ARTICLE

Growth, Lipid Content, Productivity, and FattyAcid Composition of Tropical Microalgae forScale-Up Production

Roger Huerlimann, Rocky de Nys, Kirsten Heimann

School of Marine and Tropical Biology, James Cook University, Townsville 4811,

Queensland, Australia; telephone: þ61-7-4781-5795; fax: þ61-7-4725-1570;

e-mail: kirsten.heimann@jcu.edu.au

Received 1 February 2010; revision received 2 May 2010; accepted 12 May 2010

Published online 20 May 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10

.1002/bit.22809

ABSTRACT: Biomass and lipid productivity, lipid content,and quantitative and qualitative lipid composition arecritical parameters in selecting microalgal species for com-mercial scale-up production. This study compares lipidcontent and composition, and lipid and biomass produc-tivity during logarithmic, late logarithmic, and stationaryphase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp.,and Rhodomonas sp. grown in L1-, f/2-, and K-medium. Ofthe tested species, Tetraselmis sp. exhibited a lipid produc-tivity of 3.9–4.8 g m�2 day�1 in any media type, with com-parable lipid productivity by Nannochloropsis sp. andIsochrysis sp. when grown in L1-medium. The dry biomassproductivity of Tetraselmis sp. (33.1–45.0 g m�2 day�1)exceeded that of the other species by a factor 2–10. Ofthe organisms studied, Tetraselmis sp. had the best drybiomass and/or lipid production profile in large-scale cul-tures. The present study provides a practical benchmark,which allows comparison of microalgal production systemswith different footprints, as well as terrestrial systems.

Biotechnol. Bioeng. 2010;107: 245–257.

� 2010 Wiley Periodicals, Inc.

KEYWORDS: aquaculture; tropical microalgae; productiv-ity; lipid composition; biodiesel

Introduction

Microalgae produce a variety of lipids of nutritionalimportance. Thus, microalgae are a critical component ofthe aquaculture food chain mainly for live feeds for larvalculture (e.g., Brown et al., 1997; Martinez-Fernandez et al.,2006). Lipid content and productivity are also sought afterin alternative applications, for example, biodiesel (Chisti,2007; Hu et al., 2008; Schenk et al., 2008), health food

Correspondence to: K. Heimann

Additional Supporting Information may be found in the online version of this article.

� 2010 Wiley Periodicals, Inc.

(Natrah et al., 2007; Plaza et al., 2008), animal feeds andfertilizers (reviewed in Pulz and Gross, 2004; Spolaore et al.,2006), and bioplastics (Murphy, 2006).

Lipid composition and productivity depend on growthconditions such as growth phase (Xu et al., 2008), mediumcomposition (Valenzuela-Espinoza et al., 2002), irradiance(Thompson et al., 1993), and temperature (Renaud et al.,2002). Qualitatively, the profiles of fatty acids (FAs), rangingin length from 10 to 24 carbons, are often similar betweenspecies of the same phyla or classes but differ greatly betweenclasses and phyla (Hu et al., 2008; Mourente et al., 1990)from low-yield cyanobacteria to oleaginous (oil-rich)species/strains from eukaryotic algal taxa (Hu et al.,2008). With the exception of cyanobacteria, lipid contentincreases significantly in all microalgal taxa under nitrogen-limiting conditions (Hu et al., 2008).

Quantitatively, the total lipid content varies betweenspecies ranging from very low (4.5%) to very high (80%)(Hu et al., 2008; Renaud et al., 1999). Similarly, lipid contentfor a given species depends on growth phase with lowestyields common for logarithmic-increasing in late logarith-mic-and stable or increasing in stationary phase (Hu et al.,2008; Xu et al., 2008). During logarithmic growth, mostlipids are glycerol-based polar membrane lipids, whichfunction to maintain cell structure (Hu et al., 2008). Incontrast, triacylglycerides (TAGs) are neutral lipids used forstorage without structural function. TAG production isenhanced under unfavorable conditions, which inducecessation of cell division and diversion of photosyntheticenergy into TAG production (Guschina and Harwood,2006; Harwood and Jones, 1989; Hu et al., 2008; Sheehanet al., 1998).

Quantitative and qualitative differences in the lipidcontent of microalgae affect the use of microalgal oil.Traditionally, microalgal lipids have been used as a dietarysource for metabolic energy and essential components forgrowth of animals in aquaculture, with the FA content andcomposition being the central factor in the selection of

Biotechnology and Bioengineering, Vol. 107, No. 2, October 1, 2010 245

microalgal species, as this regulates the productivity of thecultured species (e.g., Renaud et al., 1999; Martinez-Fernandez et al., 2006). Long chain v-3 polyunsaturatedFAs (PUFAs), in particular eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), are essential for the growth,development, and reduced mortality of marine fish larvae(Eamta et al., 2003), shrimp (Cavalli et al., 1999), andmollusks (Knaur and Southgate, 1999; Langdom andWaldock, 1981). Furthermore, ratios of v-3/v-6 FAs >2are considered to be optimal for larvae and juvenile oysters(Enright et al., 1986; Fidalgo et al., 1998). The same essentialv-3 PUFAs for aquaculture are also used as nutraceuticals toimprove human health. EPA and DHA reduce bloodpressure and viscosity, low-density lipoprotein (LDL) count,platelet aggregation, cardiac arrhythmia, and plasmatriglyceride levels, which is beneficial for the immunesystem and in the prevention of cardiovascular diseases (e.g.,Das, 2008; Ruxton et al., 2004). They are also proposed toalleviate neuropsychiatric disorders (Young and Conquer,2005). As with aquaculture feeds, ingesting larger amountsof v-6 FAs compared to v-3 FAs can have adverse effects onhealth, especially on cardiovascular diseases (Lands, 2005).PUFAs are mainly obtained from fatty fish species such asherring, mackerel, sardine, and salmon (Gunstone, 1996),and the demand will soon exceed current yields ofaquaculture and fisheries (Sijtsma and de Swaaf, 2004),demanding new sources of supply. Microalgae produce highlevels of v-3 PUFAs and are an obvious source given theselection of appropriate species and large-scale culturetechnologies (Brown et al., 1997; Molina-Grima et al., 2003;Zhukova and Aizdaicher, 1995).

Microalgal lipids are also being targeted as a feedstock forthe sustainable production of biodiesel (Chisti, 2007; Schenket al., 2008). The production of biodiesel from microalgaerequires the transesterification of TAGs to FA alkyl esters(Sheehan et al., 1998). The fuel quality of biodiesel (i.e.,cetane number, exhaust emission, heat of combustion, coldflow, oxidative stability, viscosity, and lubricity) (Knothe,2005) depends on the characteristics of the individual FAalkyl esters, which are mainly determined by the structuralfeatures of the FAs (chain length and alcohol rest, number ofdouble bonds or unsaturation, and chain branching)(Knothe, 2005; Schenk et al., 2008). The quality of biodieselto be produced can be directed by selecting an appropriatemixture of the different types of FAs through the selection ofproduction organisms, enriching the oil with desired FAs orby genetically modifying the microalgal species (Knothe,2005). One recommended optimal ratio of FAs for goodbiodiesel properties (low oxidative potential while retaininggood cold flow characteristics and a high cetane number) is5:4:1 for C16:1, C18:1, and C14:0 (Schenk et al., 2008). Asbiodiesel has to be competitive with conventional fossilfuels, value-adding products, which are mainly derived afterlipid extraction (e.g., animal feeds, Spolaore et al., 2006; orbiochar, which stores CO2 and enhances soil properties forincreased fertility, Marris, 2006) are important to decreaseproduction cost.

