 � 0.12

0.26 � 0.02 0.24 � 0.02 0.27 � 0.01

0.71 � 0.03 0.61 � 0.04 0.69 � 0.04

0.26 � 0.03 0.24 � 0.02 0.27 � 0.02

0.55 � 0.04 0.58 � 0.04 0.61 � 0.05

0.32 � 0.03 0.34 � 0.04 0.38 � 0.02

0.35 � 0.03 0.35 � 0.02 0.38 � 0.03

0.42 � 0.03 0.44 � 0.03 0.46 � 0.04

0.42 � 0.02 0.44 � 0.04 0.50 � 0.03

0.73 � 0.04 0.76 � 0.04 0.78 � 0.06

7.56 � 0.18 7.46 � 0.25 7.19 � 0.26

35.12 � 0.88 32.73 � 0.47 29.85 � 0.53

54.55 � 0.79 57.16 � 1.42 59.91 � 0.72

45.41 � 0.47 42.83 � 0.87 39.89 � 0.24

3.32 � 0.12 3.49 � 0.15 3.54 � 0.09

96.64 � 1.02 96.50 � 0.27 96.26 � 0.52

4.65 � 0.19 4.39 � 0.10 4.15 � 0.08

L. Zhu et al. / Process Biochemistry 42 (2007) 210–214 213

(w/v). However, when salinity decreased from 0.9% (w/v) to 0,

lipids content lowered from 48.97 to 30.55%.

The variation of salinity also induced many changes of

cellular fatty acids of S. limacinum as shown in Table 2. 16:0

decreased from 43.38 to 38.05% when salinity decreased from

3.6 to 0.9% (w/v), and then decreased to 32.62% at 0 salinity.

And 14:0 showed behaviors similar to 16:0 with various salinity

except that 14:0 decreased by 1.97-fold when salinity decreased

from 0.9% (w/v) to 0. In contrast, the amounts of the odd-

numbered fatty acids 15:0 and 17:0 only showed slight changes

with the changing salinity from 3.6 to 0.9% (w/v), and then

increased by 3.55- and 3.92-times, respectively, when the

salinity decreased from 0.9% (w/v) to 0. In addition, the

contents of 18:0 and these traced SFAs including 12:0, 20:0,

21:0 and 22:0 all exhibited similar changes which decreased

slightly with the lowered salinity from 3.6 to 0.9% (w/v) and

then raised by 1.12-, 1.36-, 1.63-, 1.52- and 1.43-times,

respectively, when salinity lowered from 0.9% (w/v) to 0.

Some differences were also observed in UFA at various

salinities. 22:6 n � 3 increased from 29.85 to 36.65% when

salinity decreased from 3.6% (w/v) to 0.9% (w/v), and then

down to 31.97% when salinity was 0. An increased trend was

observed in 22:5 n � 6 with the decreased salinity. Thus, the

ratio of 22:6 n � 3/22:5 n � 6 increased from 4.15 to 4.80 when

salinity decreased from 3.6% (w/v) to 0.9% (w/v), and then

down to 3.87 at salinity of 0. Additionally, the changing pattern

of the traced UFAs at different salinities was similar to those of

the traced SFAs.

As a result, the amount of total SFA decreased from 59.91 to

53.11% when salinity decreased from 3.6% (w/v) to 0.9% (w/

v), and increased to 55.57% at 0 salinity, however, the total UFA

changed at the opposite direction. And a 3.51-times increase

was observed for the odd-numbered fatty acids when salinity

decreased from 0.9% (w/v) to 0.

4. Discussions

S. limacinum is a genus of marine fungi isolated from the

coastal seawater in the west Pacific Ocean. Yokochi reported

that temperature at 20–30 8C and salinity at 50–200% of

seawater were appropriate for the growth of S. limacinum SR21

[13]. Similar results were obtained in this study. When cultured

at 16–30 8C and 0.9–3.6% salinity (w/v), the strain grew better,

lipids content was higher and fatty acid composition kept

relatively stable. However, when the temperature increased

from 30 to 37 8C, the biomass and lipids content of the cultures

reduced significantly by 2.60- and 1.63-fold; the percentages of

most fatty acids increased significantly, especially 15:0 and

17:0 which exhibited notable increases by 4.62- and 4.82-times,

respectively, while the amounts of 14:0, 16:0 and 22:6 n � 3

decreased significantly. And similar changes were also

observed in this strain when salinity of the culture medium

fell from 0.9% (w/v) to 0. The decreased cell growth at 37 8Cand 0 salinity indicates that S. limacinum cells encounter

