Journal of Applied Phycology 9: 19–24, 1997. 19c 1997 Kluwer Academic Publishers. Printed in Belgium.

Characterization of the growth and lipid content of the diatom Chaetocerosmuelleri

K. M. McGinnis, T. A. Dempster & M. R. Sommerfeld�Department of Botany Arizona State University, Box 871601, Tempe, AZ 85287-1601, USA(�Author for correspondence)

Received 28 October, 1996; revised 7 January, 1997; accepted 7 January, 1997

Key words: Chaetoceros, growth, neutral lipid, total lipid, nitrogen deprivation, Nile Red

Abstract

Chaetoceros muelleri (Schutt) was cultured on a thermal gradient plate, subjected to two media types with a rangeof specific conductances, and evaluated for growth and neutral lipid accumulation. Growth was measured directlyby daily changes in cell numbers and indirectly by changes in optical density at 750 nm. C. muelleri exhibiteda growth rate of at least two doublings day�1 over broad temperature (20 to 35 �C) and conductance ranges (10to over 60 mS cm�1) and the optimum growth rate approached 4.0 doublings day�1 at 30 �C and a conductanceof 25 mS cm�1. Intracellular neutral lipid storage was evaluated with fluorometry and epifluorescent microscopyusing the fluorochrome Nile Red. Gravimetric analysis revealed a total lipid content in nitrogen-depleted culturesof C. muelleri of over 400 mg L�1, five to seven times that observed in nitrogen-replete cultures. Based on its highgrowth rate, tolerance to a broad range of temperatures and specific conductances, and large quantity of intracellularlipid, C. muelleri may have potential for exploitation as a renewable precursor to liquid fuels or as a lipid source.

Introduction

In 1979 the (USA) Department of Energy (DOE) andthe Solar Energy Research Institute (SERI) initiated aresearch program to investigate the feasibility of pro-ducing liquid fuels from microalgae. This topic wasof initial interest due to the rising cost and limited liq-uid fuel resources and, subsequently, for the potentialfor reduction of atmospheric CO2 by microalgal pho-tosynthesis (Campbell, 1983; Steeg, 1994). Microal-gae were selected as a potential feedstock because theenergy-rich storage lipid product of these plants wasamong the more useful natural products for conver-sion to fuels such as gasoline and diesel fuel (Neenanet al., 1986) and because microalgae were known to beextremely efficient biomass producers (Cheremisinoffet al., 1980; Hall & House, 1995; Meier, 1955).

For microalgal lipids to be cost competitive as aliquid fuel source, a feedstock organism must be capa-ble of high photosynthetic efficiencies (12–16%) andlarge lipid yields (50–60% of biomass weight in theform of lipids) (Neenan et al., 1986). Assuming these

requirements were met, it was projected that gasolinefrom microalgal lipids could be produced in the US for$1.60–$2.00 gallon�1 ($ 0.42–$ 0.53 L�1) by the year2010 (Neenan et al., 1986).

The feasibility of using microalgal lipids as a fuelprecursor on a large scale was based not only on a pro-ductive, lipid-producing alga but on an environmentconducive to supporting microalgal culture. The south-western United States, with its relatively inexpensiveland, abundant sunlight, moderate temperatures andlarge reservoirs of saline groundwater, was considereda prime location for large-scale efforts (Neenan et al.,1986).

The economic feasibility of algal mass culture forfuel production was greatly enhanced by the abilityto increase microalgal biomass productivity (Vonshak& Richmond, 1985) and by demonstrating that cer-tain culture conditions could result in higher quantitiesof storage lipids, such as triglycerides (Opute, 1974).For example, it was demonstrated that a significantincrease in lipid content occurred in several species ofunicellular algae after subjecting them to a nitrogen-

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deficient environment (Ben-Amotz et al., 1985; Shifrin& Chisholm, 1981; Chelf, 1990). Light was also shownto be an important environmental factor: blue lightwas reported to cause important differences in the fattyacid profiles of Chaetoceros sp. (Saavedra & Voltotina,1994). Others reported changes in cellular lipid charac-teristics during light-dark cycles (Shifrin & Chisholm,1981) and from variations in media salinity and con-ductivity (Renaud & Parry, 1994; Chelf, 1990).

