Dunaliella salina Sinecosyfis sp.,Chroomonas sp. at 20 C Sinecosys sp. at 20 C Dunaliella salina at 32 C Chroomonas sp. at 32 C Sinecosys sp. at 32 C F :Comparativegrowthcurvesof Dunaliellasalina

Research ArticleEvaluation of Culture Conditions to Obtain Fatty Acids fromSaline Microalgae Species Dunaliella salina Sinecosyfis spand Chroomonas sp

D A Castilla Casadiego12 A R Albis Arrieta1 E R Angulo Mercado2

S J Cervera Cahuana1 K S Baquero Noriega1

A F Suaacuterez Escobar3 and E D Morales Avendantildeo4

1Engineering Faculty Chemical Engineering Program Bioprocess Research Group Universidad del AtlanticoKm 7 Antigua Vıa a Puerto Colombia Barranquilla Colombia2Basic Sciences Faculty Chemistry Program Biotechnology of Microalgae Research Group Universidad del AtlanticoKm 7 Antigua Vıa a Puerto Colombia Barranquilla Colombia3Engineering Faculty Universidad de Bogota Jorge Tadeo Lozano 4 Avenue No 22-61 Bogota Colombia4Sciences Experimental Faculty Universidad del Zulia Calle 61 con Prolongacion Avenida 77 Maracaibo 4011 Venezuela

Correspondence should be addressed to D A Castilla Casadiego davidcastillaupredu

Received 3 January 2016 Revised 26 April 2016 Accepted 12 May 2016

Academic Editor Wenguang Zhou

Copyright copy 2016 D A Castilla Casadiego et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

The use of the saline microalgae Dunaliella salina Sinecosyfis sp and Chroomonas sp was explored as an alternative source forthe production of fatty acids using fertilizer and glycerol as culture media The nutrient medium used contained ldquoNutrifoliarrdquo acommercial fertilizer andor glycerol in natural sea water The microalgae were placed in cultures with different conditions Theparameters that favored the largest production of fatty acids were 24 hours of agitation and illumination 1620 Lday of air supply225 L of airmin and a temperature of 32∘C using ldquoNutrifoliarrdquo as the culture media Results indicated that from 3 g of microalgaein wet base of Chroomonas sp 5443mg of oil was produced The chromatographic characterization of oil obtained revealed thepresence of essential fatty acids such as 91215-octadecatrienoic acid (omega-3) and 4710-hexadecatrienoic acid (omega-6) fromthe species Dunaliella salina On the other hand 912-octadecadienoic acid (omega-6) and cis-11-eicosenoic acid (omega-9) wereidentified from the species Chroomonas sp The temperature variations played an important role in the velocity of growth or theproduction of the algae biomass the amount of oil and the ability to produce fatty acids

1 Introduction

Essential fatty acids are divided into two main familiesomega-3 (n-3) and omega-6 (n-6) which are denominatedas polyunsaturated fatty acids (PUFAs)This is due to the factthat its carbon chains are not saturated with hydrogen atomsand have more than one double bond between carbon atoms[1] There are certain fatty acids that our organism is not ableto synthesize called essential fatty acids These are of vitalimportance contributing to the health of human beings andtherefore must be administered through a personrsquos diet [2]

A normal consumption of PUFAs has been associatedwith good health of human beings [3] Fatty acids play a role

in the development and maintenance of correct cerebralfunction especially in the gestation period and childhood [4]Several researches show that PUFAs protect and help reducerisk of contracting cardiovascular illnesses [5] diseases inpremature babies [6] in the lactation and pregnancy period[7] ocular diseases associated with age [8] Alzheimerrsquosdisease [9] inflammatory illnesses [10] diabetes prevention[11] and certain types of cancer [12] Finally these lipids canbe used for treatment of mental illnesses [13] and weight loss[14]

Currently omega-3 fatty acids are obtained from sourcessuch as fish oil salmon tuna halibut anchovy or herringshrimp and dried fruit oils [15] Also omega-6 fatty acids can

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 5081653 7 pageshttpdxdoiorg10115520165081653

2 BioMed Research International

be found in foods such as vegetable oil like soy safflower orcorn in dried fruit and seeds [16] and in small amounts inmeat birds and eggs [17]

One of the biggest problems to obtain these fatty acidsfrom fish oil is their residual taste which is not pleasantfor the human palate reducing its consumption and marketsales Due to the refinement process of this oil necessaryto eliminate the residual taste the final product is moreexpensive and on account of this alternative ways to obtainthese lipids have been proposed in order to replace PUFAsfrom fish oil using as feedstock another type of marine lifelike microalgae [15]

Recently it has been proved that certain species ofmicroalgae accumulate under specific culture conditionsa good amount of fatty acids [15] In particular somesaline strains present high percentages of omega oils thatallow the exploration of obtaining on a commercial scaleessential fatty acids such as docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA) Various studies developed withdifferentmicroalgae used to obtain these fatty acids are shownin Table 1

With the purpose of understanding which other speciesof saline algae can be good precursors to obtain fatty acidsthis study presents as the principal objective to determine thepotential of the species of salinemicroalgaeDunaliella salinaSinecosyfis sp and Chroomonas sp as sources of essentialfatty acids evaluating the best culture conditions that couldhelp generate a larger production of algae biomass amount ofoil and the ability to produce fatty acids of interest Besidesthis study explores the alternative of using commerciallyavailable feedstock such as commercial fertilizers and glyc-erol for the formulation of the growthmedium of the cultureof saline microalgae in order to reduce the cost associatedwith nitrogen and carbon sources for the production of fattyacids omega-3 using microalgae