stressed growth conditions, and these significant changes in

fatty acid composition is implicated in tolerance to temperature

and salt stress. In general, the growth and metabolite of

microorganism are inhibited under conditional stress and some

changes in lipid composition occur to enable microbes survive

poor environments [4]. Previously reported strategies for

adaptive response of microbes in terms of fatty acid changes

include: (1) increasing the degree of fatty acid unstauration, (2)

shortening fatty acid chain length, and (3) increasing the

proportion of branched fatty acids [5,16]. These changes in

lipid composition are associated with maintaining the

membrane fluidity since unsaturated, shortened and branched

fatty acids have lower melting points than saturated straight

long-chain fatty acids [17]. An increase in odd-numbered fatty

acid content under conditional stress has not been reported

previously. Though we did not determine if 15:0 and 17:0 are in

the membranes, considering the unusual behaviors of 15:0 and

17:0 under the environment of high temperature and low

salinity, it is presumed that the two odd-numbered fatty acids

may play important roles in the tolerance of S. limacinum cells

to temperature stress, as well as to salt stress.

The temperature affects the degree of fatty acid unsaturation

of S. limacinum. The proportion of UFA in total fatty acid fell

from 46.96 to 40.68% when temperature increased from 16 to

37 8C. These results were in accord with previous reports

[2,17–19]. In addition, as previous studies have shown

[2,18,19], the degree of fatty acid unsaturation of S. limacinum

cell was also affected by salinity which showed a decreased

tendency in response to increased salinity with an exception

when salinity was 0. The variation of fatty acid unsaturated

degree was regarded as an alternate response to provide an

appropriate degree of membrane fluidity for growth of microbe.

The fatty acid of S. limacinum OUC88 contains a high

proportion of polyunsaturated fatty acids as reported previously

[10,13]. However, our study also reached some different results.

Previous fatty acid study of Schizochytrium sp. analyzed major

fatty acids including 16:0, 22:6 n � 3, 20:5 n � 6 and 14:0 [10]

or 15:0 [13]. However, the main fatty acids of S. limacinum

OUC88 in our study were in order 16:0, 22:6 n � 3, 14:0 and

20:5 n � 6. In addition, traces of 18:2 n � 6, 18:3 n � 6, 20:0,

21:0 and 22:0 were also detected. Those variations are most

likely caused by different growing conditions although

different species and strain may be a reason too.

The synthesis of 22:6 n � 3 in Schizochytrium have attracted

interests from some researchers. It was reported that unlike the

discovered way of PUFA synthesis which required desaturation

and elongation of saturated fatty acids, the synthesis of 22:6

n � 3 in Schizochytrium were catalyzed by a novel polyketide

synthase [20]. The behavior of 22:6 n � 3 that is different from

other UFA under different environmental conditions also

indicates a particular way of synthesis for 22:6 n � 3. Nakahara

et al. concluded that 22:5 n � 6 was the direct precursor of 22:6

n � 3 on the basis of a fairly constant ratio of 22:6 n � 3 to 22:5

n � 6 under a variety of culture conditions which were not

described in their article [9]. However, in this study the

temperature and the salinity both affect the ratio of 22:6 n � 3

to 22:5 n � 6, for example, the ratio was 5.58 at 16 8C while

2.41 at 37 8C. Moreover, 22:6 n � 3 exhibited a different

changing tendency from other UFAs while behavior of 22:5

n � 6 was similar to other UFAs under different culture

L. Zhu et al. / Process Biochemistry 42 (2007) 210–214214

conditions. Thus, the hypothesis that 22:6 n � 3 was the

precursor of 22:5 n � 6 seemed more reasonable.

Overall, this study provides detailed understanding for the

effects of temperature and salinity on the growth, lipid and fatty

acid composition of S. limacinum. According to the present

work, S. limacinum shows thermal and salt adaptation process

by regulating the degree of fatty acid unsaturation to maintain

the ideal membrane lipid physical state. Once environmental

conditions cause stress for this organism, additional regulation

of increasing amounts of odd-numbered fatty acid 15:0 and

17:0 would be induced. This study provides additional

understanding of the relationship between environmental

conditions and fatty acid composition of fungal, and these

results will also be helpful to study physiological and

biochemical characteristics of Schizochytrium to further

improve its biotechnological potential.