246 Biotechnology and Bioengineering, Vol. 107, No. 2, October 1, 2010

The geographic advantage of the tropics in terms of solarenergy and temperature provides an impetus for massproduction of tropical microalgae for the production ofbiocommodities. Of particular interest is the selection ofspecies that combine high levels of lipid and biomassproductivity with lipid content and FA profiles suitable forthe production of nutraceuticals based on v-3 PUFAs, or theproduction of saturated lipids for biodiesel production.Many studies target the lipid content and FA composition inlate logarithmic phase and a single medium (Martinez-Fernandez et al., 2006; Natrah et al., 2007; Renaud et al.,2002; Zhukova and Aizdaicher, 1995). This limits the abilityto determine quantitative and qualitative effects of thelogarithmic and stationary phase as well as media type onlipid content and productivity.

This study aimed to quantify the effect of growth mediaand growth phase on the qualitative and quantitative lipidcontent, dry biomass, and lipid productivity of four selectedtropical marine microalgae, Isochrysis sp. (Haptophyta),Nannochloropsis sp. (Eustigmatophyceae), Tetraselmis sp.(Prasinophyceae), and Rhodomonas sp. (Cryptophyta), withthe objective of selecting species for scale-up production.

Materials and Methods

Organisms and Growth Conditions

Isochrysis sp. (CS-177) was obtained from CSIRO, whileNannochloropsis sp. (NQAIF010), Tetraselmis sp.(NQAIF012), and Rhodomonas sp. (NQAIF040) wereobtained from the North Queensland Algal Identification/Culturing Facility (NQAIF) at James Cook University (JCU)in Townsville, Australia, which had been isolated from theNelly Bay, Magnetic Island, Townsville, Australia(NQAIF010 and NQAIF012) and at the mouth of RossCreek, Townsville, Australia (NQAIF040). As axeniccultures often perform poorly in terms of biomassproductivity (Lorenz et al., 2005), xenic, monoclonalcultures were maintained in 150 mL L1-medium(Andersen et al., 2005) in 250 mL Erlenmeyer flasks at248C, an L/D cycle of 12:12 h, and a photon flux intensity of43mmol m�2 s�1 provided by cool white fluorescent lightsand were sub-cultured every 3 weeks. Cultures wereroutinely examined for bacterial loads using differentialinterference contrast with a 100� oil immersion objectiveon an Olympus BX51 fitted with a CCD-cooled digitalcamera (Olympus DP70), as per Heimann et al. (2002).Cultures were maintained at a bacteria to algal ratio of lessthan 1:104 cells, which was verified using a hemocytometerand Evans Blue (1%; Sigma-Aldrich, Sydney, New SouthWales, Australia) stained culture material. Seawater forscale-up cultures was filtered through a 1.2mm WhatmanGF/C and stored in a non-transparent carboy and wascomplemented with nutrient stocks followed by autoclaving.Seawater for media of 10 L cultures in 15 L carboys wasfiltered through a filter system consisting of a 1.0mm filter, a

0.35mm filter, and an activated charcoal filter (9.75 in.cartridges), followed by UV irradiation (Wedeco LCP-8).

For growth and lipid analyses, triplicate replicates of eachspecies were cultured in 10 L of L1-, f/2-, and K-medium(Andersen et al., 2005), respectively. Cultures were main-tained at 24� 18C, a L/D photoperiod of 12:12 h and a lightintensity of 250mmol photons m�2 s�1 provided by coolwhite fluorescent tubes. Cell density was determined as %transmission at 750 nm (turbidity) on a UV/Vis SpectramaxPlus in 96-well microtiter plates (A5P96; LivingstoneInternational, Rosebery, New South Wales, Australia) intriplicate daily between 7:30 am and 9:00 am. Linearregression curve fit equations were used to calculate celldensity from % transmission at 750 nm.

Scale-Up to 10 L Aerated Batch Cultures in 15 L Carboys

Based on an inoculation density (ID) of 1.0� 105 cells mL�1,required scale-up procedures varied with species anddepended on maximal cell densities of stock and inter-mediate scale-up cultures. Three replicate 1 L cultures ofNannochloropsis sp., Isochrysis sp., and Tetraselmis sp. wereinoculated with an ID of 1.0� 105 cells mL�1 from 150 mLstock cultures per medium type. Pooled 1 L replicates ofeach species and medium were used to inoculate threereplicate 10 L cultures with an ID of �1.0� 105 cells mL�1.Media were complemented with autoclaved nutrient stocks.

Slow growth of Rhodomonas sp. required smaller scale-upsteps to maintain an ID of 1.0� 105 cells mL�1 for eachscale-up step. Therefore, three replicate 0.5 L cultures wereinoculated from 150 mL stock cultures per medium type,which were used to inoculate 1 L cultures. The 1 L batchcultures were scaled up to 2.0–3.5 L in 15 L carboys,depending on the medium type and then to 10 L after 10days. All scale-up cultures were supplemented withautoclaved media stocks.

Sampling and Harvesting for Lipid and FattyAcid Analysis

Approximately, 1 g of wet biomass per medium type,species, and growth phase was harvested for accurate lipidand FA analyses from the three replicate cultures percondition and species and pooled (Supplementary MaterialTable I). No samples were harvested for cultures ofNannochloropsis sp. in the logarithmic phase in K-mediumdue to poor growth. Samples were centrifuged followingMartinez-Fernandez et al. (2006) at 3,000g at 158C for30 min. To remove salt, the cell pellets were resuspended in200 mL ammonium formate (0.5 M, pH 8.0, adjusted with1 M NaOH) and centrifuged again at 3,000g at 158C for15 min. Cell pellets were kept under nitrogen in 50 mLcentrifuge tubes (Iwaki, Lomb Scientific, Taren Point, NewSouth Wales, Australia) and stored at �808C until analysis.Samples, shipped on dry ice, were analyzed by theDepartment of Primary Industry (DPI) Queensland fordry weight, total lipid content, and FA composition.

Lipid, Fatty Acid Methyl Ester, and Dry Matter Analyses

Lipids were extracted from samples with chloroform/methanol by the method of Folch et al. (1957). Total lipidwas determined gravimetrically on an aliquot of the extractby drying for 4 h at 808C in a pre-weighed glass vial. Afurther aliquot of the extract was taken for FA analysis. Thelipid FAs in the extract were derivativized to their FA methylesters (FAME) using 14% boron trifluoride–methanol (VanWijngaarden, 1967). FAME were analyzed on an AgilentTechnologies 6890 gas chromatograph using split injectionwith helium carrier gas and a flame ionization detector. Thecolumn used was a DB23 fused silica capillary column,30 m� 0.25 mm, with a 0.25mm coating (AgilentTechnologies, Forest Hill, Victoria, Australia). Columnoven temperature was held at 1408C for 5 min and thenelevated at 38C/min to 2108C where it was held until allFAME of interest had been eluted. FAME were identified bycomparing their retention times with those of authenticstandards (Sigma–Aldrich, Sydney, New South Wales,Australia), and were quantified by comparison with theresponse of an internal standard, heneicosanoic acid. Drymatter was determined by oven drying a sub-sample toconstant weight at 1058C.

