Journal of Applied Phycology (2005) 17: 61–65DOI: 10.1007/s10811-005-5525-4 C© Springer 2005

Differences in growth, total lipid content and fatty acid composition among60 clones of Cylindrotheca fusiformis

Ying Liang∗, Kangsen Mai & Shichun SunMariculture Research Laboratory, Ocean University of China, Qingdao, People’s Republic of China

∗Author for correspondence: e-mail: yliang@mail.ouc.edu.cn; fax: +86-532-2894024

Received 19 March 2004; revised and accepted 31 August 2004

Key words: fatty acid, genetic variation, Cylindrotheca fusiformis, relative growth rate, total lipid

Abstract

Differences between clones of the diatom Cylindrotheca fusiformi were studied with respect to growth rate, totallipid content and fatty acid composition. Sixty clones were isolated and cultivated under batch conditions. All cloneswere grown under identical conditions (temperature 22 ± 1◦C, light intensity 100 µmol photon m−2 s−1, salinity28, F/2 medium) and were harvested in the late exponential growth phase for lipid and fatty acid analysis. Theresults show a wide variation in growth, total lipid content and fatty acid profiles among clones (p < 0.05). Themajor fatty acids in the 60 clones were 14:0 (4.6–9.1%), 16:0 (18.2–32.0%), 16:1n-7 (21.6–33.1%), 20:4n-6 (4.1–13.5%) and 20:5n-3 (6.2–17.2%), with the highest proportion of 20:4n-6 in clone CF13 (13.5%), and the highestproportion of 20:5n-3 in clone CF5 (17.2%). The results support the view that some microalgal fatty acid variationis not restricted to interspecific variation and external factors, but also varies from clone to clone within the samespecies.

Abbreviations: AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturatedfatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.

Introduction

Microalgal fatty acids play an important role in animalnutrition as energy sources, membrane constituents,and metabolic intermediates (Yongmanitchai & Ward,1989; Brown et al., 1996). Many marine animals ap-pear to have limited ability to synthesize the long-chainpolyunsaturated fatty acids, especially EPA (20:5n-3)and DHA (22:6n − 3), to maintain good growth andsurvival. They must be obtained from food. AlthoughEPA and DHA are not essential to some animals, addi-tion of these two fatty acids to their food can increasegrowth rate and survival rate (Yongmanitchai & Ward,1989; Renaud et al., 1991; Reitan et al., 1994). Fattyacids also play a beneficial role in human health dueto their association with the prevention and treatmentof several diseases such as atheroclerosis, thrombosis,cancers and so on (Yongmanitchai & Ward, 1989).

The fatty acids of many microalgal species havebeen examined previously. However, most publisheddata focus on external factors (e.g. culture condi-tions, analytical methods, harvest time, etc.), whichcould modify the fatty acid composition of microalgae(Yongmanitchai & Ward, 1991; Brown et al., 1996;Xu & Beardall, 1997; Fidalgo et al., 1998; Zhukova& Aizdaicher, 2001; Lourenco et al., 2002; Mansouret al., 2003). Only a few authors (Shaw et al., 1989;Alonso et al., 1992a, b, 1994) reported that differencesin fatty acid content could also be explained by ge-netic (internal) differences between strains of the samespecies. For example, Shaw et al. (1989) reported sig-nificant fatty acid variation between 4 clones of Skele-tonema costatum from coastal, estuarine and oceaniclocations. Alonso et al. (1992a) monitored the EPA andDHA content of 59 clones separated from Isochrysisgalbana Parke, the results showed significant different

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EPA and DHA content among different clones. Fattyacids of 42 isolates of a single selected strain of I.galbana were also analysed by Alonso’s group (Alonsoet al., 1992b) and the data showed an evident betweenisolates variability in fatty acid content. In addition,heritability of fatty acid content in culture collectionstrains and 40 new isolates of I. galbana were in-vestigated and similar results were obtained (Alonsoet al., 1994). However, a similar experiment with fiveUTEX strains of Phaeodactylum tricornutum did notshow significant difference in EPA content (Alonsoet al., 1994). It is indicated that some microalgal fattyacid variation is not restricted to interspecific variationbut also varies from clone to clone within the samespecies.

