analytical methods for microalgae

7/27/2019 Analytical Methods for Microalgae

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Journalof Applied Phycology 3: 43-53, 1991. 1991 Kluwer Academic Publishers. Printed n Belgium. 43

Effect of light intensity on the proximate biochemical and fatty acidcomposition of Isochrysis sp. and Nannochloropsis oculata for use intropical aquacultureS.M. Renaudla, D.L. Parryla, Luong-Van, Thinh b, C. Kuo 2 , A. Padovanla.b & N. Sammy 3i a. School of Chemistry and Earth Sciences, b. School of Biological Sciences, Faculty of Science,Northern Territory University, P.O. Box 40146, Casuarina, N.T. 0811, Australia;2DepartmentofPrimaryIndustry and Fisheries, G.P.O. Box 990, Darwin, N.T. 0801, Australia;3Office of the Ministerfor Conservation, G.P.O. Box 3146, Darwin, N.T. 0801, Australia.Received 28 November 1990; accepted 12 December 1990

Key words: microalgae, fatty acids, mariculture, nutrition, capillary gas chromatography

AbstractThe total protein, carbohydrate, lipid and ash compositions, and fatty acid contents of two species ofmarine microalgae, the eustigmatophyte Nannochloropsisoculata (formerly 'Chlorellasp., Japan') and thechrysophyte Isochrysis sp. (Tahitian) used in tropical Australian mariculture, were studied. The micro-algae were grown under a range of culture conditions (4 1and 60 1 aboratory culture, 300 1bag culture,and 8000 1outdoor culture) and four light regimes (100 to 107 ktE m - 2 - , 240 to 390 E m- 2 s - ', 340to 62 0 iE m- 2 s -1, and 1100 to 1200 ,tE m-2 s- 1 respectively) to determine the effect of light intensityon the chemical composition of large scale outdoor cultures. Laboratory and bag cultures were axenicand cultured in Walne medium while outdoor cultures were grown in a commercial medium designed foroptimum nutrition in tropical outdoor aquaculture operations. Change in growth medium and photon fluxdensity produced only small changes in the proximate biochemical composition of both algae. N. oculataand Isochrysis sp. both showed a trend towards slightly lower carbohydrate and higher chlorophyll a inshaded outdoor culture. Isochrysis sp. showed significant concentrations of the essential polyunsaturatedfatty acid 22:6(n-3) (docosahexaenoic acid) from 5.3 to 10.3% of total fatty acid, and 20: 5(n-3)(eicosapentaenoic acid) ranged from 0.6 to 4.1%. In contrast, N. oculata had high concentrations of20: 5(n-3) (17.8 to 39.9%) and only traces of 22: 6(n-3). The fatty acid composition of Isochrysis sp.grown at high photon flux density (1100-1200 #tE m-2 s- ') under outdoor culture showed a decreasein the percentage of several highly unsaturated fatty acids, including 20: 5(n-3), and an increase in22 : 6(n-3). N. oculata showed a similar decrease in the percentage of 20: 5(n-3). High light intensitycaused a decrease in the ratio of total C, 6 unsaturated fatty acids to saturated 16: 0 in N. oculata, anda decrease in the ratio of total C,8 unsaturated fatty acids to saturated 18 :0 together with a decreasein the ratio of total unsaturated fatty acids to total saturated fatty acids in both microalgae.Introduction culture organisms, in spite of attempts by aqua-culturists to substitute artificial food sources. TheMicroalgae are the primary source of feed for at nutritional value of the microalgae is related toleast some stages in the life cycle of most aqua- their biochemical composition, especially the lipid


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45Japan' (Maruyama et al., 1986) and Isochrysis sp.(Chrysophyceae), also known as 'Tahitian Iso-chrysis', used in tropical Australian mariculture.The microalgae were grown under a range of cul-ture conditions, including laboratory culture, bagculture, and large scale outdour culture, underdifferent light regimes. We consider that one fac-tor that may be manipulated in tropical aqua-culture projects, where, typically there are manyhours of sunlight throughout the day for most ofthe year, is the photon flux density that reachesthe surface of the culture tank. Our aim was todetermine whether the modification in chemicalcomposition associated with changes in lightintensity, observed in laboratory cultures, couldbe demonstrated in large scale outdoor cultures.