Variability of Dry Matter, Lipid Content, and FAComposition Analyses

To obtain an estimate of the variability of dry matter, lipidcontent, and FA composition, two sample replicates wereharvested for Nannochloropsis sp. grown in L1- and f/2-medium on subsequent days in late logarithmic phase. Thevariability (coefficient of variation, which is the standarddeviation divided by the mean, expressed in %) dry matter,lipid content, and FA analyses ranged from 12.8 to 12.9%CV, 4.0 to 4.1% CV, and 0.0 to 10.1% CV, respectively (datanot shown). The variability was considered sufficiently lowand average values were reported for those samples.

Productivity

Biomass and lipid productivity during logarithmic phasewas calculated as follows. The specific growth rate wascalculated using the equation m ¼ LnðNy=NxÞ=ðty � txÞ(Wood et al., 2005) with Ny and Nx being the number ofcells (N) at the start (tx) and the end (ty) of the logarithmicgrowth phase. The specific growth rate (m [day�1]), drymatter (DM [g m�3]), and diameter of the carboys(d¼ 0.21 m) were used to calculate culture productivity(PDM) according to the equation PDM ¼ DM� m� d

(g m�2 day�1) (modified from Lanier, 1986). Lipid content(LC [%]) and productivity (PDM [g m�2 day�1]) were usedto calculate lipid productivity (PL) according to theequation PL ¼ PDM � LC=100% (g m�2 day�1). For com-parison with other studies which report their results inmg L�1 day�1, DM (g m�3) was converted into DM(mg L�1) and lipid productivity was calculated according

Huerlimann et al.: Lipid Productivity of Tropical Microalgae 247

Biotechnology and Bioengineering

Table I. Specific growth rates (m) of Nannochloropsis sp., Isochrysis sp.,

Tetraselmis sp., and Rhodomonas sp. in L1-, f/2-, and K-medium.

L1-medium f/2-medium K-medium

Nannochloropsis sp. 0.41 0.33 0.32

Isochrysis sp. 0.25 0.18 0.10

Tetraselmis sp. 0.19 0.14 0.24

Rhodomonas sp. 0.28 0.26 0.30

to the following equation PL ¼ DM� m� LC=100%(mg L�1 day�1).

Results and Discussion

Specific Growth Rates and Maximal Cell Densities

Nannochloropsis sp. exhibited the fastest specific growthrates (0.32–0.41) in all media (Table I) and reached thehighest cell density (�2.5� 107 cells mL�1 in L1- and f/2-medium) (Fig. 1a), while Rhodomonas sp. with the secondfastest specific growth rates (0.26–0.30) in all media(Table I) reached the lowest cell densities (�1.0�106 cells mL�1) (Fig. 1d). Isochrysis sp. showed the thirdfastest specific growth rates in L1- and f/2-medium (0.18and 0.25, respectively), but the lowest in K-medium(Table I) and reached the second highest cell density

Figure 1. Growth curves ofNannochloropsis sp. (a), Isochrysis sp. (b), Tetraselmis sp.

Mean� SE is shown, n¼ 27 (error bars smaller than symbols), y-axes scaling differ in figure

density. Arrows indicate where fresh medium was added.

248 Biotechnology and Bioengineering, Vol. 107, No. 2, October 1, 2010

(�6.4� 106 cells mL�1 in L1- and f/2-medium) (Fig. 1b).Finally, Tetraselmis sp. grown in K-medium and L1-mediumshowed similar specific growth rates (0.19 and 0.24) toIsochrysis sp. grown in L1-medium and f/2-medium,respectively (Table I), and reached the third highest celldensity (�1.9� 106 cells mL�1 in L1- and f/2-medium)(Fig. 1c).

Specific growth rates did not vary between media types forRhodomonas sp. but were lower in f/2- and K-medium forNannochloropsis sp. and Isochrysis sp., while Tetraselmis sp.showed the highest growth rate in K-medium (Table I).Maximal cell densities did not vary greatly between mediatypes for Rhodomonas sp. For Isochrysis sp., maximal celldensities were highest in L1-medium, lower in f/2-medium,and lowest in K-medium. For Nannochloropsis sp. andTetraselmis sp., maximal cell densities were considerablylower in K-medium than in L1- and f/2-medium (Fig. 1).

While slightly different in K-medium, nitrate, phosphate,iron, or vitamin supplies were identical for L1- and f/2-medium. Thus, observed differences in cell densities andgrowth rates were most likely due to the more refined traceelemental composition of L1, in particular with regard to theprovision of selenium, nickel, vanadium, and chromium,not present in either f/2- or K-medium, and approximately3� higher molybdenum content. Rhodomonas sp. did notshow a medium preference in regard to maximal celldensities achieved. All other microalgal strains grown in

(c), and Rhodomonas sp. (d) grown in L1- (circle), f/2- (triangle), and K-medium (square).

s (a) (30� 106), (b) (8� 106), and (c and d) (3� 106) due to larger differences in final cell

K-medium performed less well, which could be the result ofthe lack of an inorganic phosphate source. As the traceelemental composition between f/2- and K-medium isalmost identical except for the additional provision ofchelated iron in K-medium, large observed differences ingrowth responses of Nannochloropsis sp., Isochrysis sp., andTetraselmis sp., between f/2- and K-medium would be bestexplained by reduced metal availability in K-medium, whichis 10� more chelated.

In general, specific growth rates and maximal celldensities found in the present study were in the medianrange of other published studies. The maximal cell densitiesof Nannochloropsis sp. and Isochrysis sp. corresponded to celldensities found by Dunstan et al. (1993). However, the celldensity of Isochrysis sp. reported by Renaud et al. (1999) isapproximately five times lower, while the cell densityreported by Fidalgo et al. (1998) is approximately threetimes higher than reported here. Maximal cell densities forTetraselmis sp. and Rhodomonas sp. corresponded to thevalues reported by Renaud et al. (1999). Most studiesinvestigating lipid content and FA composition in