Diatoms, in particular, were regarded as useful neu-tral lipid sources, both as liquid fuel precursors andas foods for marine culture of zooplankters (Ahlgrenet al., 1990), larval and postlarval shrimp (Chu, 1989),copepods (Bourdier & Amblard, 1989), and juvenileoysters (Tsitsa-Tzardis et al., 1993). This is not onlydue to characteristically high lipid and fatty acid con-tent, but also to the abundance of certain polyunsatu-rated fatty acids (PUFAs) in diatoms (Renaud & Parry,1994). Diatoms are known to contain large amounts ofeicosapentaenoic acid (Renaud et al., 1994), a PUFArequired in the diet of many marine animals that maynot be able to synthesize the compound (Sicko-Goad& Andresen, 1991; Dunstan et al., 1993). The abilityto manipulate microalgal or diatom lipid quantity andquality is necessary for effective use of this renewableresource as fuel or food.

The diatom, C. muelleri Schutt, was isolated fromthe surface waters of the Southwest, cultured in twomedia types, and subjected to a range of temperaturesand specific conductances, and nutrient stress (nitrogendeprivation), to determine whether this organism mightachieve growth rates and lipid yields necessary to serveas a potential feedstock for the production of liquidfuels or for other applications.

Materials and methods

Culture

C. muelleri Schutt was isolated from AlamogordoPlaya, New Mexico and grown in two media types(SERI Types I and II). These media types were for-mulated to simulate the natural groundwater types inthe Southwestern United States (Barclay et al., 1985).Type I was a magnesium chloride water, whereasType II was a sodium chloride/sulfate water. Variousspecific conductances were prepared according to Bar-clay et al. (1985).

Triplicate cultures were grown on a thermogradientplate designed to provide a temperature range from

5 to 45 �C and an uniform incident illumination ofca. 150 �mol cm�2 s�1.

Growth analysis

Each experiment typically lasted six days.The first fourdays consisted of daily monitoring of growth rates. At24-h intervals, 3 mL of each culture were removed,cells counted using a hemocytometer and optical den-sity measured with a Beckman Model DU-64 spec-trophotometer at 750 nm. Spectrophotometric valuesfor optical density were transformed to logarithm basetwo and growth rates (doublings day�1) were calcu-lated from linear regressions of cell numbers, opticaldensity and time (Dempster, 1994).

Nitrogen-depletion

On day four, the cultures were subjected to nitrogendeprivation. Cultures were decanted into 50-mL dis-posable centrifuge tubes and pelleted in a MSE Super-Minor Centrifuge at 1500� g for 5 min. The super-natant was drawn off and the cells were rinsed withnitrogen-deplete media. Centrifugation was repeated,and the cells were resuspended in 25 mL of nitrogen-deplete media.

Lipid analysis

To estimate lipid accumulation fluorescence mea-surements were recorded on day four and at dailyintervals for the final two days of the experiments.For fluorescence (lipid) measurements, a 4 mL cellsuspension was placed into a 5 mL (12� 75 mm)borosilicate round cuvette. The cells were stainedwith 50 �L Nile Red (100 �g 9-diethylamino- 5H-benzo[�]phenoxazine-5-one per 1 mL acetone). Flu-orometric analysis occurred ca. 10 min. after stainingusing a Sequoia-Turner Model 450 Digital Fluorom-eter with a 450 nm narrow band excitation filter anda 540 nm narrow band emission filter. With this tech-nique, cellular storage or neutral lipids fluoresced yel-low/gold (Cooksey et al., 1987).

Lipid extractions were performed on unstressedcultures on day four and after an additional two daysin nitrogen-deplete media using a modified (Demp-ster, 1994) Bligh and Dyer method (1959). Eight25 mL cultures were centrifuged, the supernatantremoved by aspiration, and the concentrate combinedto yield concentrated samples of 32 mL. These sam-ples were sonicated and placed into a separatory fun-

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Figure 1. Growth of C. muelleri in Media Type I at various conduc-tances and temperatures.

nel with methylene chloride and methanol in a 1:2:0.8methylene chloride:methanol:water mixture. Mediawas added dropwise until separation occurred. Afterremaining overnight, the bottom layer of methylenechloride with extracted lipid was removed and evapo-rated under a stream of nitrogen.