Acknowledgement

This work was supported by the Science and Technology

Program of Qingdao, China (Grant No. 04-2-HH-76).

References

[1] Hazel JR, Williams EE. The role of alterations in membrane lipid

composition in enabling physiological adaptation of organisms to their

physical environment. Prog Lipid Res 1990;29:167–227.

[2] Sajbidor J. Effect of some environmental factors on the content and

composition of microbial membrane lipids. Crit Rev Biotechnol 1997;

17:87–103.

[3] Chihib NE, Tierny Y, Mary P, Hornez JP. Adaptational changes in cellular

fatty acid branching and unsaturation of Aeromonas species as a response

to growth temperature and salinity. Int J Food Microbiol 2005;102:113–9.

[4] Russell NJ. Mechanisms of thermal adaptation in bacteria: blueprints for

survival. Trends Biochem 1984;9:108–12.

[5] Yumoto I, Hirota K, Iwata H, Akutsu M, Kusumoto K, Morita N.

Temperature and nutrient availability control growth rate and fatty acid

composition of facultatively psychrophilic Cobetia marina strain L-2.

Arch Microbiol 2004;181:345–51.

[6] Simopoulos AP. Omega-3 fatty acids in health and disease and growth and

development. Am J Clin Nutr 1991;54:438–63.

[7] Das UN, Fams MD. Long-chain polyunsaturated fatty acids in the

growth and development of the brain and memory. Nutrition 2003;

19:62–5.

[8] Barclay WR, Meager KM, Abril JR. Heterotrophic production of long

chain omega-3 fatty acids utilizing algae and algae-like microorganisms. J

Appl Phycol 1994;6:123–9.

[9] Nakahara T, Yokochi T, Hifashihara T, Tanaka S, Yaguchi T, Honda D.

Production of docosahexaenoic and docosapentaenoic acids by Schizo-

chytrium sp. isolated from Yap Islands. J Am Oil Chem Soc 1996;73:

1421–6.

[10] Fan KW, Chen F, Jones EBG, Vrijmoed LLP. Eicosapentaenoic and

docosahexaenoic acids production by and okara-utilizing potential of

thrarstochytrids. J Ind Microbol Biotechnol 2001;27:199–202.

[11] Jiang Y, Fan KW, Wong RTY, Chen F. Fatty acid composition and

squalene content of the marine microalga Schizochytrium mangrovei. J

Agric Food Chem 2004;52:1196–200.

[12] Wu ST, Yu ST, Lin LP. Effect of culture conditions on docosahexaenoic

acid production by Schizochytrum sp. S31. Process Biochem 2005;40:

3103–8.

[13] Yokochi T, Honda D, Nakahara T. Optimization of docosahexaenoic acid

content by Schizochytrium limacinum SR21. Appl Microbol Biotechnol

1998;49:72–6.

[14] Chi Z, Liu J, Zhang W. Trehalose accumulation from soluble starch by

Saccharomycopsis fibuligera sud. Enzyme Microb Technol 2001;28:

240–5.

[15] Somashekar D, Venkateshwaran G, Sambaiah K, Lokesh BR. Effect of

culture conditions on lipid and gamma-linolenic acid production by

mucroaceous fungi. Process Biochem 2002;38:1719–24.

[16] Gounot AM, Russell NJ. Physiology of cold-adapted microorganisms. In:

Margesin R, Schinner F, editors. Cold-adapted organisms. Ecology,

physiology, enzymology and molecular biology. New York: Springer;

1999. p. 33–55.

[17] Suutari M, Laakso S. Microbial fatty acids and thermal adaptation. Crit

Rev Microbiol 1992;20:285–328.

[18] Monteoliva-Sanchez M, Ferrer MR, Ramos-Cormenzana A, Quesada E,

Montoeliva M. Cellular fatty acid composition of Deleya halophila: effect

of growth temperature and salt concentration. J Gen Microbiol 1998;134:

199–203.

[19] Chihib NE, Silva MR, Delattre G, Laroche M, Federighi M. Different

cellular fatty acid pattern behaviours of two strains of Listeria

monocytogenes Scott A and CNL 895807 under different tem-

perature and salinity conditions. FEMS Microbiol Lett 2003;218:

155–60.

[20] Metz JG, Roessler P, Facciotti D, Levering C, Dittrich F, Lassner M.

Production of polyunsaturated fatty acids by polyketide synthetases in

both prokaryotes and eukaryotes. Science 2001;293:290–5.