Table II. Cell density, dry matter, and total lipid content for cultures of Nann

L1-, f/2-, and K-medium and harvested in logarithmic, late logarithmic, and s

Species Medium Growth phase Cell density (cells m

Nannochloropsis sp. L1 L 1.32� 107

Nannochloropsis sp. L1 LLa 2.19� 107

Nannochloropsis sp. L1 S 2.55� 107

Nannochloropsis sp. f/2 L 1.26� 107

Nannochloropsis sp. f/2 LLa 2.24� 107

Nannochloropsis sp. f/2 S 2.52� 107

Nannochloropsis sp. K LL 1.16� 107

Nannochloropsis sp. K S 1.33� 107

Isochrysis sp. L1 L 2.60� 106

Isochrysis sp. L1 LL 5.90� 106

Isochrysis sp. L1 S 6.38� 106

Isochrysis sp. f/2 L 2.38� 106

Isochrysis sp. f/2 LL 5.54� 106

Isochrysis sp. f/2 S 6.09� 106

Isochrysis sp. K L 2.34� 106

Isochrysis sp. K LL 4.56� 106

Isochrysis sp. K S 5.20� 106

Tetraselmis sp. L1 L 1.14� 106

Tetraselmis sp. L1 LL 1.63� 106

Tetraselmis sp. L1 S 1.94� 106

Tetraselmis sp. f/2 L 1.16� 106

Tetraselmis sp. f/2 LL 1.67� 106

Tetraselmis sp. f/2 S 1.86� 106

Tetraselmis sp. K L 6.35� 105

Tetraselmis sp. K LL 1.02� 106

Tetraselmis sp. K S 1.29� 106

Rhodomonas sp. L1 L 4.83� 105

Rhodomonas sp. L1 LL 1.01� 106

Rhodomonas sp. L1 S 9.71� 105

Rhodomonas sp. f/2 L 5.20� 105

Rhodomonas sp. f/2 LL 8.91� 105

Rhodomonas sp. f/2 S 9.83� 105

Rhodomonas sp. K L 4.62� 105

Rhodomonas sp. K LL 5.76� 105

Rhodomonas sp. K S 8.10� 105

aValues represent mean of two replicate samples.

microalgae do not report specific growth rates. Isochrysissp. and Tetraselmis sp. showed about half the growth ratesreported by Renaud et al. (1999), while Rhodomonas sp.showed a similar value. For Nannochloropsis sp., no valueswere found in the literature. Differences in growth rates andmaximal cell densities compared to the other studies couldbe due to differences in culturing method and/or system(e.g., Borowitzka, 1999; Wen and Chen, 2000), strain (e.g.,Illman et al., 2000), prior culture history (e.g., Miyamotoet al., 1988), and culture conditions (e.g., Yongmanitchaiand Ward, 1991). Even though growth rates and maximalcell densities can differ greatly, the selection of species forscale-up culture for biocommodities also depends onbiomass and lipid productivity (a function of specificgrowth rate, biomass concentration, and lipid content) aswell as FA composition.

Lipid Content

A large variation in lipid content (% dry weight) wasobserved between species and culture media used (Table II).

ochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in

tationary growth phase.

L�1) Dry matter (mg L�1) Total lipid content (% of dry matter)

42.3 21.3

156.8 31.3

273.8 32.7

63.5 21.9

174.1 29.2

202.4 33.2

67.1 35.7

97.4 37.8

360.8 23.5

852.5 34.1

258.7 28.6

292.5 23.5

803.2 30.1

542.5 26.8

281.2 22.0

683.0 24.1

472.4 28.6

1129.0 10.6

977.0 8.7

612.9 10.1

1126.9 11.8

889.2 8.2

1243.5 11.4

690.1 13.3

1116.2 26.6

694.8 33.0

226.9 9.5

563.2 13.0

305.0 12.5

190.3 11.4

501.5 12.9

586.6 13.3

191.0 17.1

243.1 20.5

680.7 14.6

Huerlimann et al.: Lipid Productivity of Tropical Microalgae 249

Biotechnology and Bioengineering

Nannochloropsis sp., the fastest growing species, had thehighest overall lipid content, followed by Isochrysis sp.,Tetraselmis sp., and Rhodomonas sp. (Table II). Except forRhodomonas sp. grown in K-medium and Isochrysis sp.grown in L1- and f/2-medium, lipid content increased in latelogarithmic and stationary phase (Table II). The increasedaccumulation with culture age has been observed in manymircoalgal taxa, as photosynthetic energy is diverted to lipidproduction instead of cell division, often initiated bynitrogen limitation (Hu et al., 2008; Xu et al., 2008). Incontrast, Rhodomonas sp. grown in K-medium and Isochrysissp. grown in L1- and f/2-medium showed the highest lipidcontent in late logarithmic phase. This observed pattern islikely due to species-specific biochemical responses inducedby the different growth media, although it is difficult toestablish which particular nutrient provision is responsiblefor the response during culture growth.

The lipid content of Nannochloropsis sp. and Rhodomonassp. grown in L1- and f/2-medium conformed to the valuesreported by Renaud et al. (1991) and values observed forIsochrysis sp. were similar to values reported by Renaudet al. (1999) and Martinez-Fernandez et al. (2006). Thelipid content of Tetraselmis sp. was slightly lower than thevalues reported by Renaud et al. (1999). To ourknowledge, the increased lipid content in Rhodomonas sp.when grown in K-medium has not been reported before.While Nannochloropsis sp. followed the generally acceptedpattern of increasing lipid content with increasingculture age (Hu et al., 2008; Xu et al., 2008), the resultsfor Isochrysis sp. showed that this was not the case for allspecies.

Previous studies mainly focused on lipid content as a wayto evaluate the nutritional value of different microalgalspecies (Renaud et al., 1999; Behrens and Kyle, 1996;Zhukova and Aizdaicher, 1995). Only in the last 5 years,studies started to focus more on lipid productivity forbiodiesel production (Griffiths and Harrison, 2009; Gouveiaet al., 2009; Chiu et al., 2008). High lipid content is oftenoffset by lower growth rates. Lower growth rates and/orsmall cell sizes lead to lower overall productivity (Rodolfiet al., 2009). Therefore, lipid content alone is an unsuitablemeasure for yield, since it also depends on growth rate andcell size. In conclusion, species which have been promotedfor their high lipid content require careful evaluation of lipidproductivity, considering that selecting a suitable species forscale-up production also depends on growth rate, biomass,and lipid productivity.

Table III. Biomass and lipid productivity (g m�2 day�1) based on dry biomas

during logarithmic growth in L1-, f/2-, and K-medium.

Species

Biomass productivity (g m�2 day�1)

L1-medium f/2-medium K-m

Nannochloropsis sp. 13.4 4.4

Isochrysis sp. 18.8 11.3

Tetraselmis sp. 45.0 33.1

Rhodomonas sp. 13.4 3.8

250 Biotechnology and Bioengineering, Vol. 107, No. 2, October 1, 2010

Productivity

In our opinion, the most crucial comparative measure islipid productivity. Although lipid productivity is mostcommonly reported in mg L�1 d�1 (Li et al., 2008; Rodolfiet al., 2009), reporting lipid productivity in g m�2 day�1 hasadditional advantages. Most importantly, aquatic produc-tivities of different footprints can be directly compared withterrestrial production systems (see Chisti, 2007). Therefore,especially for industrial-scale production, both volumetricand area productivities should be reported, as presented inthis study.

Even though Tetraselmis sp. showed the lowest specificgrowth rate, it had the highest biomass productivityirrespective of growth medium, which was 2–10 timeshigher than for the other species (Table IV). Furthermore,Tetraselmis sp. demonstrated the highest lipid production,irrespective of growth medium (3.9–4.8 g m�2 day�1).Isochrysis sp. showed the second highest biomass produc-tivity, being highest in L1- and lowest in K-medium. Lipidproductivity was slightly lower than for Tetraselmis sp. inL1-medium but 30–60% lower in f/2- and K-medium.Rhodomonas sp. showed the third highest overall biomassproductivity. Conversely, it showed the lowest lipidproductivity, with the highest value in K-medium(1.5 g m�2 day�1). Nannochloropsis sp. showed the lowestbiomass productivity, despite the fast specific growth rate.Lipid productivity was similar to Tetraselmis sp. andIsochrysis sp. when grown in L1-medium, but much lowerin f/2-medium (Table III).