Results

Growth of C. muelleri in Media Type I was greatest(1.6 doublings day�1) at temperatures between 30 �Cand 35 �C and in conductances between 25 and 40 mScm�1 (Figure 1). Growth in Media Type II was approx-imately twice that in Media Type I at all temperatures(Figure 2). In Media Type II, C. muelleri grew at a rategreater than one doubling day�1 in all conductances(10 to 70 mS cm�1) and at all temperatures higherthan 15 �C. C. muelleri grew optimally at 25 mS cm�1

between 30 �C and 35 �C (Figure 2).Optimum growth rates in the two media types over

the four day growth experiment is illustrated in Fig-ure 3. C. muelleri cultures grew exponentially with lit-tle or no lag phase during the first two days (Figure 3).By Day 3, growth reached stationary phase. Obser-vations of the daily growth rate revealed that whileC. muelleri averaged over 1.5 doublings day�1 over afour day period, a growth rate of 3.5 doublings day�1

occurred during the the first day of the culture cycle(Figure 4). Individual cultures, however, approached 4doublings day�1 during the first day. During the secondday, growth had slowed to about 2.5 doublings day�1,and by the fourth day it slowed to less than half a dou-bling day�1. When cultures were subjected to nitrogen

Figure 2. Growth of C. muelleri in Media Type II at various con-ductances and temperatures.

Figure 3. Typical growth curve for C. muelleri in Media Type IIwith a specific conductance of 25 mS cm�1 and a temperature of30 �C.

deprivation, growth rates were greatly reduced, but didnot completely cease.

Figure 4. Growth of C. muelleri in doublings day�1 in Media Type IIwith a specific conductance of 25 mS cm�1 and a temperature of30 �C.

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Figure 5. Comparison of Nile Red fluorescence for C. muellericultures grown in Media Type II at 30 �C at various specific con-ductances prior to and 2 days after nitrogen-deprivation.

Table 1. Nile Red relative fluorescence for neutral lipid and gravi-metric analysis for total lipid content of Chaetoceros muelleri beforeand after nitrogen-depletion.

Before nitrogen- Relative Extracted

depletion Fluorescence Units lipid (mg L�1)

1 835 73.3

2 771 66.7

After nitrogen-

depletion

1 >2000 400.1

2 >2000 462.2

3 >2000 497.8

Based on Nile Red fluorescence, intracellular neu-tral lipid accumulation in C. muelleri was easily quan-tified (Figure 5). The highest Nile Red relative fluores-cence was exhibited under those conditions in whichthe best growth occurred (i.e., in Media Type II, aconductance of 25 mS cm�1 and 30 �C). Relative flu-orescence of cultures was similar over a broad specif-ic conductance range (25 to 55 mS cm�1). Nitrogendeprivation stimulated up to a five-fold increase in flu-orescence, depending on the conductance.

The relationship between Nile Red fluorescenceand extractable lipid before and after nitrogen-depletion is shown in Table 1. Relative fluorescencemore than doubled and the extractable lipid increasedfrom five to seven-fold following 2 days of nitrogendeprivation. The lipid content of unstressed culturesranged from 66.7 to 73.3 mg L�1, whereas the lipidcontent of nitrogen-stressed cultures ranged from 400.1to 497.8 mg L�1 (Table 1).

Discussion

C. muelleri exhibited an extremely high growth rate attemperatures very near the median temperature (opti-mum growth occurred at 30 �C) of the saline surfacewaters of the Southwest, and also showed the capacityto grow at a range of temperatures (up to 35 �C) nor-mally considered to be intolerable for many diatoms(Hutchinson, 1967). Blinn (1984) collected C. muellerifrom southwestern water sources only when the tem-perature exceeded 15 �C and most commonly whentemperatures exceeded 25 �C. The median water tem-perature of 127 sites reported in a study of the salinewaters of the Southwest was 27 �C (Ellingson, 1990).Daytime temperatures in desert areas, however, canaverage 40–50 �C in the summer (Vonshak & Rich-mond, 1989).