2 Materials and Methods

21Microorganisms Themicroalgae used correspond to threespeciesDunaliella salina Chroomonas sp and Sinecosyfis spThese species were donated by the Ceparium of Photosyn-thetic Microorganismrsquos Laboratory of the Biology Depart-ment of the Experimental Science Faculty the Universidaddel Zulia Maracaibo VenezuelaThese strains were providedin test tubes with 10mL of algal culture medium containing4mM of nitrogen and a salinity of 35

22 Nutrients and Culture Conditions The algae were sub-merged in different conditions of growth alternating thenutrient medium to obtain biomass commercial ldquoNutrifo-liarrdquo fertilizer with a 4mM concentration of nitrogen andglycerol at 4mM of carbon both in filtrated sea waterand autoclaved The cultures were at controlled temper-atures of 20∘C and 32∘C The artificial illumination wassupplied through fluorescent lamps (Sylvania Daylight plusF96T12DLP) of 75W for all the cultures with a luminousflow of 16880 lm (16 lamps) The illumination source wasregulated a continuous exposition was supplied for selected

Table 1 Values () of EPA and DHA fatty acid contents withrelations to total lipids in some microalgae [16]

Microalgae of EPA and DHAproduction Reference

Nannochloropsis salinasim28 EPA [17]

Thraustochytrium sp 451 EPA + DHA [18]Dunaliella salina 214 EPA [19]Pavlova viridis 360 EPA + DHA [20]Isochrysis galbana

sim280 EPA + DHA [21]

cultures and others were submerged at a photoperiod oflight darkness 12 h 12 h The agitation and supply of airwere provided through air pumps (PowerLife P -500) whichprovide 225 L of airmin with the purpose of avoiding thesedimentation of algae and permit culture homogenization

Natural sea water was obtained from Salgar Beach in theAtlantic Coast Colombia coordinate latitude 11∘11015840166210158401015840Nlongitude 74∘56101584072410158401015840W The salinity of the sterile sea waterwas measured with conductivity electrodes WTW brandand Multi 3420 model The registered value was 375 and562mScm for salinity and conductivity respectively All thematerials usedwere previously sterilized in autoclave at 121∘C

23 Cell Growth Cell growth was determined by takingdaily samples of 5mL from each of the different culturesand determining the concentration of microalgae spectro-scopically (Spectronic Genesys 2) measuring the absorbanceat a wavelength of 647 nm and monitoring each culturein fixed 24-hour intervals Moisture content in centrifugedmicroalgae was obtained drying samples of microalgae at105∘C until constant weight

24 Lipid Extraction The lipid fraction of microalgae wasextracted using Soxhlet method which is based on theconstant evaporation and condensation through stages of avolatile solvent for posterior contact with the sample studiedQuantities of the biomass between 15 and 24 grams wereweighed for each microalga and were subjected to Soxhletextraction using analytic hexane (JT Baker) as solvent

A mixture of hexane and lipids was obtained from theprocess of extraction This mixture was separated through arotary evaporator Buchi Heating Bath Model B-490 at 48∘Cwith a rotation velocity 64ndash66 rpm for 45 minutes

25 Determination of the Fatty Acids Profile The extractedoils were transesterified adding 1mL of amethanolic solutionof NaOH 1 The mixture was heated to 55∘C for 15minand then 2mL of a methanol solution containing 2 ofHCl was added and heated for another 15min [22] Laterthe obtained FAMEs were dissolved in 1mL of hexane andinjected to an Agilent 7890 A gas chromatograph coupledto a selective mass detector 5975C (Agilent) with an AgilentSelect Biodiesel for FAME column (30m 020mm 025 120583m)using Helium as a carrier gas with a flow of 1mLmin and avolume of injection of 1 120583L with a temperature programmedat 60∘C for 2 minutes and then incremented in 15∘Cmin

BioMed Research International 3

Table 2 Variables and factors of experimentation

Factors (119865) Variables Levels

1198651 nutrient type Nutrifoliar minus

Glycerol +

1198652 temperature (∘C) 20 minus

32 +

1198653 agitation time (h) 12 minus

24 +

1198654 photoperiod light darkness (h) 12 12 minus

24 0 +

1198655 air supply (Lday) 810 minus

1620 +

Table 3 Experimental factors level combinations

Experimental condition 1198651 1198652 1198653 1198654 1198655

1 minus + + + +2 minus + minus minus minus

3 minus minus + + +4 + + + + +5 + + minus minus minus

6 + minus + + +

until a final temperature of 240∘C was reached with a timeduration of 10minutesThe injector and detector temperaturewere of 250∘C according to EN 14103 The total time ofanalysis was 363 minutes

26 Experimental Design The experimental conditions eval-uated are presented in Table 2

Six experiments were established to develop this investi-gation and these are presented inTable 3 alternating the levels(minus or +) which symbolize the different conditions or factorsof experimentation presented in Table 2 Experiments weredone in triplicate

3 Results and Discussion

Some of the conditions studied according to the experimentaldesign were no favorable for the algae growth In Table 4 theconditions where the algae showed an increase of populationare marked with ldquoYESrdquo and those where the algae was notable to adapt are marked with ldquoNOrdquo

Table 4 allows concluding that the microalgae have ahigher affinity for the media formulation composed by thecommercial fertilizer ldquoNutrifoliarrdquo when compared to theculture media enriched with glycerol Clearly this can beobserved for experimental conditions 4 and 6 reported inTable 4 where it is demonstrated that none of the microalgaespecies were able to survive in the media containing glycerolunder these experimental conditions but an acceptance ofglycerol was seenwhen the experimental conditions 5 showedTable 3 were employed which refers to the conditions of agi-tation illumination and air supply in low levels The strainsDunaliella salina and Chroomonas sp were able to show a

00

02

04

06

08

10

12

14

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Time (day)

Abso

rban

ce (A

U)