The diatom Cylindrotheca fusiformis can grow welland be used in abalone culture. However, only a few au-thors (Dunstan et al., 1994; Tan & Johns, 1996; Lianget al., 2001, 2002) have followed changes in the fattyacid composition of this species, and to our knowledge,no one has studied the fatty acid variation among dif-ferent clones of this species.

The aim of our study was to elucidate whether dif-ferences in fatty acid content are determinated geneti-cally during a traditional strain improvement method.“Random screening” was used to study wide vari-ations in the growth, total lipid content and fattyacid composition among 60 clones of Cylindrothecafusiformis (obtained from the Microalgae Culture Cen-ter, Ocean University of China) cultured under identicalconditions.

Materials and methods

Organism and culture conditions

The ‘parent’ culture used was obtained from the Mi-croalgae Culture Center (MACC), Ocean Universityof China, it is Cylindrotheca fusiformis MACC/B200.Cells were isolated with a micropipet under a mi-croscope. Micromanipulation must be utmost carefulto ensure single-cell isolates have been made in thismethod. Each isolated cell was transferred to a test tubefor cultivation; 120 clones were established in this man-ner, we selected 60 clones that grew well in test tubesfor further cultivation.

All clones were grown under identical conditionsof nutrient level, light intensity and temperature. Thecultures used were uni-algal but not axenic, althoughbacterial numbers were minimal at all stages. The alga

was grown in 2-L conical flask with 1.5 L mediumat 22 ± 1 ◦C under continuous illumination providedfrom cool white fluorescent lamps and given a photonflux at the culture surface of 100 µmol photon m−2s−1.Salinity was 28 ppt and pH was 8.2. Cultures weregrown in sterilized natural seawater enriched with f/2medium (Guillard & Ryther, 1962). Cell density wasdetermined using an improved Neubauer haemocy-tometer. The maximum specific growth rate (divisionsd−1) was calculated from the slope of the linear por-tion of a curve showing log cell number as a function oftime.

µ = (ln Nt − ln N0)/(t − t0)

where Nt = cell density at time (t) and N0 = cell densityat the start of the exponential phase (t0).

Total lipid and fatty acid determination

Each clone of C. fusiformis was harvested at the lateexponential growth phase by centrifugation at 3000 gfor 10 min. Total lipid was extracted and determined bythe modified method of Bligh (1959). Fatty acid extrac-tion and methylation were performed on freeze-driedsamples according to the modified method of Zhukovaand Aizdaicher (1995). Fatty acid methyl esters wereanalyzed by a HP5890II gas chromatograph fitted witha carbowax capillary column (30 m × � 0.25 mm).High purity N2 was used as the carrier gas at a flowrate of 2 mL min−1, injector and detector temperatureof 280 ◦C. The oven was programmed from 150 ◦C to200 ◦C at 15 ◦C min−1, then to 250 ◦C at 2 ◦C min−1 andheld at 250 ◦C until all peaks had appeared. Fatty acidmethyl esters were identified by comparing the reten-tion times of experimental samples to those of knownstandards.

Fatty acids were designated as the number of carbonatoms: number of double bonds. The position of thefirst double bond (n–x) was counted starting from theterminal methyl group.

Statistical analysis

Statistics were performed using SPSS (version 11.5)statistical software with possible differences amonggroups being tested by one-way ANOVA. Differencesare reported as significant when p < 0.05. Values arethe mean of three replicates.

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Results

Growth

The cell density at harvest ranged between 6.4 and17.2 × 106 cells mL−1 in the 60 clone, with the low-est value in clone CF6 and the highest in clone CF32).The maximum specific growth rate was 0.59–0.79, withthe lowest value in clone CF6 and the highest in cloneCF32. The total lipid content (% dry weight) rangedbetween 7.3% and 23.4% (Table 1).

Fatty acid composition

Tables 1 and 2 show that the major fatty acids were14:0 (4.6–9.1%), 16:0 (18.2–32.0%), 16:1n−7 (21.6–33.1%), AA (20:4n−6) (4.1–13.5%) and EPA (6.2–17.2%). SFA subtotal represented 24.6–37.8% of thetotal fatty acids while MUFA subtotal represented22.9–39.2%, and PUFA subtotal represented 21.3–40.2% of the total fatty acids. The major PUFA were

Table 1. Relative growth rate (d−1), cell density at harvest (105 cellsmL−1), total lipid content (% of dry weight) and fatty acid compo-sitions (% total fatty acids) in 60 clones of C. fusiformis. Values aremean of three replicates.

