Chinese Journal of Oceanology and Limnology   2016, Vol. 34 issue(2): 391-398     PDF
Institute of Oceanology, Chinese Academy of Sciences

Article Information

WU Hualian(吴华莲), LI Tao(李涛), WANG Guanghua(王广华), DAI Shikun(戴世鲲), HE Hui(何慧), XIANG Wenzhou(向文洲)
A comparative analysis of fatty acid composition and fucoxanthin content in six Phaeodactylum tricornutum strains from different origins
Journal of Oceanology and Limnology, 34(2): 391-398

Article History

Received Nov. 18, 2014
accepted in principle Mar. 10, 2016
A comparative analysis of fatty acid composition and fucoxanthin content in six Phaeodactylum tricornutum strains from different origins
WU Hualian(吴华莲), LI Tao(李涛), WANG Guanghua(王广华), DAI Shikun(戴世鲲), HE Hui(何慧), XIANG Wenzhou(向文洲)        
Key Laboratory of Tropical Marine Bio-Resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
ABSTRACT: Phaeodactylum tricornutum is a potential livestock for the combined production of eicosapentaenoic acid(EPA) and fucoxanthin. In this study, six marine diatom strains identified as P. tricornutum were cultured and their total lipid, fatty acid composition and major photosynthetic pigments determined. It was found that the cell dry weight concentration and mean growth rate ranged between 0.24-0.36 g/L and 0.31-0.33/d, respectively. Among the strains, SCSIO771 presented the highest total lipid content, followed by SCSIO828, and the prominent fatty acids in all strains were C16:0, C16:1, C18:1, and C20:5(EPA). Polyunsaturated fatty acids, including C16:2, C18:2, and EPA, comprised a significant proportion of the total fatty acids. EPA was markedly high in all strains, with the highest in SCSIO828 at 25.65% of total fatty acids. Fucoxanthin was the most abundant pigment in all strains, with the highest in SCSIO828 as well, at 5.50 mg/g. The collective results suggested that strain SCSIO828 could be considered a good candidate for the concurrent production of EPA and fucoxanthin.
Key words: Phaeodactylum tricornutum     fatty acid composition     polyunsaturated fatty acids     eicosapentaenoic acid     fucoxanthin    

Marine microalgae are not only the primary producers at the base of the marine food chain,but they are also important sources of marine bioactive materials(Samarakoon and Jeon,2012). From their varieties and great numbers,marine diatoms account for ~40% of primary productivity in marine ecosystems(Yool and Tyrrell,2003; Rabosky and Sorhannus,2009). Moreover,marine diatoms play a key role in aquatic animal husbandry for their abundant bioactive components with high nutritional value(Patil et al.,2007). Valuable compounds,such as polyunsaturated fatty acids(PUFAs)and extracellular polymeric substances,are also found in diatom metabolites,which have diverse potential applications,including as food for humans and animals,products for health and cosmetology,and even energy sources(Yang et al.,2013). Phaeodactylum tricornutum,the lone species of its genus,is an excellent marine diatom whose high PUFA content is largely responsible for its high nutritional value(Kim et al.,2012a). PUFAs are considered essential in many marine animal diets to promote high growth and survival rates(Marshall et al.,2010). Thus,this species has been investigated as a desirable larvae-based feed in aquaculture ventures.