Dunaliella salina at 20∘CChroomonas sp at 20∘CSinecosyfis sp at 20∘C


Figure 1 Comparative growth curves ofDunaliella salina Sinecosy-fis sp and Chroomona sp microalgae employing the commercialfertilizer in conditions 20 and 32∘C of temperature agitation andillumination of 24 hours and air supply of 1620 Lday

pattern of effective growthThe opposite effect was presentedunder the fourth-experimental conditions with high levelsof agitation time illumination and air supply which led tothe death of this microalga The species Sinecosyfis sp didnot grow at any of the experimental conditions with glycerolThese can be explained either by the rapid depletion of theinitial nitrogen contained in sea water when no additionalnitrogen is added as it was done in the experiment that usesthe culturemedia enriched with glycerol or by osmotic stressdue to the presence of glycerol in the enriched media or bythe combination of both phenomena

Evaluating the effect of the variable temperature exper-iments 1 and 3 reported that microalgae can grow underestablished conditions of 20 and 32∘C On the other hand thespecies Chroomonas sp was able to live through the estab-lished conditions of low levels of agitation time illuminationand air supply whichmake reference to experiment 2 Table 4

Figures 1 and 2 show kinetics of growth of the microalgaespecies that were able to adapt and grow under differentconditions established in Table 4

Figure 1 shows that the temperature at which themicroal-gae culture grew is an influential and selective parameter forthemicroalgae species exerting an effect in the concentrationof biomass in time The microalgae culture of Sinecosyfis spand Chroomonas sp at 32∘C showed the highest concen-trations of biomass generating a reason for accumulationeach day of 153 and 123 times more for the Chroomonas spand Sinecosyfis sp microalgae respectively in the 11 days ofculture in comparison with the same strains of microalgaeexposed to a growth below 20∘C The growth of Dunaliellasalinamicroalgae under these two conditions of temperaturedemonstrated that this microalga is selective at temperaturesaround 20∘C presenting a higher concentration of almostdouble the biomass in comparison with cultures exposed at32∘C Other researchers that had studied the Chlorella sp


Table 4 Results of culture condition evaluations and treatment of the microalgae species subject to studies

Experimental condition Microalgaeevaluation of culture conditions and treatmentDunaliella salina Sinecosyfis sp Chroomonas sp

1 YES YES YES2 NO NO YES3 YES YES YES4 NO NO NO5 YES NO YES6 NO NO NO

Table 5 Kinetic growth behavior of microalgae presented in Figure 1

Microalgae Behavior at 20∘C Behavior at 32∘CDunaliella salina Linear exponential stationary Adaptation exponential stationarySinecosyfis sp Linear stationary Linear exponential stationaryChroomonas sp Linear stationary Linear exponential stationary

00

02

04

06

08

10

12

14

0 1 2 3 4 5 6 7 8 9 10 11 12

Abso

rban

ce (A

U)

Time (day)

Chroomonas sp with glycerolDunaliella salina with glycerol

Chroomonas sp with NutrifoliarDunaliella salina with Nutrifoliar

Figure 2 Comparative growth curves that represent absorbanceversus time in days of microalgaeDunaliella salina and Chroomonassp employing glycerol and ldquoNutrifoliarrdquo as nutrient mediums at20∘C an agitation and illumination of 24 hours and an air supplyof 1620 Lday

Nannochloropsis sp [23] and Isochrysis galbana microalgae[24] have reported that cultures exposed to moderately warmtemperatures and high levels of light are able to grow signif-icantly more This is due to the increment in temperaturewhich favorably contributes to the photosynthesis [25] andrespiration [26] reflecting an accelerated algal growth [2327] Table 5 describes the behavior or stages of kinetic growthofmicroalgae seen in Figure 1These cultures were recollectedonce they achieved the stationary growing phase to proceedwith the oil extraction One control for each culture wasallowed to continue its growing showing the final deathphase for every culture After centrifugation wet microalgaeshowed average moisture of 688

Figure 2 shows that microalgae have a favorable accep-tance of ldquoNutrifoliarrdquo as its nutrient medium This is due

to the fact that the adaptation phase of the culture was lessrelevant in the kinetics described in Table 5 going straightto the linear phase followed by the exponential phase (insome cases) stationary phase and death phase The highestabsorbance data was achieved at 32∘C with 1187 absorbanceunits with Chroomonas sp microalgae and 1127 absorbanceunits with Sinecosyfis sp Both results were reported after 15days of culture growth At 32∘C the microalgae Dunaliellasalina demonstrated little acceptance due to the fact thatit presented an adaptation phase for more than seven daysstarting with an absorbance of 017 at the first day to 0317after seven days at 32∘C Evaluating its growth at 20∘Cits concentration went from 018 of absorbance the firstday to 077 after the seven days of culture At 20∘C theexponential phase was not projected for Sinecosyfis sp andChroomonas sp microalgae causing it not to be able to growwith vitality and firmness for this reason the lowest biomassconcentration was generated

Figure 2 shows glycerol as a promising nutrient mediumto achieve high concentrations of biomass in a short periodof time Observing Figure 2 in detail the amount of biomassobtained in three days using this nutrient source was ableto surpass the amount obtained using a Nutrifoliar culturein 11 days for Dunaliella salina cultures with a ratio ofbiomass concentration of about 3 times more per day TheChroomonas spmicroalgae cultures nourishedwith glycerolalso showed the same behavior observed in the Dunaliellasalina cultures the amount of biomass that was accumulatedusing ldquoNutrifoliarrdquo in 11 days was obtained in only 5 days in aglycerol culture at a growth ratio of about twice as much