Major fatty acids Minimum Maximum Average S.D.

14:0 4.6 9.1 6.2 1.05

16:0 18.2 32.0 25.1 3.22

16:1(n − 7) 21.6 33.1 27.0 2.58

16:2(n − 4) 0.3 1.5 0.8 0.27

16:3(n − 4) 0.6 2.9 1.7 0.42

16:4(n − 1) 1.3 8.4 3.5 2.17

18:0 0.2 1.6 0.7 0.35

18:1(n − 9) 0.9 3.4 2.0 0.74

18:1(n − 7) 0.8 2.4 1.8 0.67

18:2(n − 6) 0 1.0 0.5 0.39

18:3(n − 3) 0 1.2 0.5 0.29

20:4(n − 6) 4.1 13.5 9.1 1.43

20:5(n − 3) 6.2 17.2 12.0 1.95

22:4(n − 6) 0 2.1 1.2 0.68

22:6(n − 3) 0.1 1.3 0.7 0.48

SFA 24.6 37.8 31.9 3.38

MUFA 22.9 39.2 31.8 3.41

PUFA 21.3 40.2 33.1 4.54

Relative growth rate 0.59 0.79 0.67 0.04

Cell density at harvest 6.4 17.2 10.7 1.51

Total lipid content 7.3 23.4 13.8 1.98

Table 2. Fatty acid composition (% total fatty acids) of eight selectedclones of C. fusiformis. Values are mean of three replicates.

Clone

Fatty acids CF5 CF9 CF13 CF16 CF27 CF34 CF46 CF51

14:0 6.2 6.4 6.3 5.1 5.6 7.1 6.7 6.5

16:0 22.2 22.0 24.3 24.9 26.7 23.2 19.9 22.8

16:1(n − 7) 24.0 29.8 27.7 24.9 28.5 25.8 32.6 30.4

16:2(n − 4) 0.8 0.6 1.0 0.9 1.3 0.9 1.1 1.2

16:3(n − 4) 2.5 2.6 2.0 2.9 1.9 2.7 1.7 1.8

16:4(n − 1) 8.4 6.3 5.1 7.1 5.2 7.8 6.1 4.5

18:0 0.5 0.8 0.5 0.4 0.7 0.4 1.6 1.5

18:1(n − 9) 1.5 2.4 1.3 1.4 2.1 1.9 1.0 1.7

18:1(n − 7) 1.7 1.4 1.8 1.0 1.6 1.1 1.7 0.9

18:2(n − 6) 0.7 0 0.5 0.5 0 1.0 0.4 0.6

18:3(n − 3) 0.8 0.7 0.6 0.5 0.5 0.2 0.3 0.3

20:4(n − 6) 7.7 11.0 13.5 10.7 7.7 6.3 6.1 8.2

20:5(n − 3) 17.2 10.8 8.7 14.6 10.2 15.4 15.3 13.0

22:4(n − 6) 1.3 1.4 1.0 0.8 1.1 1.3 0.9 1.1

22:6(n − 3) 0.8 0.3 0.5 0.4 0.7 1.0 1.0 0.4

SFA 28.9 29.2 31.1 30.4 33 30.7 28.2 30.8

MUFA 27.2 33.6 30.8 27.3 32.2 28.8 35.3 33

PUFA 40.2 33.7 32.9 38.4 28.6 36.6 32.9 31.1

AA and EPA, with the highest proportion of AA inclone CF13 (13.5%), the highest proportion of EPA inclone CF5 (17.2%).

Despite this overall description, marked variationamong clones was observed. For example, The 16:1n-7 in 60 microalgal clones had significant difference(p < 0.01), ranged between 21.6% and 33.1%. In sev-eral clones, 16:0 represented over 30% of total fattyacids while in others it was less than 19%. As anotherexample, clone CF5 was the richest in EPA with 17.2%total fatty acids and, at the other end, clone CF21 hadonly 6.2% EPA (Tables 1 and 2).