Recently,P . tricornutum has drawn attention for another excellent bioactive component,fucoxanthin(Kim et al.,2012a). Fucoxanthin is a primary marine carotenoid found in brown seaweeds and some microalgae,including diatoms,that exhibits a variety of biological activities,such as antioxidant,anticancer,anti-obese,antidiabetic,antiangiogenic,antimalarial,and anti-inflammatory activities. In addition,it has been shown to have protective effects on liver,brain blood vessels,bones,skin,and eyes(Peng et al.,2012). Most notably,fucoxanthin effectively promotes fat consumption in the body,resulting in weight loss,and might prove a panacea for weight-loss drugs(Hosokawa et al.,2010). Interestingly,fucoxanthin and its metabolite fucoxanthinol exhibit few adverse effects on normal,uninfected cells both in vitro and in vivo(Yamamoto et al.,2011). Extensive studies have been carried out regarding the separation,purification,bioavailability,and biological activity of fucoxanthin from macroalgae(Wang et al.,2005; Yang et al.,2008; Fung et al.,2013; Molina et al.,2014; Wang et al.,2014; Ye et al.,2014). However,the fucoxanthin content in macroalgae is very low,requiring complex processes for purification and high production costs. Some microalgae have high fucoxanthin content that is dozens or even hundreds of times that in macroalgae(Kim et al.,2012ab; Xia et al.,2013). Isolation and selection of promising strains that display favorable growth characteristics and high fucoxanthin content is an important activity for cultivating microalgae as a fucoxanthin source. Fortunately,in searching for natural fucoxanthin sources in microalgae,P . tricornutum has been found to be fucoxanthin rich(Kim et al.,2012ab). It is well known that P . tricornutum also contains high proportions of eicosapentaenoic acid(EPA),which belongs to the PUFAs,and represents an interesting alternative source for industrial EPA production(Domergue et al.,2002). Therefore,P . tricornutum could be utilized for the combined production of such fatty acids and fucoxanthin,yielding it a beneficial source for obtaining high value natural compounds. However,different strains in the same species appear to possess different physiological and biochemical characteristics,even when grown under the same conditions. Extensive screening of microalgae obtained from culture collections or newly isolated cultures is especially important in obtaining the best strains for microalgal applications.

In this study,six P . tricornutum strains from different origins were chosen to compare their growth characteristics,fatty acid compositions,and fucoxanthin contents to screen for a promising strain with good potential for production of both EPA and fucoxanthin. 2 MATERIAL AND METHOD

2.1 Strains and culture conditions

The six P . tricornutum strains used in this study were isolated by our laboratory. Strains SCSIO140 and SCSIO771 were collected from the Western Pacific,SCSIO431,SCSIO433,and SCSIO766 from Daya Bay,and SCSIO828 from the Fuzhou coast. The six strains were maintained in 2-L flasks in sterilized F/2 medium supplemented with sodium metasilicate(18.76 mg/L)at a constant temperature of 25±1℃ with light intensity of 40 μE/(m 2 ∙s),provided by coolwhite fluorescent tubes with constant lighting. After cultivation for 11 d,until the late exponential growth phase,all cultures were harvested by centrifugation(3 000× g,5 min)and the sedimented cells washed twice with distilled water. Cell pellets were then freeze-dried and stored at -20℃ until analyses.

2.2 Microscopic observation

The morphological characteristics of these strains were observed under an inverted microscope(Nikon Corp.,Tokyo,Japan)at 400× magnification.

2.3 PCR amplification of the ITS

For molecular identification,microalgal DNA was extracted by the CTAB method(Doyle and Doyle,1987). The primers for amplification of internal transcribed spacers(ITS)included the forward(5′-TCCGTAGGTGAACCTGCGG-3′)and reverse oligonucleotide(5′-TCCTCCGCTTATTGATGC-3′; White et al.,1990). The amplification protocol consisted of an initial denaturation step at 95℃ for 5 min,then thirty thermal cycles of 94℃ for 45 s,58℃ for 45 s,and 72℃ for 45 s were performed,followed by 72℃ for 10 min extension time. PCR products were sequenced using automatic nucleic acid sequencing equipment. Searches for similar sequences were carried out using the BLAST program and multiple alignments run using MEGA5.10. A phylogenetic tree was constructed using the maximum-likelihood method based on 1 000 bootstrap values calculated in MEGA5.10.

2.4 Determination of cell dry weight concentration

and growth rate Microalgal cell growth was monitored by measuring the OD 700 in a spectrophotometer(Rial et al.,2013; Shing et al.,2013; Barros et al.,2014). Microalgal cell dry weight concentration was calculated based on an optical density versus cell dry weight calibration curve. The growth rate of each strain was characterized based on the OD 700 . Growth rates were calculated using the Guillard Equation shown here(Avendaño-Herrera and Riquelme,2007):

where A 1 and A 2 represent the OD 700 at the experiment’s beginning and end,respectively,and t 1 and t 2 the culture’s initial and final time,respectively.

2.5 Total lipid extraction and determination

Intracellular total lipid was extracted and determined as previously described(Yang et al.,2014a). Total lipid content(% of dried cell wt)was calculated according to lipid weight and biomass.