To enable a comparison with existing literature, theresults reported in this study were converted tomg L�1 day�1 (Table IV). The lipid productivity in thisstudy, ranging from 2.1 to 22.7 mg L�1 day�1, was lowercompared to published data for other organisms. The lipidproductivity of nutrient-limited microalgae can range fromas little as 0.24 mg L�1 day�1 for Chlorella protothecoides(Illman et al., 2000) to 134 mg L�1 day�1 in Neochlorisoleoabundans with an average of 35.0 mg L�1 day�1

(Table IV). Heterotrophically grown C. protothecoidesexhibits an incredible lipid productivity of up to1,214 mg L�1 day�1 (Table IV). However, this is unsuitablefor commercial-scale outdoor cultures, since heterotrophicgrowth requires the maintenance of axenic culture condi-tions, which requires the use of expensive fully enclosedphotobioreactors which would not be commercially viablefor the production of low value products, such as biodiesel.

s of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp.

Lipid productivity (g m�2 day�1)

edium L1-medium f/2-medium K-medium

N/A 4.2 1.0 N/A

6.1 4.4 2.7 1.4

35.1 4.8 3.9 4.7

12.0 1.3 0.4 2.0

Table IV. Lipid productivity (mg L�1 day�1) for different species of

freshwater and marine microalgae.

SpeciesLipid productivity

(mg L�1 day�1) References

Chaetoceros calcitrans 17.6 Rodolfi et al. (2009)Chaetoceros muelleri 21.8 Rodolfi et al. (2009)Chlorella emersonii 50.0 Illman et al. (2000) d

Chlorella emersonii 8.08 Illman et al. (2000) e

Chlorella minutissima 8.96 Illman et al. (2000) e

Chlorella minutissima 10.2 Illman et al. (2000) e

Chlorella protothecoides 5.42 Illman et al. (2000) e

Chlorella protothecoides 0.24 Illman et al. (2000) e

Chlorella protothecoides 1214 Xiong et al. (2008) d

Chlorella sorokiniana 0.63 Illman et al. (2000) e

Chlorella sorokiniana 1.00 Illman et al. (2000) e

Chlorella sorokiniana 44.7 Rodolfi et al. (2009)Chlorella sp. 42.1 Rodolfi et al. (2009)Chlorella vulgaris 14.7 Illman et al. (2000) d

Chlorella vulgaris 5.27 Illman et al. (2000) e

Chlorella vulgaris 36.9 Rodolfi et al. (2009)Chlorella vulgaris 32.6 Rodolfi et al. (2009)Chlorococcum sp. 53.7 Rodolfi et al. (2009)Ellipsoidion sp. 47.3 Rodolfi et al. (2009)Isochrysis sp. 21.1a Current studyIsochrysis sp. 12.7b Current studyIsochrysis sp. 6.44c Current studyIsochrysis sp. 37.7 Rodolfi et al. (2009)Isochrysis sp. 37.8 Rodolfi et al. (2009)Monodus subterraneus 30.4 Rodolfi et al. (2009)Nannochloris sp. 76.5 Yamaberi et al. (1998)d

Nannochloropsis sp. 49.7 Rodolfi et al. (2009)Nannochloropsis sp. 20.0a Current studyNannochloropsis sp. 4.59b Current studyNannochloropsis sp. 61.0 Rodolfi et al. (2009)Nannochloropsis sp. 48.2 Rodolfi et al. (2009)Nannochloropsis sp. 54.8 Rodolfi et al. (2009)Nannochloropsis sp. 37.6 Rodolfi et al. (2009)Nannochloropsis sp. 60.9 Rodolfi et al. (2009)Neochloris oleoabundans 125 Li et al. (2008)Neochloris oleoabundans 133 Li et al. (2008)Neochloris oleoabundans 98.0 Li et al. (2008)Neochloris oleoabundans 44.0 Li et al. (2008)Neochloris oleoabundans 38.0 Li et al. (2008)Pavlova lutheri 50.2 Rodolfi et al. (2009)Pavlova salina 49.4 Rodolfi et al. (2009)Phaeodactylum tricornutum 44.8 Rodolfi et al. (2009)Porphyridium cruentum 34.8 Rodolfi et al. (2009)Rhodomonas sp. 6.04a Current studyRhodomonas sp. 2.06b Current studyRhodomonas sp. 9.74 Current studyScenedesmus quadricauda 35.1 Rodolfi et al. (2009)Scenedesmus sp. 53.9 Rodolfi et al. (2009)Scenedesmus sp. 40.8 Rodolfi et al. (2009)Skeletonema costatum 17.4 Rodolfi et al. (2009)Skeletonema sp. 27.3 Rodolfi et al. (2009)Tetraselmis sp. 22.7a Current studyTetraselmis sp. 18.6b Current studyTetraselmis sp. 22.2c Current studyTetraselmis sp. 43.4 Rodolfi et al. (2009)Tetraselmis suecica 27.0 Rodolfi et al. (2009)Tetraselmis suecica 36.4 Rodolfi et al. (2009)Thalassiosira pseudonana 17.4 Rodolfi et al. (2009)

Values in italics represent results from low nitrogen experiments and arefollowed by values for replete growth. Values with gray backgroundrepresent cultures grown with 24 h light and air enriched with5% CO2.aL1-medium.bf/2-medium.cK-medium.dValues calculated by Liet al. (2008).eValues calculated according to Li et al. (2008).

Enhanced productivity in terms of biomass, andconsequently also lipids, can be achieved by enrichingcultures with CO2 or lengthening the period of lightavailability. Li et al. (2008) and Rodolfi et al. (2009) used airenriched with 5% CO2 and 24 h lighting conditions for theirexperiments (Table IV, values with gray background). CO2

can be easily added and, if the production facility is placedclose to an energy plant, it would aid biological carboncapture and sequestration or recycling. However, 24 hlighting conditions would make up scaled production morecostly and may not be feasible for low-value products. Theavailability of nitrogen also affects productivity of biomass,with generally higher biomass when nitrogen is not limited(Li et al., 2008). In contrast lipid productivity, as opposed tobiomass productivity, is generally higher when cultures arenitrogen limited (Illman et al., 2000; Li et al., 2008).In contrast, Li et al. (2008) found that there is an optimalnitrogen concentration for maximal lipid productivity inNeochloris oleoabundans, while concentrations below orabove this concentration led to decreased lipid productivity.Using the same organism Pruvost et al. (2009) found thatvolumetric total lipid productivity was higher undernutrient replete conditions, but TAG-specific productivitywas also higher under nutrient-limiting conditions. Thishighlights the importance to differentiate between generallipid productivity and TAG-specific productivity, as thisimpacts on the usefulness of culture conditions for themaximization of biofuel production, which is TAG-driven.