Materials and methodsOrganismsIsochrysis sp. (Tahitian), T.ISO. (CS177) andNannochloropsis oculata (CS179) were obtainedfrom Dr S. Jeffrey, CSIRO Algal Culture Collec-tion, C.S.I.R.O. Division of Fisheries, Hobart,Tasmania.

enriched with commercial fertilisers. The com-mercial fertilizer was potassium nitrate-based forthe culture of Isochrysis sp., and ammonium sul-phate-based for the culture of N. oculata.Laboratory 41 , 601 and 300 1 cultures wereilluminated with 18 watt cool-white fluorescentlights on a 12: 12 h light/dark cycle at 25 + 1 C.Photon flux density (PFD) was measured with aLicor Model Li 1000 datalogger. PFD at depthwas measured using an underwater quantumsensor. Outdoor cultures were shaded using open-weave polypropylene shade-cloth supported onperspex sheeting. Constant mixing was achievedby use of an air lift system from the bottom of eachtank. Darwin (12 25 ' S, 130 52 ' E) has an aver-age of twelve hours of daylight with maximumPFD 2100 to 2200 uE m - 2 s - , and twelve hoursof darkness. The temperature within each tankranged from a minimum of 25 C to a maximumof 29 C over a 24 h period.Microalgal cells were harvested in late log-phase of growth by centrifugation using aBeckman Model J2-21M/E refrigerated cen-trifuge. Samples were lyophilized and stored at- 80 C prior to chemical analysis.

Analytical methodsGrowth conditionsThe experimental culture conditions, includingcell count at harvest, for each of the cultures areshown in Table 1.Microalgae were cultured in 4 1Erlenmeyer flasks, 60 1 ectangular aquaria, 300 1polythene bags, or 8000 1circular (diameter 3.8 m,depth 0.8 m) fibre glass tanks. Cell counts weremade daily, using a Neubauer haemacytometer,and algal cells harvested at late log-phase. Thesalinity of all culture media was adjusted to 25 pptwith distilled water before sterilization. Salinity ofoutdoor cultures was readjusted daily. Axeniclaboratory culture media were obtained by auto-claving at 103.4 kPa for 15 minutes. Culture bagswere sterilized by chlorination followed bysodium thiosulphate rinse. Outdoor culture mediawas prepared using 0.2 pm filtered seawater,

Total protein was determined using the Buchisemi-micro kjeldahl system with potentiometricendpoint detection, and protein calculated asnitrogen x 6.25 (Horwitz, 1980).Carbohydrates were analysed by the colori-metric phenol-sulphuric acid method with themeasurement of absorbance at 485 nm followinghot water extraction of the algal cells for 1 h on asteam bath (Dubois et al., 1956), using a VarianDMS 200 UV/Visible spectrophotometer.Total lipids were determined gravimetricallyafter extraction of the microalgae with chloro-form-methanol (2: 1) using the method of Folchetal., (1957) as modified by Bligh and Dyer(1959).Chlorophyll a was determined in the pigmentsextracted from 10 mg of lyophilized cells by soni-cation for 5 min in 1 ml of methanol, AR. This


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46Table 1. Algal identification, culture conditions and cell counts at harvest.Species & i.d. CSIRO Volume Medium Cells ml- Photon

culture (1) at harvest flux density(x 10) (E m- 2s- )