2.6 Analysis of fatty acid composition

Fatty acids were transesterified to methyl esters(FAME)according to the method of Lepage and Roy(1984). Then,the FAME were resolved and quantitated using a GC-MS(6890N-5975,Agilent Technologies,Tokyo,Japan)with an Omegawax 250 polyethylene glycol capillary column(length,30 m; diameter,0.25 mm; film thickness,0.25 μm; Supelco Inc.,Bellefonte,PA,USA)following a previously reported method(Yang et al.,2014b). Briefly,a 1-μL sample was injected into the capillary column with a split ratio of 5/1. The inlet and detector temperatures were set at 250℃ and the oven temperature programmed from 130 to 250℃ at 5℃/min and then held at maximum for 5 min. The fatty acid components were identified by comparison with retention times of authentic standards. Fatty acid percentages were calculated according to the area normalization method.

2.7 Extraction and quantitative analysis of pigments

Lyophilized microalgal powder(10 mg)was extracted with methanol(4 mL)in a conical centrifugation tube with a magnetic stirrer in an ice bath in darkness for 2 h. Pigments were collected in the supernatant by centrifugation(8 000× g,4℃,and 30 min)of the extracted solution. The supernatant was then filtered through a 0.22-μm millipore organic membrane and diluted appropriately with methanol prior for HPLC analysis. The entire process was carried out under low light conditions.

The pigment content determination was carried out by HPLC according to the method reported by Vidussi et al.(1996). Briefly,extracted pigment samples(200 μL aliquots)were separated and analyzed on an HPLC system(Waters Corp.,Milford,MA,USA)equipped with reversed-phase C 8 column(4.6× 250 mm,5 μm). The mobile phase consisted of solvent A(methanol/ 0.5 mol/L ammonium acetate,70/30,v/v)and solvent B(methanol). Elution was performed at 1.0 mL/min using a linear binary gradient between

solvents A and B,programmed according to the following procedure(min,solvent A/B,v/v):(0,75/25),(1,50/50),(15,0/100),(18.5,0/100),and(19,75/25). Pigment absorption spectra were collected from 210 to 700 nm. Peaks for chlorophylls and carotenoids were identified according to their absorption at 450 nm.

2.8 Statistical analysis

Unless otherwise indicated,data were expressed as mean±standard deviation. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post - hoc test. Significance was considered at P<0.05 and P<0.01. All statistical analyses were carried out with OriginPro 8.6 software.

3 RESULT AND DISCUSSION 3.1 Morphology and Identification of P. tricornutum

P . tricornutum can exist in three different morphotypes(fusiform,triradiate,and ova)depending upon environmental conditions(Bojko et al.,2013). The morphological features under light microscopy of these strains are shown in Fig. 1. Under the present culture conditions these strains’ morphotypes were fusiform,~20 μm in length,and contained two or three chloroplasts in each cell.

Figure 1 Cell morphology of six P . tricornutum strains

These P . tricornutum strains were further identified by PCR amplification of the ITS sequences with specific primers. The resulting ITS sequences were aligned together with sequences from other diatom species and Dunaliella bardawil . Homology(sequence identity)searches confirmed a close relationship of these strains and that these strains belonged to the genus Phaeodactylum with high confidence. A maximum likelihood phylogenetic tree was generated and the results consistent with the morphology described above(Fig. 2).

Figure 2 Maximum-likelihood tree of six P . tricornutum strains inferred from ITS gene sequences Numbers above branches were bootstrap values of 1 000 replications.
3.2 Biomass production and growth rate

The cell dry weight concentration and growth rate of six P . tricornutum strains were determined at the late exponential phase(Fig. 3). The dry weight concentration of these strains was found to range from 0.24 to 0.36 g/L,with SCSIO140 having the highest concentration,reaching 0.36 g/L after 11 d of culture,and SCSIO828 having the lowest concentration,at 0.24 g/L. The mean growth rate among these strains ranged from 0.31 to 0.33/d,with the highest mean growth rate observed in SCSIO431,which was significantly higher than other groups except for SCSIO140(P<0.05). Various factors,including nutritional and physical,exert positive effects on P . tricornutum growth(Yongmanitchai and Ward,1991; Zhao et al.,2014). Biomass,storage lipids,and saturated and monounsaturated fatty acids of P . tricornutum under autotrophic,heterotrophic,and mixotrophic growth conditions differ significantly(Cerón García et al.,2006; Morais et al.,2009). Using glycerol as an organic carbon source,P . tricornutum biomass productivity increases by 30%(Morais et al.,2009). Although the strains’ biomasses in the present study were not very high,presumably their biomass in production might be improved by culture condition optimization.