It could also be appreciated in the useful culture lifetimeThe strains were only able to bear 4 to 5 days for the culturesnourished with glycerol in comparison with ldquoNutrifoliarrdquowhich kept them alive for 11 days The beginning of thecellular death phase occurred at an early stage for cultureswith glycerol This suggests that the microalgae studied dueto itsmixotrophic character can use this substance as a sourceof carbon for growth causing a vertiginous growth duringthe exponential phase with the absence of the adaptation


stage However this rapid growth also caused an early disap-pearance of other nutrients in the culture medium (possiblynitrogen) and therefore its unviability generating a rapiddecay in biomass concentration Gallardo and Cobelas [28]presented that one of the factors for the massive productionof algae is the source of carbon and nitrogen due to the factthat the absence of these would generate disequilibrium inthe cultures For this reason even though it generated greatdensity of microalgae biomass the reported results of thiswork with the use of glycerol (carbon source) as a culturemedium did not have the capacity necessary to maintain theculture without invigorated nitrogen

Table 6 presents the results of the oil characterizationproduced by each microalgae biomass using different cultureconditions revealing the fatty acids that were identified in thesamples

Table 6 shows the results of the chromatographic charac-terization of the experiments that succeeded in the produc-tion of oil These correspond to experimental conditions 12 and 3 of Table 3 in which the nutrient medium employedwas ldquoNutrifoliarrdquo Those cultures that reported growth andgeneration of high levels of biomass with glycerol as anutrient source did not provide a significant amount of oilafter the extraction process This led to the conclusion thatthe series of culture conditions expressed in experiment 5 ofTable 3 even though a high level of biomass was produced ina short period of time were not able to stress the microalgaeto concentrate oil in its interior

From the experimental conditions 1 (Table 3) only theresults for Chroomonas sp and Dunaliella salina microalgaeare shown Sinecosyfis sp did not produce a significantamount of oil From the experimental conditions 2 only theresults for Chroomonas sp are showed For the conditionsstudied in experiment 3 Chroomonas sp and Sinecosyfissp cultures that were able to survive did not guarantee aconsiderable production of oil

Temperature was a crucial variable that favored theproduction of oil and the extraction of fatty acids because bysubmitting the cultures at 20∘C the amount of oil obtainedand the extraction of fatty acids decreased in comparisonwith the conditioning of the cultures at 32∘C Our resultsreported that for each gram of wet microalgae biomass ofChroomonas sp at 32∘C (experimental conditions 1) 1814mgof oil was produced and 5 different fatty acids were extractedwhile for each gram of biomass of Chroomonas sp at 20∘C(experimental condition 3) 262mg of oil was obtained andonly 3 types fatty acids were extracted For the experimentalconditions 2 where only the agitation and illuminationperiod were fixed at 12 12 h supplied with 810 Lday of airand at 32∘C the generation of oil was 1666mg for eachgram of wet microalgae obtaining a very small variation incomparison with results obtained in experiment 1 stressingthe fact that the temperature has more influence in theproduction of oil than the other variables The productionof oil of the Sinecosyfis sp microalga was also affectedby temperature reporting that for each gram of biomass

369mg of oil was obtained This is due to the fact that theincrement in temperature was able to stress the microalgaefavoring a greater generation in the amount of oil

Within the culture conditions evaluated the one thatfavored the production of essential fatty acids with microal-gae was 24 hours of agitation and 1620 Lday of air sup-ply 32∘C employing Nutrifoliar at 4mM of nitrogen asthe culture medium (experimental conditions 1) At theseconditions microalgae Chroomonas sp andDunaliella salinaproduced 4 and 5 different fatty acids (Table 6) respectivelyamong them 91215-octadecatrienoic acid (omega-3) and4710-hexadecatrienoic acid (omega-6) for Dunaliella salinathese fatty acidswere also identified in the research developedby Bhosale and collaborators in 2010 [19] with this samemicroalgae and 912-octadecadienoic acid (omega-6) withChroomonas sp microalgae Also hexadecanoic acid andheptadecanoic acid were the most common fatty acidsreported in all the analysis for the samples of differentmicroalgae at diverse culture conditions

4 Conclusions

The nutrient source is the main variable that affected thegrowth andbiomass production of themicroalgaeThe resultsshowed that the glycerol which only provides the carbonsource to the cultures was useful to produce around of thesame microalgae biomass concentration that ldquoNutrifoliarrdquogained in 11 days in only 3 dayswith themicroalgaeDunaliellasalina and 5 days to Chroomonas sp Low yields of oilproduction were obtained using glycerol as nutrient sourcetherefore the extraction of fatty acids was not successfulldquoNutrifoliarrdquo was the nutrient source that all the studiedmicroalgae accepted the most with almost all the differentexperimental conditions being favorable for the culturesThetemperature was the most influential variable generatingsignificant contributions on the microalgae biomass andoil production and for obtaining fatty acids At 32∘C themicroalgae Sinecosyfis sp and Chroomonas sp were able togrow and produced oil At 20∘C the microalgae Dunaliellasalina grew but did not produce oil in a quantifiable amountThe parameters that favored the largest production of oiland extraction of essential fatty acids were 24 hours ofagitation and illumination 1620 Lday of air supply and 225 Lof airmin and a temperature of 32∘C using Nutrifoliar asculturemediumAt these conditions theChroomonas sp andDunaliella salina produced polyunsaturated fatty acids of theomega-3 and omega-6 families

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was financially supported by the Universidad delAtlantico Colombia and by the ldquoPrograma de Jovenes Inves-tigadores e Innovadores de COLCIENCIASrdquo The authoracknowledge Universidad del Zulia Experimental Faculty of


Table6Identifi

edfatty

acidsa

ndqu

antityof

oilprodu

cedforthe

studied

microalgae

Experim

entn

umber

12

3

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationfor2

4ho

urssupp

lied1620

Ldayof

airtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationof

1212ho

urssupp

lied810L

day

ofairtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nand

illum

inationof

24ho

urssupp

lied1620

Ldayof

air

temperature

20∘

C

Microalgaes

pecies

Chroom

onas

sp

Dun

aliellasalin

aCh

room

onas

sp

Chroom

onas

sp

Sinecosyfis

sp

Quantity

ofwet

microalgae(g)