Isochrysis sp.A CS177 4 Walnea 5.08 13 5B 4 Walne 11.26 140C 4 Walne 12.06 140D 60 Walne 4.52 107E 300 Walne 2.21 107F 8000 Comm. Ib 0.41 1200G 8000 Comm. I 3.81 1200H 8000 Comm. I 0.78 620I 8000 Comm. I 1.24 620J 8000 Comm. I 1.11 620K 8000 Comm. I 0.98 390L 8000 Comm. I 1.27 390M 8000 Comm. I 1.44 390Nannochloropsis oculataA CS179 4 Walne 8.49 107B 4 Walne 5.69 100C 4 Walne 49.1 100D 60 Walne 59.0 100E 300 Walne 8.50 107F 300 Walne 10.49 107G 8000 Comm. IIF 6.55 1100H 8000 Comm. II 9.17 340I 8000 Comm. II 8.22 340J 8000 Comm. II 7.97 340K 8000 Comm. II 5.88 243L 8000 Comm. II 4.56 243M 8000 Comm. II 6.40 243aWalne, 1966.b Commercial I Potassium nitrate (100-150 mg 1- ), sodium dihydrogen phosphate (10-15 mg 1- ), ferric chloride (5 mg 1-'),

EDTA (5 mg 1- ) + Walne medium trace metals.c Commercial II Ammonium sulphate (150 mg 1- ), urea (7.5 mg 1-), superphosphate (25 mg 1-), ferric chloride (5 mg 1- ),EDTA (5 mg 1-').

was followed by 24 h extraction in the dark at4 C after the addition of 10 ml of acetone, AR,and 10 mg of magnesium carbonate, AR. Chloro-phyll a was determined spectrophotometricallyand calculated using the equations of Jeffrey andHumphrey (1975).

Total ash was determined gravimetrically afterashing at 540 C.Fatty acid methyl esters (FAME) were pre-pared by transesterification of the saponifiedlipids with 14% methanol-BC13 at 60 C for

15 minutes. FAME samples were analysed usinga Varian 6000 gas chromatograph with FIDdetector using a fused-silica column (SGEBP225, 50% cyanopropyl 50% phenyl dimethyl-siloxane, 25 m x 0.22 mm i.d.). Data were col-lected and manipulated using the Delta chromato-graphy data system. Fatty acids were identified bycomparison with retention times of known stand-ards obtained from Sigma Chemical Co. andAldrich Chemical Co. and using cod liver oil as asecondary standard. Identities were verified using


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47a second column (Alltech RSL150, polymethyl-siloxane, 25 m x 0.35 mm i.d.) and calculationsof relative retention (Ackman, 1986). The molecu-lar weights of fatty acids were confirmed byGC-MS analysis with an HP5890 GC and 5790MSD using the SGE, BP225 capillary column.The shorthand notation used in fatty acid identifi-cation is L: B(n-x), where L is the chain length,B is the number of double bonds, and x is theposition of the ultimate double bond from theterminal methyl group.

Results and discussionSurface irradianceThe surface irradiance (PFD) of microalgal cul-tures, measured in late log-phase when cell den-sities and self-shading were at a maximum, isgiven in Table 1. Irradiance at the bottom of all8000 1 outdoor cultures averaged 5.68 + 1.48 /MEm-2 s-.

Table 2. Fatty acid composition of Nannochloropsis oculatacultured in two different nitrogen-based media, at PFD1100#E m- 2 s- lFatty acid Outdoor culture 80001(% of totalfatty acids) Commercial I Commercial II

NO3 NH 412:0 1.7 0.614:0 6.3 6.414: 1 0.1 0.315:0 0.2 2.016:0 26.0 30.016 : (n-9) 21.7 21.416:2 1.3 3.418:0 4.0 2.218: 1(n-9) 11.6 6.518: 2(n-6) 5.2 2.818: 3(n-6) 0.2 0.518: 3(n-3) nd 0.318:4(n-3) 3.1 0.420:0 nd 0.220: l(n-9) 3.6 5.120: 5(n-3) 13.0 17.822: 6(n-3) nd 0.2Total lipid, % OW 30.3 36.6% OW percentage of organic matter.