Figure 3 Cell dry weight concentration and growth rate of six P . tricornutum strains
3.3 Total lipid content and fatty acid composition

Neutral and polar lipids,including wax esters,sterols,and hydrocarbons,as well as prenyl derivatives,such as tocopherols,carotenoids,terpenes,and quinines,and phytylated pyrrole derivatives,such as the chlorophylls,are classed as lipids(Hu et al.,2008). The total lipids of the six P . tricornutum strains examined here ranged from 15.91% to 30.75% of dry cell weight(Fig. 4). The highest total lipid content was observed in SCSIO771,at >30% of dry weight and significantly higher than the other strains(P<0.01). SCSIO433 had the lowest lipid content,at only 15.91%,while the remaining four strains did not vary significantly,ranging between 22.79% and 24.22%. This was similar to data from algal nutritive assessments that reported P . tricornutum total lipid at ~22.8% of dry weight(Okauchi and Tokuda,2003). Generally,macroalgal biomasses have lower lipid content compared to microalgae,only accounting for 0.3%–6% of the dry weight(Milledge et al.,2014). P . tricornutum total lipid content in this study varied over 15.91%– 30.75%,which was significantly higher than the 21– 63 mg/g detected in the macroalgae Undaria pinnatifida sporophyll,as reported by Boulom et al.(2014).

Figure 4 Lipid content of six P . tricornutum strains

Most lipids in microalgae are considered to be useful biodiesel or bioactive substances,according to their hydrocarbon chain carbon numbers and unsaturated bonds. PUFAs containing two or more double bonds have been an intense research area because of their potential applications in pharmaceutical,nutraceutical,and cosmetic industries. As a diatom containing abundant PUFAs,P . tricornutum has been studied as a nutraceutical source and,for its rich fatty acid content,including EPA,as a nutrient source for marine animal diets. The fatty acid compositions in the six P . tricornutum strains were determined here and all strains contained fatty acids ranging from C14 to C20(Table 1). The predominant fatty acids were C14:0,C16:0,C16:1,C18:1,and C20:5(EPA),at 5.55%– 7.25%,13.70%–31.91%,25.88%–44.41%,2.21%– 25.51%,and 12.43%–25.65% of total fatty acids(TFA),respectively. These results were consistent with findings reported by Okauchi and Tokuda(2003). Saturated fatty acid profiles were similar in these strains,except for the absence of C18:0 in SCSIO828,which also showed the lowest proportion of C16:0. Among monoenoic fatty acids,C16:1 was the major fatty acid in these strains,being present at a highest proportion in SCSIO431. Among these strains,SCSIO828 contained the highest percentage of unsaturated fatty acids(UFAs),accounting for 77.39%,followed by SCSIO433 and SCSIO766 at 74.96% and 74.18%,respectively. PUFAs,including C16:2,C18:2,C18:3,C20:4,C20:5,and C22:6,constituted 15.52%–34.29% of TFA. EPA was the major component of these PUFAs and present at the highest proportion in SCSIO828 among these strains. C22:6(docosahexaenoic acid,DHA)was only observed in SCSIO140,SCSIO766,and SCSIO828,at <3%. In comparison,C18:2 and C20:4 are the major PUFAs in Undaria sporophylls,accounting for 6% and 14% of total fatty acids,respectively(Boulom et al.,2014). However,C18:2 and C20:4 were extremely low here,even undetectable,in P . tricornutum . These results showed that there were distinct differences in fatty acid composition among different strains in the same species,which was similar to findings in which different fatty acid proportions and compositions exists among different Chaetoceros strains(Liang et al.,2000). Different factors,including algal species,strains,culture temperature,salinity,light density,medium composition,harvest stage,preservation,cultivation modes,and physical/chemical mutation impact the relative EPA contents in Chaetoceros as well as in other diatoms(Cao et al.,2008). The most likely cause of this condition in the present study was because genetic differences exist among these strains from the same species.