34

15155

24

Oilprod

uced

(mg)

5443

7314

2499

4069

8876

mgof

oilg

ofwet

biom

ass

1814

1829

1666

262

369

Extractedfatty

acids

(1)H

exadecanoica

cidmethyl

ester(palmitica

cid)

(2)9

12-O

ctadecadieno

icacid

methyleste

rLino

leicacid

omega-6

(3)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(4)16-Octadecenoica

cid

methyleste

r(ste

aricacid)

(1)H

exadecanoica

cidmethyl

ester

(2)4

710-H

exadecatrie

noicacid

methyleste

rOmega-6

(3)O

ctadecadieno

icacid

methyleste

r(4)9

1215

-Octadecatrie

noic

acidm

ethyleste

r(Z

ZZ)

alph

a-lin

olenicacidomega-3

(5)16-Octadecenoica

cid

methyleste

r

(1)H

exadecanoica

cidmethyl

ester

(2)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(3)C

is-11-eicosano

icacid

omega-9

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r


Sciences Maracaibo Venezuela for the donation of microal-gae and the Universidad Libre Faculty of EngineeringBogota Colombia for the execution of chromatographicanalysis

References

[1] A Lopez Farre and C Macaya ldquoEfectos antitromboticos y anti-inflamatorios de los acidos grasos omega-3rdquo Revista Espanolade Cardiologıa vol 6 supplement pp 31ndash37 2006

[2] V Dıaz-Arguelles Ramırez-Corrıa ldquoDeficiencia de acidos gra-sos esenciales en el feto y en el recien nacido preterminordquoRevista Cubana de Pediatria vol 73 no 1 pp 43ndash50 2001

[3] M Rodrıguez-Cruz A R Tovar M del Prado and N TorresldquoMecanismosmoleculares de accion de los acidos grasos poliin-saturados y sus beneficios en la saludrdquo Revista de InvestigacionClınica vol 57 no 3 pp 457ndash472 2005

[4] M S Reece J A McGregor K G D Allen and M A HarrisldquoMaternal and perinatal long-chain fatty acids possible roles inpreterm birthrdquo American Journal of Obstetrics and Gynecologyvol 176 no 4 pp 907ndash914 1997

[5] J J Carrero E Martın-Bautista L Baro et al ldquoEfectos car-diovasculares de los acidos grasos Omega-3 y alternativas paraincrementar su ingestardquo Nutricion Hospitalaria vol 20 no 1pp 63ndash69 2005

[6] N I Velazquez Quintana J L Masud Yunes Zarraga and RAvila Reyes ldquoRecien nacidos con bajo peso causas problemasy perspectivas a futurordquo Boletın Medico del Hospital Infantil deMexico vol 61 no 1 pp 73ndash86 2004

[7] V Dıaz-Arguelles Ramırez-Corrıa ldquoLactancia materna eval-uacion nutricional en el recien nacidordquo Revista Cubana dePediatrıa vol 77 no 2 2005

[8] J P SanGiovanni and E Y Chew ldquoThe role of omega-3 long-chain polyunsaturated fatty acids in health and disease of theretinardquo Progress in Retinal and Eye Research vol 24 no 1 pp87ndash138 2005

[9] R Valenzuela B K Bascunan G and A Valenzuela B ldquoAcidodocosahexaenoico (DHA) una perspectiva nutricional para laprevencion de la enfermedad de Alzheimerrdquo Revista Chilena deNutricion vol 35 pp 250ndash260 2008

[10] C Perez Tarrago A Puebla Maestu and A Mijan de laTorre ldquoTratamiento nutricional en la enfermedad inflamatoriaintestinalrdquo Nutricion Hospitalaria vol 23 no 5 pp 418ndash4282008

[11] A Nasiff-Hadad EMeri and EMerino-Ibarra ldquoAcidos grasosomega-3 pescados de carne azul y concentrados de aceites depescado Lo bueno y lo malordquo Revista Cubana de Medicina vol42 no 2 pp 128ndash133 2003

[12] D Cardona ldquoTratamiento farmacologico de la anorexia-caquexia cancerosardquo Nutricion Hospitalaria vol 21 pp 17ndash262006

[13] J Quintero and J Rodrıguez-Quiros ldquoAspectos nutricionalesen el trastorno por deficit de atencionhiperactividadrdquo RevueNeurologique vol 49 pp 307ndash312 2009

[14] M Sekiya N Yahagi T Matsuzaka et al ldquoPolyunsaturated fattyacids ameliorate hepatic steatosis in obese mice by SREBP-1suppressionrdquo Hepatology vol 38 no 6 pp 1529ndash1539 2003

[15] J Singh and S Gu ldquoCommercialization potential of microalgaefor biofuels productionrdquo Renewable and Sustainable EnergyReviews vol 14 no 9 pp 2596ndash2610 2010

[16] T C Adarme-Vega D K Y Lim M Timmins F Vernen YLi and P M Schenk ldquoMicroalgal biofactories a promisingapproach towards sustainable omega-3 fatty acid productionrdquoMicrobial Cell Factories vol 11 article 96 2012

[17] J Van Wagenen T W Miller S Hobbs P Hook B Crowe andM Huesemann ldquoEffects of light and temperature on fatty acidproduction inNannochloropsis salinardquo Energies vol 5 no 3 pp731ndash740 2012

[18] S D Scott R E Armenta K T Berryman and A W NormanldquoUse of raw glycerol to produce oil rich in polyunsaturated fattyacids by a thraustochytridrdquo Enzyme and Microbial Technologyvol 48 no 3 pp 267ndash272 2011

[19] R A Bhosale M P Rajabhoj and B B Chaugule ldquoDunaliellasalina Teod as a prominent source of eicosapentaenoic acidrdquoInternational Journal on Algae vol 12 no 2 pp 185ndash189 2010