Culture solutions nd no t detected.Axenic laboratory cultures grown under optimumconditions in potassium nitrate-based Walnemedium (Walne, 1966) provided the baseline forou r study of the effect of increased culture vol-ume, commercial growth media and natural sun-light in larger scale out-door culture. Growthmedia derived from fertilizers that are readilyavailable commercially were used in the largescale outdoor cultures. Outdoor cultures of Iso-chrysis sp. were grown in a potassium nitrate-based medium (Commercial I). Large scale cul-tures of Nannonochloropsisoculata were grown inan ammonium sulphate-based medium (Com-mercial II). This ammonium sulphate-based cul-ture solution was chosen for outdoor culture ofN. oculata over a nitrate-based culture mediumdue to results of preliminary outdoor trials(Table 2) which showed a higher total lipid con-tent (36.6% of organic weight compared with30.3% OW) and higher percentage composition

of the essential, highly unsaturated fatty acideicosapentaenoic acid, 20:5(n-3) (17.8% OWcompared with 13.0% OW) when the microalgawas grown in the Commercial II medium.

Proximatecellularchemical compositionThe proximate chemical composition of Isochrysissp. and N. oculata are shown in Tables 3 and 4.a. Comparison of laboratory cultures with out-door

cultures grown at low irradianceLaboratory cultures of Isochrysis sp. and largescale outdoor cultures, grown at lowest PFD(K, L and M in Table 3), showed little differencein gross biochemical composition, except that theconcentrations of chlorophyll a in laboratory cul-tures were an average of 1% (organic weight)lower than those of the out-door cultures. The


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50Table 5. Fatty acid composition of Isochrysis sp. under four different culture conditions and four light regimes.Fatty acid Laboratory culture Outdoor culture(% of totalfatty acids) 4 1 60 1 300 1 8000 1

A B C D E F G H I J K L M1200IE m-2 s- ' 620uEm-2 s - ' 390pE m-2 s -

12 : 0 tr 0.6 tr 0.4 tr 3.1 1.9 tr 0.1 tr tr 0.3 tr14:0 21.5 21.6 17.5 18.5 14.0 15.3 15.9 14.8 14.6 15.0 17.7 19.2 18.914:1 0.6 0.5 tr 0.4 0.5 1.3 1.5 0.9 0.4 1.1 0.5 0.1 0.415:0 1.1 0.7 0.8 0.4 1.7 2.6 1.9 nd nd nd nd nd nd16:0 11.2 13.3 9.1 9.1 10.1 16.6 16.3 9.1 9.2 8.3 9.1 9.2 8.616: 1(n-7) 1.8 3.8 6.0 8.2 6.0 6.4 8.7 6.8 6.3 6.6 7.4 8.4 7.816: 2(n-4) 5.1 1.2 4.3 0.8 2.1 3.1 3.2 4.7 3.6 2.8 3.8 3.5 4.118:0 0.8 0.6 0.8 0.8 2.2 2.9 2.0 1.8 0.6 2.2 0.5 0.7 0.418: (n-9) 13.3 11.1 10.6 8.3 10.1 9.2 6.3 7.4 9.2 9.6 8.8 9.1 9.218: 2(n-6) 3.5 3.8 5.0 9.8 2.8 3.0 3.2 4.3 5.1 5.6 6.6 7.2 8.318: 3(n-6) 0.8 1.0 1.3 3.5 tr 2.1 1.6 1.8 1.6 1.8 2.0 2.8 3.518: 3(n-3) 5.5 5.4 5.7 5.0 6.8 4.1 4.7 7.0 6.0 5.7 5.2 4.8 5.118: 4(n-3) 20.8 22.7 23.9 20.5 28.4 16.7 14.3 24.7 24.0 24.9 24.4 20.6 21.018: 5(n-3) 3.4 3.8 2.8 2.6 tr 7.6 7.2 6.1 4.9 6.8 4.7 5.1 3.220 : 1(n-9) 1.0 0.6 1.0 1.3 0.8 nd nd nd nd nd nd nd nd20 : 5(n-3) 0.7 0.6 0.9 4.1 2.0 1.5 1.6 1.6 1.2 1.3 0.8 0.9 1.122: 5(n-6) tr 1.1 1.3 0.7 1.4 nd nd nd nd nd nd nd nd22 : 6(n-3) 8.7 7.7 9.0 5.3 8.7 5.5 10.0 9.2 10.3 8.2 9.0 7.6 7.716 unsat./716:0 0.61 0.39 1.13 0.97 0.80 0.57 0.73 1.27 1.06 1.13 1.22 1.29 1.39I18 unsat./i18 :0 60.1 83.9 63.1 59.9 22.2 12.0 18.7 48.2 160 24.3 95.0 76.3 12020:5/22:6 0.08 0.08 0.10 0.77 0.23 0.28 0.13 0.15 0.08 0.16 0.08 0.12 0.14Z unsat./Z sat. 3.05 3.28 2.54 2.39 2.53 1.74 1.65 2.76 2.41 2.92 2.65 2.33 2.49tr = trace.nd = no t detected.