Table 1 Fatty acid composition of the six strains of P . tricornutum
3.4 Pigment composition and fucoxanthin content

Chlorophylls and carotenoids have been quantified in biomass cultured under various conditions,and an indirect method,HPLC,has been developed to rapidly estimate pigment content. In this study,the methanol extracts of P . tricornutum was analyzed by HPLC to detect the pigment composition and content. The profiles and amounts of major pigments of the six strains of P . tricornutum were shown as Table 2. The results exhibited that fucoxanthin was the predominant carotenoid in P . tricornutum . The fucoxanthin contents in dry weight sample ranged from 2.14 mg/g in SCSIO431 to 5.50 mg/g in SCSIO828. The amount of fucoxanthin in SCSIO828 was significantly higher than that of other strains(P<0.01). These strains contained more amount of fucoxanthin compared to brown seaweed reported by Zailanie and Purnomo(2011). Clearly,these results suggested SCSIO828 had more potential as source of fucoxanthin. Not only the cultivation conditions but also the origin of the microalgae can give effect on constituents of microalgae. In this study,these strains were cultured under the same conditions,but the differences of pigment contents were significant. Maybe the origin of these microalgae is the reason for the difference of fucoxanthin content existing in different strains in our results.

Table 2 The major pigment composition and contents of the six strains of P . tricornutum

Six strains of marine diatom isolated from different origins were identified as P . tricornutum . EPA and fucoxanthin were the dominant PUFAs and carotenoids of P . tricornutum,respectively. Among the six strains of P . tricornutum,the strain SCSIO828 was found to contain highest amount of fucoxanthin and unsaturated fatty acids,especially essential fatty acids EPA. The results from this study suggest that SCSIO828 should be a candidate for combined production of EPA and fucoxanthin.