[20] C Hu M Li J Li Q Zhu and Z Liu ldquoVariation of lipid andfatty acid compositions of the marine microalga Pavlova viridis(Prymnesiophyceae) under laboratory and outdoor cultureconditionsrdquo World Journal of Microbiology and Biotechnologyvol 24 no 7 pp 1209ndash1214 2008

[21] T YagoHArakawa TMorinaga Y Yoshie-Stark andMYosh-ioka ldquoEffect of wavelength of intermittent light on the growthand fatty acid profile of the haptophyte Isochrysis galbanardquo inGlobal Change Mankind-Marine Environment Interactions H-J Ceccaldi I Dekeyser M Girault and G Stora Eds pp 43ndash45 Springer Dordrecht Netherlands 2011

[22] A Sahu I Pancha D Jain et al ldquoFatty acids as biomarkers ofmicroalgaerdquo Phytochemistry vol 89 pp 53ndash58 2013

[23] C M James S Al-Hinty and A E Salman ldquoGrowth and 1205963fatty acid and amino acid composition of microalgae underdifferent temperature regimesrdquo Aquaculture vol 77 no 4 pp337ndash351 1989

[24] L A Velasco J Barros-Gomez G H Ospina-Salazar andC A Trujillo ldquoEfecto de la intensidad lumınica temperaturay salinidad sobre el crecimiento de la microalga isochrysisgalbana (clon T-ISO)rdquo INTROPICA vol 4 no 1 pp 93ndash992009

[25] C Lobban P Harrison and M Duncan The PhysiologicalEcology of Seaweeds Cambridge University Press New YorkNY USA 1985

[26] E Y Dawson Marine Botany An Introduction Holt Rinehartand Winston New York NY USA 1966

[27] S M Renaud and D L Parry ldquoMicroalgae for use in tropicalaquaculture II effect of salinity on growth gross chemicalcomposition and fatty acid composition of three species ofmarine microalgaerdquo Journal of Applied Phycology vol 6 no 3pp 347ndash356 1994

[28] T Gallardo and M A Cobelas ldquoUna revision sobre la biotec-nologıa de las algasrdquo Botanica Complutensis vol 15 p 9 1989

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


be found in foods such as vegetable oil like soy safflower orcorn in dried fruit and seeds [16] and in small amounts inmeat birds and eggs [17]

One of the biggest problems to obtain these fatty acidsfrom fish oil is their residual taste which is not pleasantfor the human palate reducing its consumption and marketsales Due to the refinement process of this oil necessaryto eliminate the residual taste the final product is moreexpensive and on account of this alternative ways to obtainthese lipids have been proposed in order to replace PUFAsfrom fish oil using as feedstock another type of marine lifelike microalgae [15]

Recently it has been proved that certain species ofmicroalgae accumulate under specific culture conditionsa good amount of fatty acids [15] In particular somesaline strains present high percentages of omega oils thatallow the exploration of obtaining on a commercial scaleessential fatty acids such as docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA) Various studies developed withdifferentmicroalgae used to obtain these fatty acids are shownin Table 1

With the purpose of understanding which other speciesof saline algae can be good precursors to obtain fatty acidsthis study presents as the principal objective to determine thepotential of the species of salinemicroalgaeDunaliella salinaSinecosyfis sp and Chroomonas sp as sources of essentialfatty acids evaluating the best culture conditions that couldhelp generate a larger production of algae biomass amount ofoil and the ability to produce fatty acids of interest Besidesthis study explores the alternative of using commerciallyavailable feedstock such as commercial fertilizers and glyc-erol for the formulation of the growthmedium of the cultureof saline microalgae in order to reduce the cost associatedwith nitrogen and carbon sources for the production of fattyacids omega-3 using microalgae

2 Materials and Methods

21Microorganisms Themicroalgae used correspond to threespeciesDunaliella salina Chroomonas sp and Sinecosyfis spThese species were donated by the Ceparium of Photosyn-thetic Microorganismrsquos Laboratory of the Biology Depart-ment of the Experimental Science Faculty the Universidaddel Zulia Maracaibo VenezuelaThese strains were providedin test tubes with 10mL of algal culture medium containing4mM of nitrogen and a salinity of 35

22 Nutrients and Culture Conditions The algae were sub-merged in different conditions of growth alternating thenutrient medium to obtain biomass commercial ldquoNutrifo-liarrdquo fertilizer with a 4mM concentration of nitrogen andglycerol at 4mM of carbon both in filtrated sea waterand autoclaved The cultures were at controlled temper-atures of 20∘C and 32∘C The artificial illumination wassupplied through fluorescent lamps (Sylvania Daylight plusF96T12DLP) of 75W for all the cultures with a luminousflow of 16880 lm (16 lamps) The illumination source wasregulated a continuous exposition was supplied for selected

Table 1 Values () of EPA and DHA fatty acid contents withrelations to total lipids in some microalgae [16]

Microalgae of EPA and DHAproduction Reference

Nannochloropsis salinasim28 EPA [17]

Thraustochytrium sp 451 EPA + DHA [18]Dunaliella salina 214 EPA [19]Pavlova viridis 360 EPA + DHA [20]Isochrysis galbana

sim280 EPA + DHA [21]

cultures and others were submerged at a photoperiod oflight darkness 12 h 12 h The agitation and supply of airwere provided through air pumps (PowerLife P -500) whichprovide 225 L of airmin with the purpose of avoiding thesedimentation of algae and permit culture homogenization

Natural sea water was obtained from Salgar Beach in theAtlantic Coast Colombia coordinate latitude 11∘11015840166210158401015840Nlongitude 74∘56101584072410158401015840W The salinity of the sterile sea waterwas measured with conductivity electrodes WTW brandand Multi 3420 model The registered value was 375 and562mScm for salinity and conductivity respectively All thematerials usedwere previously sterilized in autoclave at 121∘C