Discussion

The study showed considerable differences in specificgrowth rate, total lipid content and fatty acid profilesamong clones in batch cultures of C. fusiformis. This isconsistent with the previous results for Isochrysis gal-bana reported by Alonso et al. (1992a,b, 1994), whichshowed significant major fatty acid variation amongstrains and among isolates. Because all Cylindrothecafusiformis clones were manipulated, cultivated and

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harvested under identical conditions and all cloneswere analysed with the same techniques, fatty acidvariation among clones cannot be explained by envi-ronmental variation, but solely by genetic differences(Shaw et al., 1989; Alonso et al., 1992a,b, 1994). How-ever, a similar experiment with five UTEX strains of P.tricornutum did not show significant difference in EPAcontent (Alonso et al., 1994). This suggests that the im-portance of genetic differences in the fatty acid compo-sition of different strains of any particular species dif-fers between microalgae (Alonso et al., 1994). Alonsoet al. (1992b), who reported the fatty acid variationamong different isolates of a single strain of I. galbana,pointed out that the existence of genetic variation in theparent culture of I. galbana merits discussion. Theysuggested that micromanipulation might not have beensufficiently careful as to exclude the establishment ofcultures of more than one cell and ensure reproductionwas asexual, just as clonal cultures were expected. So,perhaps, isolates were found with several cells that in-tercrossed, originating a genetically heterogeneous cul-ture. Thus the parent clone was likely to be not geneti-cally homogeneous (Alonso et al. 1992b). However, inthe present study, the much larger cell of C. fusiformis(length 100–120 µm, width 4–6 µm) was used insteadof the smaller one I. galbana (length 5–6 µm, width2–4 µm ), making micromanipulation more easier thanthat for I. galbana, and could ensure single-cell isolateshad been made.

Despite differences in the relative proportions ofthe main fatty acids among clones, the fatty acid com-position remained typical of diatoms, high proportionsof 14:0, 16:0, 16:1n − 7 and 20:5n − 3 fatty acids.C18 and C22 PUFA were minor constituents. However,wide changes appear in different studies on the fattyacid composition of C. fusiformis (Dunstan et al.,1994; Tan & Johns, 1996; Liang et al., 2001, 2002).For example, in the present study, EPA represented6.2–17.2% of the total fatty acids (Table 1), which isconsistent with the results reported by Tan and Johns(1996) (EPA 7.7–18.8%), but a little lower than theresult of Dunstan et al. (1994) (EPA 20.3%). Theproportions of AA in C. fusiformis in our present studywas 4.1–13.5%, which agrees with that for the samespecies reported by Dunstan et al. (1994) (AA 4.9%),but differs from the result analyzed by Tan and Johns(1996) (not detectable AA) in the same species. Themost significant difference in the fatty acid profile ofC. fusiformis in different studies was the value of 18:0fatty acid, 18:0 had high proportions (28.4–38.9%)in three C. fusiformis strains in Tan and Johns’ report

(1996), which quite different from the results publishedby Dunstan et al. (1994) (0.4% 18:0 fatty acid) andour present data which showed a total clone average of0.7% 18:0 (Table 1). These differences could be relatedto the different culture medium, an artificial sea-watermedium supplemented with 0.01 gL−1 tryptone and1 mg L−1 yeast extract was used by Tan and Johns(1996) whereas Dunstan et al. (1994) and the presentstudy used natural seawater enriched with F/2 medium,or they could result from the different methods forfatty acid analysis (Dunstan et al., 1994; Tan & Johns,1996).

Cylindrotheca fusiformis, which could be used asabalone food in aquaculture, has a high percentage ofPUFA (21.3–40.2%), especially AA (4.1–13.5%) andEPA (6.2–17.2%) according to our present data. It alsohas many good characteristics, such as rapid growthand multiplication rate, easy to culture and harvest, andendurance to contamination. The AA and EPA contentcould be increased by changing external factors suchas culture conditions, harvest stage and preservationmethods (Liang et al., 2001, 2002), it also could beincreased by strain improvement, both modern tech-niques of genetic engineering and traditional methodsof mutation and random screening selection have beenconsidered for this purpose. Therefore, it can be consid-ered as a potentially important species for aquaculture.

Acknowledgments

This work was supported by the National High Tech863 Project (863-819-02-01). The authors wish tothank anonymous reviewers for the critical commentsand suggests for the manuscript.

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