Avendaño-Herrera R E, Riquelme C E, 2007. Production of a diatom-bacteria biofilm in a photobioreactor for aquaculture applications. Aquacult. Eng., 36 (2) : 97 –104. Doi: 10.1016/j.aquaeng.2006.08.001
Barros M U G, Da Cruz Coelho A A, Da Sliva J W A, Bezerra J H C, Moreira R T, Farias W R L, Moreira R L, 2014. Lipid content of marine microalgae Chaetoceros muelleri Lemmermann(Bacillariophyceae) grown at different salinities. Biotemas, 27 (2) : 1 –8. Doi: 10.5007/i2079
Bojko M, Brzostowska K, Kuczyńska P, Latowski D, Olchawa-Pajor M, Krzeszowiec W, Waloszek A, Strzałka K, 2013. Temperature effect on growth, and selected parameters of Phaeodactylum tricornutum in batch cultures. Acta Biochim. Pol., 60 (4) : 861 –864.
Boulom S, Robertson J, Hamid N, Ma Q, Lu J, 2014. Seasonal changes in lipid, fatty acid, α-tocopherol and phytosterol contents of seaweed, Undaria pinnatifida, in the Marlborough Sounds, New Zealand. Food Chem., 161 : 261 –269. Doi: 10.1016/j.foodchem.2014.04.007
Cao X H, Li S Y, Wang C L, Lu M F, 2008. Effects of nutritional factors on the growth and heterotrophic eicosapentaenoic acid production of diatom Nitzschia laevis. J. Ocean Univ. Chin., 7 (3) : 333 –338. Doi: 10.1007/s11802-008-0333-5
Cerón García M C, Camacho F G, Mirón A S, Sevilla J M F, Chisti Y, Grima E M, 2006. Mixotrophic production of marine microalga Phaeodactylum tricornutum on various carbon sources. J. Microbiol. Biotechnol., 16 (5) : 689 –694.
Domergue F, Lerchl J, Zähringer U, Heinz E, 2002. Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. Eur. J. Biochem., 269 (16) : 4105 –4113. Doi: 10.1046/j.1432-1033.2002.03104.x
Doyle J J, Doyle J L, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull., 19 (1) : 11 –15.
Fung A, Hamid N, Lu J, 2013. Fucoxanthin content and antioxidant properties of Undaria pinnatifida. Food Chem., 136 (2) : 1055 –1062. Doi: 10.1016/j.foodchem.2012.09.024
Hosokawa M, Miyashita T, Nishikawa S, Emi S, Tsukui T, Beppu F, Okada T, Miyashita K, 2010. Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic/obese KK-A y mice. Arch.Biochem. Biophys., 504 (1) : 17 –25. Doi: 10.1016/
Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A, 2008. Microalgal triacylglycerols as feedstocks for biofuel production:perspectives and advances. Plant J., 54 (4) : 621 –639. Doi: 10.1111/j.1365-313X.2008.03492.x
Kim S M, Jung Y J, Kwon O-N, Cha K H, Um B-H, Chung D, Pan C-H, 2012a. A potential commercial source of fucoxanthin extracted from the microalga Phaeodactylum tricornutum. Appl. Biochem. Biotechnol., 166 (7) : 1843 –1855. Doi: 10.1007/s12010-012-9602-2
Kim S M, Kang S-W, Kwon O-N, Chung D, Pan C-H, 2012b. Fucoxanthin as a major carotenoid in Isochrysis aff. galbana:characterization of extraction for commercial application. J. Korean Soc. Appl. Biol. Chem., 55 (4) : 477 –483.
Lepage G, Roy C C, 1984. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J. Lipid Res., 25 (12) : 1391 –1396.
Liang Y, Mai K S, Sun S C, 2000. Total lipid and fatty acid composition of seven Chaetoceros strains. Transaction of Oceanology and Limnology (3) : 29 –33.
Marshall R, McKinley S, Pearce C M, 2010. Effects of nutrition on larval growth and survival in bivalves. Rev.Aquacult., 2 (1) : 33 –55. Doi: 10.1111/raq.2010.2.issue-1
Milledge J J, Smith B, Dyer P W, Harvey P, 2014. Macroalgaederived biofuel:a review of methods of energy extraction from seaweed biomass. Energies, 7 (11) : 7194 –7222. Doi: 10.3390/en7117194
Molina N, Morandi A C, Bolin A P, Otton R, 2014. Comparative effect of fucoxanthin and vitamin C on oxidative and functional parameters of human lymphocytes. Int.Immunopharmacol., 22 (1) : 41 –50. Doi: 10.1016/j.intimp.2014.06.026
Morais K C C, Ribeiro R L L, Santos K R, Taher D M, Mariano A B, Vargas J V C, 2009. Phaeodactylum tricornutum microalgae growth rate in heterotrophic and mixotrophic conditions. Thermal Engineering, 8 (1) : 84 –89.
Okauchi M, Tokuda M. 2003. Trophic value of the unicellular diatom Phaeodactylum tricornutum for larvae of Kuruma prawn, Penaeus japonic u s. In:Symposium on Aquaculture and Pathobiology of Crustaceans and Other Species in Conjunctions with the 32nd UJNR Aquaculture Panel Meeting. 18p.