Fatty Acid Composition

The FA composition of lipids varied markedly betweenmicroalgal species, with a lower level of variation betweenmedia types and different growth phases. The FA profile ofNannochloropsis sp. was essentially identical for all mediaand growth phases with some small quantitative differencesbetween media and growth phase (Table V). Palmitic acid,palmitoleic acid, and EPA together accounted for more than70% of the FAs. The only variation in the quantitative profilewas EPA, which in L1- and f/2-medium made up the largestportion of FAs in logarithmic phase, but decreased in latelogarithmic and was reduced to only half the amount instationary phase. EPA was considerably lower in K-medium,but the concentration of palmitoleic acid was significantlyhigher compared to L1- and f/2-medium (Table V). The FAprofile of Nannochloropsis sp. was consistent with otherpublished profiles, including the decrease of EPA withincreased culture age (Dunstan et al., 1993).

Myristic acid, palmitic acid, oleic acid, stearidonic acid,and DHA accounted for more than 60% of the FAsindependent of culture phase and medium type in Isochrysissp. (Table VI). In contrast to the significant FA profile shiftobserved for Nannochloropsis sp. when grown in K-medium,no large media-induced FAs changes were observed(Table VI). Like Nannochloropsis sp., the FA profile of

Huerlimann et al.: Lipid Productivity of Tropical Microalgae 251

Biotechnology and Bioengineering

Table V. Fatty acid composition in dry weight % of Nannochloropsis sp. grown in L1-, f/2-, and K-medium during logarithmic, late logarithmic, and

stationary phase.

Fatty acid Common name

L1 f/2 K

L LLa S L LLa S LL S

14 Myristic acid 4.5 5.1 5.0 4.6 5.1 4.9 3.9 3.8

14:1n-5 Myristoleic acid — — — — — — — —

15 0.3 0.4 0.5 0.3 0.4 0.5 0.3 0.3

16 Palmitic acid 25.3 30.8 37.5 27.3 33.2 38.6 34.6 35.0

16:1n-7 Palmitoleic acid 23.4 21.3 23.3 24.1 21.4 23.1 40.8 39.7

17 Margaric acid 0.2 0.3 0.4 0.2 0.3 0.4 0.3 0.3

17:1n-8 — — — — — — — —

18 Stearic acid 0.9 0.8 0.9 0.8 0.9 1.0 0.9 1.1

18:1n-9 Oleic acid 4.8 7.5 11.6 5.1 7.1 10.7 4.7 7.3

18:1n-7 Vaccenic acid 0.4 0.3 0.3 0.6 0.3 0.3 1.5 1.6

18:2n-6 Linoleic acid 2.2 2.2 1.5 2.1 2.1 1.6 0.7 0.9

18:3n-6 g-Linolenic acid N/A N/A N/A N/A N/A N/A N/A N/A

18:3n-3 a-Linolenic acid — 0.1 — — 0.1 — 0.1 0.1

18:4n-3 Stearidonic acid — — — — — — — —

19 — — — — — — — —

20 Eicosanoic acid 0.1 — 0.1 0.1 — 0.1 0.1 0.1

20:1n-11 — — — — — — — —

20:1n-9 — — — — — — — 0.1

20:1n-7 — — — — — — 0.1 0.1

20:2n-6 — — — — — — — —

20:3n-6 — — — — — — — —

20:3n-3 — — — — — — — —

20:4n-6 Arachidonic acid (AA) 6.3 5.0 3.3 6.0 4.8 3.2 2.0 1.8

20:4n-3 — — — — — — — —

20:5n-3 Eicosapentaenoic acid (EPA) 30.8 26.1 15.3 28.1 24.3 15.2 9.7 7.6

22 0.6 0.4 0.4 0.6 0.4 0.3 0.1 0.2

22:1n-11 — — — — — — — —

22:1n-9 — — — — — — — —

22:1n-7 — — — — — — — —

22:3n-3 — — — — — — — —

22:4n-6 — — — — — — — —

22:5n-6 0.1 0.1 — — — — 0.1 —

22:5n-3 Docosapentaenoic acid — — — — — — — —

22:6n-3 Docosahexaenoic acid (DHA) — — — — — — — —

24 — — — — — 0.1 — —

24:1n-9 — — — — — — — —

Total saturated 31.9 37.7 44.8 33.9 40.2 45.9 40.2 40.8

Total monounsaturated 28.6 29.1 35.2 29.8 28.7 34.1 47.1 48.8

Total polyunsaturated 39.4 33.4 20.1 36.2 31.3 20.0 12.6 10.4

v-3 30.8 26.2 15.3 28.1 24.4 15.2 9.8 7.7

v-6 8.6 7.3 4.8 8.1 6.9 4.8 2.8 2.7

v-3/v-6 3.6 3.6 3.2 3.5 3.5 3.2 3.5 2.9

L, logarithmic phase; LL, late logarithmic phase; S, stationary phase.aValues represent mean of two replicate samples.

Isochrysis sp. corresponded with other published profiles(Dunstan et al., 1993).

The FA composition of Tetraselmis sp. was similar acrossgrowth phases and across all media, with the largestquantitative changes being between growth phases ratherthan media (Table VII). Palmitic acid, oleic acid, linoleicacid, and a-linolenic acid made up 85–90% of total lipidsdepending on media type and growth phase (Table VII).Oleic acid increased with culture age, while a-linolenic aciddecreased. The FA composition of Tetraselmis sp. differedfrom other published profiles. Zhukova and Aizdaicher(1995) analyzed Tetraselmis sp. and Tetraselmis viridis andfound only half the amount of palmitic acid, and between

252 Biotechnology and Bioengineering, Vol. 107, No. 2, October 1, 2010

one-fifth and one-tenth of oleic acid reported here. Incontrast, hexadecatetraenoic acid was present (Zhukova andAizdaicher, 1995), which was not detected in this study.Furthermore, linoleic acid was present in lower quantities,compared to the present study, while stearidonic acidwas present in higher quantities of approximately 20%(Zhukova and Aizdaicher, 1995).

The FA profiles of Rhodomonas sp. grown in L1- and f/2-medium were similar for each growth phase, with theexception of the FA profile for cultures grown in K-medium(Table VIII). Palmitic acid, a-linolenic acid, stearidonicacid, and EPA together accounted for 60–75% of the FAs.The most significant changes were a threefold decrease in

Table VI. Fatty acid composition in dry weight % of Isochrysis sp. grown in L1-, f/2-, and K-medium during logarithmic, late logarithmic, and stationary

phase.