Blinn (1984) reported that his isolate of C. muel-leri divided most rapidly in culture between 20 and30 �C. The isolate was reported to achieve a maximumgrowth rate of 1.5 divisions day�1; however, cell divi-sion did not occur at conductances lower than 1.5 mScm�1 or higher than 33 mS cm�1. The mean specif-ic conductance reported for 127 surface water sites inthe southwest was 22.7 mS cm�1 (Ellingson & Som-merfeld, 1992). In this study, our isolate of C. muel-leri divided, on average, slightly more than one timeper day over a specific conductance range from 20 to70 mS cm�1 and the maximum growth rate exceed-ed 3.5 doublings per day under optimum conditions(30 �C, 25 mS cm�1). This growth was more typicalof that seen for the C. muelleri cultured by Johansenet al. (1990). High lipid content and growth rates ofC. muelleri in SERI Media Type II with a specific con-ductance of 25 mS cm�1 were also observed by Chelf(1990).

Increased Nile Red relative fluorescence and gravi-metric yield of cellular lipids clearly illustratedthat nitrogen deprivation stimulated lipid storage inC. muelleri. When sufficient nutrients are available,proteins are synthesized; however, when nutrients arelimited, cell division is suppressed, and a greateramount of carbon is available for lipid storage (Sukenik& Wahnon, 1991). In many algae, lipid storage hasbeen induced by the depletion or removal of nitrogenfrom the culture media (Roessler, 1990; Sukenik &Wahnon, 1991). Johansen et al. (1990) reported thatall except one strain of C. muelleri studied exhibitedincreases in lipid content under nutrient stress and thatin most cases nitrogen stress, in particular, led to ahigher lipid content.

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In our investigation, the growth rate of Chaeto-ceros was greatly reduced but did not completely ceasein nitrogen-deprived cultures. This agrees with previ-ous observations on Chaetoceros species (Parrish &Wangersky, 1990).

Earlier research indicated that an increase in totalcellular lipid was due to an increase in neutral lipids(Opute, 1974). Specifically, it appears that nitrogendeficiency leads to an increase in production of triacyl-glycerol in diatoms (Parrish & Wangersky, 1990; Lom-bardi & Wangersky, 1991, 1995; Sukenik & Wahnon,1991). Triacylglycerols have been reported to increasefrom 5% of total cell volume in unstressed culturesto 70% of total cell volume in nitrogen-stressed cul-tures of Chaetoceros gracilis (Lombardi & Wanger-sky, 1995). This increase was not observed in Isochry-sis galbana and Dunaliella tertiolecta, utilized in thesame study (Lombardi & Wangersky, 1995).

An increase in triacylglycerol production undernutrient stress in Chaetoceros species is well-documented. Although nutrient deficiencies, suchas silicon and phosphorus (Lombardi & Wangersky,1991; Roessler, 1990), were able to induce this shift,nitrogen depletion appeared to be more effective formost species of Chaetoceros. Johansen et al. (1990)reported that lipid content in nitrogen-stressed cultureswas over twice that in unstressed cultures. In our studythe increase was up to seven-fold.

In addition to possible exploitation as a renewablefuel source, microalgal lipids could be useful in med-ical applications, aquaculture and as lubricants andsurfactants (Roessler, 1990). Some marine species ofChaetoceros are also critical nutritional sources for avariety of fish. Chaetoceros gracilis, for example, hasbeen shown to be an adequate exclusive feed source forlarvae and postlarvae of the shrimp Metapenaeus ensis(Chu, 1989) and is commonly used as a food source forlarval molluscs (Parrish & Wangersky, 1990). Tsitsa-Tzardis et al. (1993) reported that cholesterol (the mainsterol in oysters), was rare in most phytoplankton, butwas the major sterol in species of Chaetoceros andSkeletonema. These species were the most effectivephytoplankton in promoting juvenile oyster growth(Enright et al., 1986). The ability to physiologicallymanipulate the quantity and quality of lipid in Chaeto-ceros species would thus be significant for several dif-ferent applications.

In summary, C. muelleri exhibited very rapidgrowth, tolerated a broad range of specific conduc-tances and temperatures, and yielded large quanti-ties of neutral lipid. Furthermore, the lipid content

in C. muelleri appeared to be very susceptible to phys-iological manipulation. These characteristics indicatethat this species might be a suitable candidate for futureexploitation as an alternative renewable fuel source oras a significant source of triglycerides for other appli-cations.

Acknowledgements

Support for this research was provided by a grantto KM from the Howard Hughes Medical Institutethrough the UndergraduateBiological Sciences Educa-tion Program at Arizona State University.We gratefullyacknowledge D. Czarnecki for providing the culture ofChaetoceros.

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