1200 E m - 2 s -1 . In particular, the concen-tration of 18: 4(n-3) was decreased by 6.5% andthe concentration of 18: 2(n-6) was decreased by4.3% of total fatty acids. Concentrations of thesaturated fatty acids 16: 0 and 18: 0 increasedwith increasing irradiance by 7.5% and 2%respectively. There were small increases in theconcentrations of two highly unsaturated fattyacids, 22: 6(n-3), and 18: 5(n-3) associated withincreasing irradiance (2% and 2.9% respec-tively).Eicosapentaenoic acid, 20: 5(n-3), had a smallbut constant concentration, suggesting that it wasuneffected by irradiance. Harrison et al . (1990)found similar results for 20: 5(n-3) and a slightlylarger increase (4%) in the concentration of

22: 6(n-3) in laboratory cultures of Isochrysisgal-bana Green (Tahitian Isochrysis) grown at50 Em-2s - ' and 255)uEm-2s -1, and adecrease of 1% between cells grown at125p m- 2 s-1 and 255, Em - 2 s-1 . The per-centage differences in fatty acid composition inour study are similar to those reported byThompson et al. (1990) for the Tahitian Isochrysisaff. galbana grown in laboratory culture withirradiances of 225 E m - 2 -1 and125 /E m- 2 s -1. One difference was that we didnot find an increase in 18: l(n-9) concentrationwith increased PFD.

Cells of N. oculata grown at high irradiance(1100 E m - 2 s-l) had decreased concentra-tions of the highly unsaturated fatty acids


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51Table 6. Fatty acid composition of Nannochloropsis oculata under four different culture conditions and four light regimes.Fatty acid Laboratory culture Outdoor culture(% of totalfatty acids) 4 1 601 300 1 80001

A B C D E F G H I J K L M1100 340 243/pE m-2 s'