Patil V, Källqvist T, Olsen E, Vogt G, Gislerød H R, 2007. Fatty acid composition of 12 microalgae for possible use in aquaculture feed. Aquacult. Int., 15 (1) : 1 –9. Doi: 10.1007/s10499-006-9060-3
Peng J, Yuan J P, Wu C F, Wang J H, 2011. Fucoxanthin, a marine carotenoid present in brown seaweeds and diatoms:metabolism and bioactivities relevant to human health. Mar. Drugs, 9 (10) : 1806 –1828.
Rabosky D L, Sorhannus U, 2009. Diversity dynamics of marine planktonic diatoms across the Cenozoic. Nature, 457 (7226) : 183 –186. Doi: 10.1038/nature07435
Rial D, Murado M A, Menduiña A, Fuciños P, González P, Mirón J, Vázquez J A, 2013. Effects of spill-treating agents on growth kinetics of marine microalgae. J.Hazard. Mater., 263 : 374 –381. Doi: 10.1016/j.jhazmat.2013.07.010
Samarakoon K, Jeon Y-J, 2012. Bio-functionalities of proteins derived from marine algae-a review. Food Res. Int., 48 (2) : 948 –960. Doi: 10.1016/j.foodres.2012.03.013
Shing W L, Heng L Y, Surif S, 2013. Performance of a cyanobacteria whole cell-based fluorescence biosensor for heavy metal and pesticide detection. Sensors, 13 (5) : 6394 –6404. Doi: 10.3390/s130506394
Vidussi F, Claustre H, Bustillos-Guzmàn J, Cailliau C, Marty J-C, 1996. Determination of chlorophylls and carotenoids of marine phytoplankton:separation of chlorophyll a from divinylchlorophyll α and zeaxanthin from lutein. J.Plankton Res., 18 (12) : 2377 –2382. Doi: 10.1093/plankt/18.12.2377
Wang S K, Li Y, White W L, Lu J, 2014. Extracts from New Zealand Undaria pinnatifida containing fucoxanthin as potential functional biomaterials against cancer in vitro. J.Funct. Biomater., 5 (2) : 29 –42. Doi: 10.3390/jfb5020029
Wang W J, Wang G C, Zhang M, Tseng C K, 2005. Isolation of fucoxanthin from the Rhizoid of Laminaria japonica Aresch. J. Integr Plant Biol., 47 (8) : 1009 –1015. Doi: 10.1111/jipb.2005.47.issue-8
White T J, Bruns T D, Lee S B, Taylor J W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In:Innis M A, Gelfand D H, Sninsky J J, White T J eds. PCR Protocols a Guide to Methods and Applications. Academic Press, London. p.315-322.
Xia S, Wang K, Wan L L, Li A F, Hu Q, Zhang C W, 2013. Production, characterization, and antioxidant activity of fucoxanthin from the marine diatom Odontella aurita. Mar. Drugs, 11 (7) : 2667 –2681. Doi: 10.3390/md11072667
Yamamoto K, Ishikawa C, Katano H, Yasumoto T, Mori N, 2011. Fucoxanthin and its deacetylated product, fucoxanthinol, induce apoptosis of primary effusion lymphomas. Cancer Lett., 300 (2) : 225 –234. Doi: 10.1016/j.canlet.2010.10.016
Yang F F, Long L J, Sun X M, Wu H L, Li T, Xiang W Z, 2014a. Optimization of medium using response surface methodology for lipid production by Scenedesmus sp. Mar. Drugs, 12 (3) : 1245 –1257. Doi: 10.3390/md12031245
Yang F F, Xiang W Z, Sun X M, Wu H L, Li T, Long L J, 2014b. A novel lipid extraction method from wet microalga Picochlorum sp. at room temperature. Mar.Drugs, 12 (3) : 1258 –1270.
Yang L Q, Li P C, Fan S J, 2008. The extraction of pigments from fresh Laminaria japonica. Chin. J. Oceanol. Limnol., 26 (2) : 193 –196. Doi: 10.1007/s00343-008-0193-2
Yang Z K, Niu Y F, Ma Y H, Xue J, Zhang M H, Yang W D, Liu J S, Lu S H, Guan Y F, Li H Y, 2013. Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation. Biotechnol.Biofuels, 6 : 67 . Doi: 10.1186/1754-6834-6-67
Ye G L, Lu Q, Zhao W D, Du D L, Jin L J, Liu Y S, 2014. Fucoxanthin induces apoptosis in human cervical cancer cell line HeLa via PI3K/Akt pathway. Tumour Bio l., 35 (11) : 11261 –11267. Doi: 10.1007/s13277-014-2337-7
Yongmanitchai W, Ward O P, 1991. Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions. Appl. Environ.Microbiol., 57 (2) : 419 –425.
Yool A, Tyrrell T, 2003. Role of diatoms in regulating the ocean's silicon cycle. Global Biogeochem. Cy cles, 17 (4) : 14 –1.
Zailanie K, Purnomo H, 2011. Fucoxanthin content of five species brown seaweed from Talango District, Madura Island. J. Agr. Sci. Tech. A 1, A 1 : 1103 –1105.
Zhao P P, Gu W H, Wu S C, Huang A Y, He L W, Xie X J, Gao S, Zhang B Y, Niu J F, Lin A P, Wang G C, 2014. Silicon enhances the growth of Phaeodactylum tricornutum Bohlin under green light and low temperature. Sci. Rep., 4 : 3958 .