Fatty acid Common name

L1 f/2 K

L LL S L LL S L LL S

14 Myristic acid 13.7 10.3 8.9 12.8 9.2 8.8 11.4 9.2 9.7

14:1n-5 Myristoleic acid — — — — — — — — —

15 0.2 0.4 0.4 0.3 0.8 0.8 0.3 0.3 0.4

16 Palmitic acid 13.4 12.3 13.7 12.8 12.8 13.1 16.5 17.1 19.2

16:1n-7 Palmitoleic acid 5.1 4.9 5.1 5.2 4.9 5.1 5.3 6.0 5.9

17 Margaric acid — — 0.1 — 0.1 — — — —

17:1n-8 — — — — — — — — —

18 Stearic acid 0.2 0.2 0.2 0.2 0.2 0.3 0.4 0.5 0.4

18:1n-9 Oleic acid 12.3 18.5 21.9 12.5 19.3 19.4 15.5 18.5 23.6

18:1n-7 Vaccenic acid 1.6 1.9 1.9 1.6 2.0 2.0 2.1 2.1 2.3

18:2n-6 Linoleic acid 4.5 2.5 2.3 3.6 2.3 2.3 4.3 3.0 3.2

18:3n-6 g-Linolenic acid 1.8 0.5 0.3 1.2 0.3 0.3 0.8 0.3 0.3

18:3n-3 a-Linolenic acid 6.5 5.1 4.5 6.3 4.8 4.7 5.8 4.9 4.1

18:4n-3 Stearidonic acid 24.7 24.3 22.5 25.8 22.1 22.0 22.2 21.1 17.6

19 N/A N/A N/A N/A N/A N/A N/A N/A N/A

20 Eicosanoic acid — 0.2 0.2 — 0.3 0.3 — — 0.2

20:1n-11 — — — — — — — — —

20:1n-9 — 0.2 0.2 — 0.2 0.3 — — —

20:1n-7 — — 0.1 — — — — — —

20:2n-6 — 0.3 0.4 — 0.4 0.4 — 0.3 0.5

20:3n-6 — — — — — — — — —

20:3n-3 — — 0.3 — 0.2 0.3 — — 0.2

20:4n-6 Arachidonic acid (AA) 0.2 — 0.1 — — 0.2 — — —

20:4n-3 — — — — — — — — —

20:5n-3 Eicosapentaenoic acid (EPA) 0.9 0.7 0.6 0.9 0.9 0.8 0.6 0.5 0.3

22 0.3 0.8 — 0.3 0.8 1.0 0.7 1.1 1.3

22:1n-11 — — — — — — — — —

22:1n-9 — 1.3 1.7 — 1.3 1.4 0.3 0.8 1.0

22:1n-7 — — — — — — — — —

22:3n-3 — — — — — — — — —

22:4n-6 — 0.2 0.2 — 0.2 0.2 — — —

22:5n-6 1.6 1.7 1.5 1.4 2.1 2.5 1.5 1.5 1.2

22:5n-3 Docosapentaenoic acid — — 0.2 — 0.2 — — — —

22:6n-3 Docosahexaenoic acid (DHA) 13.0 13.8 12.7 15.0 14.7 14.1 12.0 12.4 8.2

24 — — — — — — — — —

24:1n-9 — — — — 0.1 — 0.3 0.3 0.4

Total saturated 27.8 24.2 23.7 26.3 24.2 24.2 29.3 28.2 31.2

Total monounsaturated 19.1 26.7 30.8 19.3 27.8 28.0 23.5 27.7 33.1

Total polyunsaturated 53.1 49.1 45.6 54.3 48.0 47.8 47.2 44.1 35.7

v-3 45.1 44.0 40.7 48.1 42.8 41.8 40.5 38.9 30.4

v-6 8.0 5.1 4.8 6.3 5.2 6.0 6.7 5.2 5.2

v-3/v-6 5.6 8.6 8.4 7.7 8.2 7.0 6.1 7.5 5.8

L, logarithmic phase; LL, late logarithmic phase; S, stationary phase.

stearidonic acid from >30% in logarithmic phase to lessthan 10% in stationary phase and a smaller increase in a-linolenic acid from logarithmic to stationary phase when theorganism was cultured in L1- and f2-medium. In contrast,stearidonic acid content (9–14%) was independent ofgrowth phase in K-medium. EPA, a minor FA component inthis organism, was higher in L1- (7–11%) and f/2-medium(10–13%) than in K-medium (4–6%). The FA compositionof Rhodomonas sp. generally followed the pattern of otherpublished profiles, with some differences in the actualamount, probably depending on growth phases and mediatypes (Dahl et al., 2009).

On average, Rhodomonas sp. had the highest content ofPUFA (45–75%), followed by Isochrysis sp. (35–55%),Tetraselmis sp. (16.1–47.3%), and Nannochloropsis sp. (10–40%) (Tables V–VIII). There was a noticeable shift fromPUFA, with increasing culture age, to monounsaturated FAsfor Isochrysis sp. and Rhodomonas sp., saturated FAs forTetraselmis sp., and both for Nannochloropsis sp.

The v-3/v-6 ratio was highest in Rhodomonas sp. (6.5–17.9), followed by Isochrysis sp. (5.6–8.6), Nannochloropsissp. (2.9–3.6), and Tetraselmis sp. (1.1–2.8) (Tables V–VIII).Growth phase and medium type had no apparent effect onthe v-3/v-6 ratio in Nannochloropsis sp., Isochrysis sp., and

Huerlimann et al.: Lipid Productivity of Tropical Microalgae 253

Biotechnology and Bioengineering

Table VII. Fatty acid composition in dry weight % of Tetraselmis sp. grown in L1-, f/2-, and K-medium during logarithmic, late logarithmic, and stationary

phase.