12 : 0 0.5 0.5 0.7 0.6 0.8 0.5 0.9 tr tr 0.4 1.0 0.63 0.914:0 5.7 5.3 6.8 5.2 8.2 5.8 5.4 5.7 4.3 4.4 4.9 4.96 4.914 : 1 0.3 0.6 1.0 0.2 0.3 0.5 tr tr 0.2 0.2 0.4 tr 0.615:0 0.3 0.4 tr 0.7 0.6 0.7 1.4 1.1 tr 0.4 0.3 0.40 1.116:0 21.2 20.5 23.9 18.6 16.0 18.3 18.4 14.7 14.7 18.0 14.5 14.21 14.016: (n-9) 25.9 25.0 28.4 26.1 23.0 29.8 19.9 22.5 22.4 22.5 19.4 21.62 19.316 : 2(n-4) 1.5 0.9 0.7 1.1 1.1 2.1 7.6 4.3 5.3 3.8 5.6 4.61 6.418:0 0.3 0.3 0.5 0.4 0.4 0.8 1.1 0.7 0.9 0.7 0.9 0.69 1.018: (n-9) 2.2 3.7 4.2 3.1 2.8 3.9 5.0 3.7 4.2 4.3 5.0 4.27 3.118: 2(n-6) 2.2 2.4 3.3 2.4 3.0 1.9 2.5 2.7 2.7 2.9 3.3 2.19 2.618: 3(n-6) 0.4 0.6 0.5 0.9 1.1 0.5 1.0 tr 1.2 0.6 tr tr tr18: 3(n-3) 0.7 0.3 tr 0.4 0.9 0.4 0.6 0.7 0.8 0.1 0.6 0.71 0.818: 4(n-3) tr tr tr 0.3 2.0 0.8 1.0 tr tr 0.5 tr 0.18 tr20: 0 tr tr tr 0.4 0.4 0.3 tr tr tr 0.2 tr 0.58 tr20 : 1(n-9) 4.5 4.8 5.6 4.2 4.1 5.9 5.1 6.5 5.9 6.6 5.1 6.05 5.620 : 5(n-3) 34.3 34.8 24.5 35.5 35.0 27.7 33.8 37.2 37.7 37.0 39.1 38.90 40.022 : 6(n-3) nd nd nd nd 0.6 nd 0.2 nd tr tr nd nd ndl16 unsat./16:0 1.29 1.27 1.22 1 .46 1.51 1.74 1.28 1.82 1.89 1.41 1.72 1.85 1.851l8 unsat./18 :0 18.4 24.0 17.8 19.3 26.9 9.68 9.18 9.53 10.4 11.48 9.44 10.6 6.79

E unsat./E sat. 2.58 2.71 2.15 2.93 2.78 2.82 2.81 3.51 4.05 3.16 3.62 3.79 3.59tr = trace.nd = not detected.

16: 2(n-4), 18: 3(n-3), and 20: 5(n-3). The con-centration of the major fatty acid, 20:5(n-3),decreased by 5.5% while concentrations of thesaturated fatty acid 16: 0 increased by 4% at highphoton flux densities. Sukenik etal. reportedsimilar results with a reduction in the percentageconcentration of 20: 5(n-3) (from 10% to 5% oftotal fatty acids) in laboratory cultures ofNannochloropsis sp. grown at irradiances of290btEm- 2 s- and 550/pEm- 2s- respec-tively.High ratios of (n-3) to (n-6) polyunsaturatedfatty acids have been used as an index of highnutritional value to aquaculture animals (Wa-tanabe et al., 1983). The total unsaturation ratios,the ratios of total unsaturated C16 fatty acids to16: 0, and the ratios of total unsaturated C18 fattyacids to 18: 0 are shown in Tables 5 and 6. Fatty

acid composition of Isochrysis sp. grown at highPFD (1200pEm-2s - 1 ) in outdoor cultureshowed decreases in the ratio of total C18 unsatu-rated fatty acids to saturated 18:0, total C16unsaturated fatty acids to 16: 0 and total unsatu-rated fatty acids to total saturated fatty acids.N. oculata showed similar decreases in theseratios when grown at high PFD in outdoor cul-ture. These indexes may be used in conjunctionwith consideration of which fatty acids are pres-ent to indicate the nutritional value of microalgalcells to aquaculture animals (Volkman et al.,1989).Increase in temperature may influence the fattyacid composition of microalgae. James et al.(1989) reported that Nannochloropsis strainMFD-2 grown at 35 C showed a decline of 4%in the content of 20.5(n-3) when comparing cul-


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52tures grown at 25 C and 30 C. Temperatures ofthe outdoor cultures in our study, ranging from25 C to 29 C have not caused a decrease in thepercentage of20: 5(n-3) and we found little differ-ence in the percentage of 20: 5(n-3) between labo-ratory cultures grown at 25 + 1 C and theoutdoor cultures.The optimum surface photon flux density forproducing the highest percentages of unsaturatedfatty acids and highest ratios of unsaturated tosaturated fatty acids is


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53James CM, Al-Hinty S, Salman AE (1989) Growth and o3fatty acid and amino acid composition ofmicroalgae under

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analytical methods for microalgae

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