Process Biochemistry 42 (2007) 210214 www.elsevier.

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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
College of Marine Life Sciences, Ocean University of China, No. 5 Yushan Road, Qingdao, Shandong 266003, PR China b 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
a

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 1630 8C and salinity at 0.93.6% (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 signicantly 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 microorganisms can exhibit considerable variations with a changing environment [13]. These changes in microbial lipid composition may result in alteration in membranes physical characteristics which enable microbes to maintain membrane uidity, integrity and functionality in the face of environmental uctuations [4]. Temperature is one of the most important environmental factors that affect all aspects of the growth and development of living organisms, and affect signicantly 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

researchers [812]. 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 prole at different temperatures and salinities has not been studied. In this work, we investigated the effects of cultivating temperatures 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

* 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

L. Zhu et al. / Process Biochemistry 42 (2007) 210214 the basal medium. Seed cultures were grown in asks containing GS medium at 23 8C with shaking rotationally (200 rpm) for 2 days.

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2.2. Effect of temperature

Cultures were grown in ask 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 concentrations (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.

Fig. 1. Effect of temperature on the growth and lipids content of Schizochytrium limacinum OUC88. (Data were means S.D. of three replicates.)

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 ushing with nitrogen and the total lipid estimated by a gravimetric method [15].

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 identied 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

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 esteried at 60 8C for 1 h in BF3/methanol (14%, w/w) reagent. The esteried 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

Results obtained for S. limacinum OUC88 growth and lipids content at different temperatures are shown in Fig. 1. When growing at 1630 8C cultures grew better, lipid contents were higher and there were only slight uctuations in biomass and lipids content with the changing temperature. However, when the temperature was raised to 37 8C, cells hardly grew, and

Table 1 Fatty acid composition (% total fatty acids) of S. limacinum OUC88 cultured at different temperature Fatty acid Temperature (8C) 16 12:0 14:0 15:0 16:0 17:0 18:0 18:2 n 6 18:3 n 3 18:3 n 6 20:0 21:0 20:3 n 6 20:4 n 6 22:0 20:5 n 3 22:5 n 6 22:6 n 3 Saturated Unsaturated Odd Even 22:6 n 3/22:5 n 6 0.20 0.02 8.95 0.16 2.19 0.05 38.01 1.22 0.85 0.03 1.47 0.04 0.23 0.01 0.59 0.02 0.23 0.02 0.52 0.02 0.33 0.03 0.33 0.02 0.49 0.03 0.39 0.02 0.72 0.04 6.74 0.22 37.63 1.25 52.91 1.17 46.96 0.78 3.37 0.25 96.50 1.54 5.58 0.07 23 0.22 0.02 8.41 0.23 2.21 0.13 39.14 1.74 0.88 0.03 1.68 0.10 0.26 0.02 0.64 0.03 0.23 0.02 0.55 0.03 0.34 0.02 0.35 0.02 0.48 0.02 0.42 0.03 0.72 0.04 7.51 0.40 36.16 0.87 53.85 0.76 46.35 0.58 3.43 0.16 96.77 1.17 4.81 0.23 30 0.26 0.02 8.35 0.12 2.23 0.07 41.58 0.86 0.93 0.05 1.93 0.07 0.30 0.02 0.78 0.03 0.26 0.03 0.63 0.05 0.36 0.02 0.41 0.04 0.49 0.04 0.48 0.03 0.87 0.03 7.76 0.18 32.12 1.64 56.75 0.89 42.99 0.47 3.52 0.21 96.22 0.98 4.14 0.10 37 0.42 0.03 6.76 0.25 10.30 0.27 32.63 0.55 4.48 0.22 2.06 0.18 0.55 0.04 1.76 0.07 0.42 0.02 0.92 0.03 0.61 0.03 0.73 0.04 0.85 0.03 0.73 0.03 1.24 0.06 10.32 0.77 24.83 0.80 58.91 0.75 40.68 1.19 15.39 0.84 84.20 1.24 2.41 0.05

Data were means S.D. of three replicates.

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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 signicantly from 39.20% at 30 8C to 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 oddnumbered fatty acids 15:0 and 17:0 at different temperatures, the contents of which did not change signicantly 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

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 signicant 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 1.83.6% (w/v). When salinity decreased from 0.9% (w/v) to 0, the growth of the cell was inhibited obviously and the biomass lowered signicantly 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%

Table 2 Fatty acid composition (% total fatty acids) of S. limacinum OUC88 cultured at different salinity Fatty acid Salinity (% w/v) 0 12:0 14:0 15:0 16:0 17:0 18:0 18:2 n 6 18:3 n 3 18:3 n 6 20:0 21:0 20:3 n 6 20:4 n 6 22:0 20:5 n 3 22:5 n 6 22:6 n 3 Saturated Unsaturated Odd Even 22:6 n 3/22:5 n 6 0.30 0.01 3.86 0.08 10.16 0.21 32.62 1.16 4.59 0.52 2.12 0.27 0.41 0.04 0.98 0.03 0.34 0.02 0.85 0.07 0.47 0.03 0.47 0.04 0.55 0.03 0.60 0.03 0.92 0.12 8.27 0.18 31.97 1.28 55.57 1.53 43.91 0.88 15.22 0.25 84.26 1.27 3.87 0.11 0.9 0.22 0.01 7.62 0.12 2.86 0.13 38.09 0.88 1.17 0.22 1.90 0.16 0.25 0.01 0.65 0.02 0.25 0.02 0.52 0.06 0.31 0.02 0.34 0.03 0.40 0.02 0.42 0.03 0.71 0.05 7.63 0.27 36.65 1.06 53.11 1.14 46.88 0.56 4.34 0.08 95.65 1.11 4.80 0.14 1.8 0.23 0.02 8.34 0.15 2.10 0.08 39.79 0.53 0.90 0.18 1.91 0.25 0.26 0.02 0.71 0.03 0.26 0.03 0.55 0.04 0.32 0.03 0.35 0.03 0.42 0.03 0.42 0.02 0.73 0.04 7.56 0.18 35.12 0.88 54.55 0.79 45.41 0.47 3.32 0.12 96.64 1.02 4.65 0.19 2.8 0.24 0.02 8.95 0.09 2.20 0.04 41.53 1.04 0.95 0.09 1.93 0.08 0.24 0.02 0.61 0.04 0.24 0.02 0.58 0.04 0.34 0.04 0.35 0.02 0.44 0.03 0.44 0.04 0.76 0.04 7.46 0.25 32.73 0.47 57.16 1.42 42.83 0.87 3.49 0.15 96.50 0.27 4.39 0.10 3.6 0.27 0.02 9.66 0.13 2.17 0.07 43.38 0.75 0.99 0.05 1.95 0.12 0.27 0.01 0.69 0.04 0.27 0.02 0.61 0.05 0.38 0.02 0.38 0.03 0.46 0.04 0.50 0.03 0.78 0.06 7.19 0.26 29.85 0.53 59.91 0.72 39.89 0.24 3.54 0.09 96.26 0.52 4.15 0.08

Data were means S.D. of three replicates.

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(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 oddnumbered 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 Pacic Ocean. Yokochi reported that temperature at 2030 8C and salinity at 50200% of seawater were appropriate for the growth of S. limacinum SR21 [13]. Similar results were obtained in this study. When cultured at 1630 8C and 0.93.6% 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 signicantly by 2.60- and 1.63-fold; the percentages of most fatty acids increased signicantly, 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 signicantly. 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 8C and 0 salinity indicates that S. limacinum cells encounter stressed growth conditions, and these signicant 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 uidity 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 [2,1719]. 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 uidity 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

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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
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