23 Cell Growth Cell growth was determined by takingdaily samples of 5mL from each of the different culturesand determining the concentration of microalgae spectro-scopically (Spectronic Genesys 2) measuring the absorbanceat a wavelength of 647 nm and monitoring each culturein fixed 24-hour intervals Moisture content in centrifugedmicroalgae was obtained drying samples of microalgae at105∘C until constant weight

24 Lipid Extraction The lipid fraction of microalgae wasextracted using Soxhlet method which is based on theconstant evaporation and condensation through stages of avolatile solvent for posterior contact with the sample studiedQuantities of the biomass between 15 and 24 grams wereweighed for each microalga and were subjected to Soxhletextraction using analytic hexane (JT Baker) as solvent

A mixture of hexane and lipids was obtained from theprocess of extraction This mixture was separated through arotary evaporator Buchi Heating Bath Model B-490 at 48∘Cwith a rotation velocity 64ndash66 rpm for 45 minutes

25 Determination of the Fatty Acids Profile The extractedoils were transesterified adding 1mL of amethanolic solutionof NaOH 1 The mixture was heated to 55∘C for 15minand then 2mL of a methanol solution containing 2 ofHCl was added and heated for another 15min [22] Laterthe obtained FAMEs were dissolved in 1mL of hexane andinjected to an Agilent 7890 A gas chromatograph coupledto a selective mass detector 5975C (Agilent) with an AgilentSelect Biodiesel for FAME column (30m 020mm 025 120583m)using Helium as a carrier gas with a flow of 1mLmin and avolume of injection of 1 120583L with a temperature programmedat 60∘C for 2 minutes and then incremented in 15∘Cmin





Glycerol +


32 +


24 +


24 0 +


1620 +





6 + minus + + +







00

02

04

06

08

10

12

14

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Time (day)

Abso

rban

ce (A

U)














00

02

04

06

08

10

12

14

0 1 2 3 4 5 6 7 8 9 10 11 12

Abso

rban

ce (A

U)

Time (day)

















4 Conclusions


Competing Interests


Acknowledgments



Table6Identifi

edfatty

acidsa

ndqu

antityof

oilprodu

cedforthe

studied

microalgae

Experim

entn

umber

12

3

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationfor2

4ho

urssupp

lied1620

Ldayof

airtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationof

1212ho

urssupp

lied810L

day

ofairtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nand

illum

inationof

24ho

urssupp

lied1620

Ldayof

air

temperature

20∘

C

Microalgaes

pecies

Chroom

onas

sp

Dun

aliellasalin

aCh

room

onas

sp

Chroom

onas

sp

Sinecosyfis

sp

Quantity

ofwet

microalgae(g)

34

15155

24

Oilprod

uced

(mg)

5443

7314

2499

4069

8876

mgof

oilg

ofwet

biom

ass

1814

1829

1666

262

369

Extractedfatty

acids

(1)H

exadecanoica

cidmethyl

ester(palmitica

cid)

(2)9

12-O

ctadecadieno

icacid

methyleste

rLino

leicacid

omega-6

(3)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(4)16-Octadecenoica

cid

methyleste

r(ste

aricacid)

(1)H

exadecanoica

cidmethyl

ester

(2)4

710-H

exadecatrie

noicacid

methyleste

rOmega-6

(3)O

ctadecadieno

icacid

methyleste

r(4)9

1215

-Octadecatrie

noic

acidm

ethyleste

r(Z

ZZ)

alph

a-lin

olenicacidomega-3

(5)16-Octadecenoica

cid

methyleste

r

(1)H

exadecanoica

cidmethyl

ester

(2)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(3)C

is-11-eicosano

icacid

omega-9

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r



References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Glycerol +


32 +


24 +


24 0 +


1620 +





6 + minus + + +







00

02

04

06

08

10

12

14

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Time (day)

Abso

rban

ce (A

U)














00

02

04

06

08

10

12

14

0 1 2 3 4 5 6 7 8 9 10 11 12

Abso

rban

ce (A

U)

Time (day)

















4 Conclusions


Competing Interests


Acknowledgments



Table6Identifi

edfatty

acidsa

ndqu

antityof

oilprodu

cedforthe

studied

microalgae

Experim

entn

umber

12

3

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationfor2

4ho

urssupp

lied1620

Ldayof

airtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationof

1212ho

urssupp

lied810L

day

ofairtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nand

illum

inationof

24ho

urssupp

lied1620

Ldayof

air

temperature

20∘

C

Microalgaes

pecies

Chroom

onas

sp

Dun

aliellasalin

aCh

room

onas

sp

Chroom

onas

sp

Sinecosyfis

sp

Quantity

ofwet

microalgae(g)

34

15155

24

Oilprod

uced

(mg)

5443

7314

2499

4069

8876

mgof

oilg

ofwet

biom

ass

1814

1829

1666

262

369

Extractedfatty

acids

(1)H

exadecanoica

cidmethyl

ester(palmitica

cid)

(2)9

12-O

ctadecadieno

icacid

methyleste

rLino

leicacid

omega-6

(3)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(4)16-Octadecenoica

cid

methyleste

r(ste

aricacid)

(1)H

exadecanoica

cidmethyl

ester

(2)4

710-H

exadecatrie

noicacid

methyleste

rOmega-6

(3)O

ctadecadieno

icacid

methyleste

r(4)9

1215

-Octadecatrie

noic

acidm

ethyleste

r(Z

ZZ)

alph

a-lin

olenicacidomega-3

(5)16-Octadecenoica

cid

methyleste

r

(1)H

exadecanoica

cidmethyl

ester

(2)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(3)C

is-11-eicosano

icacid

omega-9

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r



References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







00

02

04

06

08

10

12

14

0 1 2 3 4 5 6 7 8 9 10 11 12

Abso

rban

ce (A

U)