Fatty acid Common name

L1 f/2 K

L LL S L LL S L LL S

14 Myristic acid 0.6 0.6 0.6 0.6 0.6 0.6 0.5 0.5 0.5

14:1n-5 Myristoleic acid — — — — — — — — —

15 — — — — — — — — —

16 Palmitic acid 27.8 31.7 32.8 27.6 31.1 32.5 31.7 33.9 34.1

16:1n-7 Palmitoleic acid — — 0.4 — — — — 0.2 0.2

17 Margaric acid — — 0.1 — — — — — —

17:1n-8 — — — — — — — — —

18 Stearic acid 0.9 1.0 1.1 1.0 1.0 1.1 1.0 1.0 1.0

18:1n-9 Oleic acid 25.3 38.7 42.7 19.6 31.5 37.4 38.5 42.4 44.2

18:1n-7 Vaccenic acid 2.9 2.6 2.3 2.7 2.8 2.4 2.5 2.2 2.3

18:2n-6 Linoleic acid 9.3 7.5 6.8 13.7 11.8 10.2 8.0 7.0 7.2

18:3n-6 g-Linolenic acid 0.7 0.6 0.4 0.9 0.8 0.6 0.4 0.3 0.3

18:3n-3 a-Linolenic acid 23.2 11.7 8.0 23.6 13.7 9.5 12.0 8.5 6.7

18:4n-3 Stearidonic acid 3.7 1.8 1.3 3.6 2.0 1.4 1.3 1.1 1.0

19 N/A N/A N/A N/A N/A N/A N/A N/A N/A

20 Eicosanoic acid — — 0.1 — — — — — —

20:1n-11 — — — — — — — — —

20:1n-9 1.3 1.2 1.2 1.3 1.1 1.1 1.7 1.5 1.5

20:1n-7 — — — — — — — — —

20:2n-6 — — 0.1 — — — — — —

20:3n-6 — — — — — — — — —

20:3n-3 — — — — — — — — —

20:4n-6 Arachidonic acid (AA) 0.9 0.6 0.5 1.4 1.2 1.0 0.7 0.3 0.2

20:4n-3 — — — — — — — — —

20:5n-3 Eicosapentaenoic acid (EPA) 3.4 2.0 1.5 3.8 2.4 1.9 1.5 1.0 0.7

22 — — — — — — — — —

22:1n-11 — — — — — — — — —

22:1n-9 — — — — — — — — —

22:1n-7 — — — — — — — — —

22:3n-3 — — — — — — — — —

22:4n-6 — — — — — — — — —

22:5n-6 — — — — — — — — —

22:5n-3 Docosapentaenoic acid — — — 0.3 — — — — —

22:6n-3 Docosahexaenoic acid (DHA) — — — — — — — — —

24 — — 0.2 — — 0.3 0.3 0.2 0.2

24:1n-9 — — — — — — — — —

Total saturated 29.3 33.3 34.8 29.1 32.7 34.4 33.5 35.5 35.8

Total monounsaturated 29.4 42.5 46.6 23.6 35.4 40.9 42.7 46.3 48.1

Total polyunsaturated 41.2 24.3 18.6 47.3 31.8 24.6 23.8 18.1 16.1

v-3 30.3 15.6 10.8 31.3 18.1 12.8 14.8 10.5 8.4

v-6 10.9 8.7 7.8 15.9 13.7 11.8 9.0 7.6 7.7

v-3/v-6 2.8 1.8 1.4 2.0 1.3 1.1 1.6 1.4 1.1

L, logarithmic phase; LL, late logarithmic phase; S, stationary phase.

Tetraselmis sp., while the v-3/v-6 ratio decreased withculture age in L1- and f/2-medium in Rhodomonas sp.(Tables V–VIII).

Rhodomonas sp., Isochrysis sp., and Nannochloropsis sp.exceed the recommended minimal v-3/v-6 ratio of >2 forlarval and juvenile oysters (Enright et al., 1986; Fidalgo et al.,1998), while Tetraselmis sp. only reached the ratio of 2 inlogarithmic phase when grown in L1- and f/2-medium.

Considering the FA composition, none of the investigatedspecies naturally contained the proposed optimal ratio forbiodiesel properties of 5:4:1 for C16:1, C18:1, and C14:0(Schenk et al., 2008). However, most of them contained oneof the three FA types in higher quantities, making mixtures

254 Biotechnology and Bioengineering, Vol. 107, No. 2, October 1, 2010

of FA derived from different species a suitable solution tothis problem.

Conclusion

Using biomass and lipid productivity as benchmarks, thisstudy identifies Tetraselmis sp. as the species of choice fromthose tested, for large-scale culture of microalgae forbiomass or lipid production (Table III). This high level ofproductivity then provides flexibility in terms of resultingproducts with high levels of CO2 sequestration in biomassfor feed stock production, or alternatively lipids as food

Table VIII. Fatty acid composition in dry weight % of Rhodomonas sp. grown in L1-, f/2-, and K-medium during logarithmic and stationary phase.

Fatty acid Common name

L1 f/2 K

L LL S L LL S L LL S

14 Myristic acid 4.7 9.5 7.8 4.5 10.2 8.5 9.5 13.3 10.4

14:1n-5 Myristoleic acid — — — — — — — — —

15 0.3 0.5 0.4 — 0.5 0.4 0.2 0.3 0.3

16 Palmitic acid 11.2 21.3 19.7 10.1 19.2 18.3 20.5 24.0 20.2

16:1n-7 Palmitoleic acid 3.6 1.9 1.5 3.7 1.7 1.4 1.0 1.0 0.8

17 Margaric acid — 0.4 0.5 — 0.5 0.6 — 0.2 0.2

17:1n-8 — — — — — — — — —

18 Stearic acid 0.7 2.4 3.0 0.6 2.0 2.5 1.2 1.4 1.2

18:1n-9 Oleic acid 0.8 4.6 5.6 0.7 3.9 5.5 8.2 10.6 7.3

18:1n-7 Vaccenic acid 6.2 3.4 2.8 5.2 3.1 2.7 4.7 4.3 4.0

18:2n-6 Linoleic acid 0.7 2.5 3.3 0.7 2.5 3.3 3.6 4.3 4.6

18:3n-6 g-Linolenic acid — — — — — — 0.3 0.3 0.2

18:3n-3 a-Linolenic acid 20.3 29.8 29.8 22.0 34.4 36.7 25.9 22.9 28.6

18:4n-3 Stearidonic acid 33.1 12.8 11.7 31.3 7.7 5.5 13.5 9.1 11.7

19 N/A N/A N/A N/A N/A N/A N/A N/A N/A

20 Eicosanoic acid — — — — — — — — —

20:1n-11 — — — — — — — — —

20:1n-9 — — — — — — — — —

20:1n-7 — — — — — — — — —

20:2n-6 — — — — — — — — —

20:3n-6 — — — — — — — — —

20:3n-3 — — — — 0.1 — — — —

20:4n-6 Arachidonic acid (AA) 0.6 0.5 0.6 0.8 0.9 1.1 0.8 0.6 0.7

20:4n-3 — — — — — — — — —

20:5n-3 Eicosapentaenoic acid (EPA) 11.1 6.8 8.6 12.6 9.2 10.0 5.5 4.1 4.6

22 — — — — — — — — —

22:1n-11 — — — — — — — — —

22:1n-9 — — — — — — — — —

22:1n-7 — — — — — — — — —

22:3n-3 — — — — — — — — —

22:4n-6 — — — — — — — — —

22:5n-6 2.5 1.4 1.5 3.0 1.8 2.0 1.0 0.8 0.9

22:5n-3 Docosapentaenoic acid — 0.2 0.2 — — — 0.2 0.2 0.2

22:6n-3 Docosahexaenoic acid (DHA) 4.1 2.1 3.0 4.7 2.1 1.6 3.7 2.8 4.0

24 — — — — 0.2 — — — —

24:1n-9 — — — — — — — — —

Total saturated 17.0 34.1 31.5 15.2 32.5 30.3 31.4 39.0 32.3

Total monounsaturated 10.6 9.8 9.9 9.7 8.7 9.5 14.0 15.9 12.1

Total polyunsaturated 72.4 56.1 58.6 75.2 58.8 60.2 54.7 45.0 55.5

v-3 68.6 51.6 53.3 70.7 53.6 53.9 48.9 39.0 49.1

v-6 3.8 4.4 5.4 4.5 5.2 6.3 5.7 6.0 6.5

v-3/v-6 17.9 11.6 9.9 15.6 10.3 8.5 8.6 6.5 7.6

L, logarithmic phase; LL, late logarithmic phase; S, stationary phase.

additives, feed ingredients, nutraceuticals, or biodiesel feedstock. Importantly, this work provides a template forcomparative assessments of productivity (g m�2 day�1)across studies on microalgae and for terrestrial biomassand lipid production platforms. In a comparative sense, themicroalgae investigated here are in the median range ofproductivities for microalgae but exceed the biomass andlipid yields of terrestrial production systems (Table I inChisti, 2007).

The authors acknowledge funding of this research by their industry

partner MBD and the culturing assistance and hands-on training

provided by Stanley Hudson, Manager of the North Queensland Algal

Identification/Culturing Facility (NQAIF).

References

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