Time (day)

















4 Conclusions


Competing Interests


Acknowledgments



Table6Identifi

edfatty

acidsa

ndqu

antityof

oilprodu

cedforthe

studied

microalgae

Experim

entn

umber

12

3

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationfor2

4ho

urssupp

lied1620

Ldayof

airtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationof

1212ho

urssupp

lied810L

day

ofairtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nand

illum

inationof

24ho

urssupp

lied1620

Ldayof

air

temperature

20∘

C

Microalgaes

pecies

Chroom

onas

sp

Dun

aliellasalin

aCh

room

onas

sp

Chroom

onas

sp

Sinecosyfis

sp

Quantity

ofwet

microalgae(g)

34

15155

24

Oilprod

uced

(mg)

5443

7314

2499

4069

8876

mgof

oilg

ofwet

biom

ass

1814

1829

1666

262

369

Extractedfatty

acids

(1)H

exadecanoica

cidmethyl

ester(palmitica

cid)

(2)9

12-O

ctadecadieno

icacid

methyleste

rLino

leicacid

omega-6

(3)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(4)16-Octadecenoica

cid

methyleste

r(ste

aricacid)

(1)H

exadecanoica

cidmethyl

ester

(2)4

710-H

exadecatrie

noicacid

methyleste

rOmega-6

(3)O

ctadecadieno

icacid

methyleste

r(4)9

1215

-Octadecatrie

noic

acidm

ethyleste

r(Z

ZZ)

alph

a-lin

olenicacidomega-3

(5)16-Octadecenoica

cid

methyleste

r

(1)H

exadecanoica

cidmethyl

ester

(2)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(3)C

is-11-eicosano

icacid

omega-9

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r



References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









4 Conclusions


Competing Interests


Acknowledgments



Table6Identifi

edfatty

acidsa

ndqu

antityof

oilprodu

cedforthe

studied

microalgae

Experim

entn

umber

12

3

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationfor2

4ho

urssupp

lied1620

Ldayof

airtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationof

1212ho

urssupp

lied810L

day

ofairtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nand

illum

inationof

24ho

urssupp

lied1620

Ldayof

air

temperature

20∘

C

Microalgaes

pecies

Chroom

onas

sp

Dun

aliellasalin

aCh

room

onas

sp

Chroom

onas

sp

Sinecosyfis

sp

Quantity

ofwet

microalgae(g)

34

15155

24

Oilprod

uced

(mg)

5443

7314

2499

4069

8876

mgof

oilg

ofwet

biom

ass

1814

1829

1666

262

369

Extractedfatty

acids

(1)H

exadecanoica

cidmethyl

ester(palmitica

cid)

(2)9

12-O

ctadecadieno

icacid

methyleste

rLino

leicacid

omega-6

(3)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(4)16-Octadecenoica

cid

methyleste

r(ste

aricacid)

(1)H

exadecanoica

cidmethyl

ester

(2)4

710-H

exadecatrie

noicacid

methyleste

rOmega-6

(3)O

ctadecadieno

icacid

methyleste

r(4)9

1215

-Octadecatrie

noic

acidm

ethyleste

r(Z

ZZ)

alph

a-lin

olenicacidomega-3

(5)16-Octadecenoica

cid

methyleste

r

(1)H

exadecanoica

cidmethyl

ester

(2)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(3)C

is-11-eicosano

icacid

omega-9

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r



References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table6Identifi

edfatty

acidsa

ndqu

antityof

oilprodu

cedforthe

studied

microalgae

Experim

entn

umber

12

3

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationfor2

4ho

urssupp

lied1620

Ldayof

airtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nandillum

inationof

1212ho

urssupp

lied810L

day

ofairtemperature

32∘

C

Nutrifoliarm

ediumperiodof

agitatio

nand

illum

inationof

24ho

urssupp

lied1620

Ldayof

air

temperature

20∘

C

Microalgaes

pecies

Chroom

onas

sp

Dun

aliellasalin

aCh

room

onas

sp

Chroom

onas

sp

Sinecosyfis

sp

Quantity

ofwet

microalgae(g)

34

15155

24

Oilprod

uced

(mg)

5443

7314

2499

4069

8876

mgof

oilg

ofwet

biom

ass

1814

1829

1666

262

369

Extractedfatty

acids

(1)H

exadecanoica

cidmethyl

ester(palmitica

cid)

(2)9

12-O

ctadecadieno

icacid

methyleste

rLino

leicacid

omega-6

(3)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(4)16-Octadecenoica

cid

methyleste

r(ste

aricacid)

(1)H

exadecanoica

cidmethyl

ester

(2)4

710-H

exadecatrie

noicacid

methyleste

rOmega-6

(3)O

ctadecadieno

icacid

methyleste

r(4)9

1215

-Octadecatrie

noic

acidm

ethyleste

r(Z

ZZ)

alph

a-lin

olenicacidomega-3

(5)16-Octadecenoica

cid

methyleste

r

(1)H

exadecanoica

cidmethyl

ester

(2)H

exadecanoica

cid1-

(hydroxymethyl)-12

-ethanediol

ester

(3)C

is-11-eicosano

icacid

omega-9

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r

(1)H

exadecanoica

cid

methyleste

r(2)O

ctadecanoica

cid

methyleste

r(3)8

11-O

ctadecadieno

icacidm

ethyleste

r



References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Dunaliella salina Sinecosyfis sp.,Chroomonas sp. at 20 C Sinecosys sp. at 20 C Dunaliella salina at 32 C Chroomonas sp. at 32 C Sinecosys sp. at 32 C F :Comparativegrowthcurvesof Dunaliellasalina