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tBiofuels and Environmental Biotechnology Biotechnology and Bioengineering

DOI 10.1002/bit.23160

Culture of Microalgae Chlorella minutissima for Biodiesel Feedstock

Production

Haiying Tang, Meng Chen, M. E. D. Garcia, Nadia Abunasser, K. Y. Simon Ng, and

Steven O. Salley

Department of Chemical Engineering and Materials Science,

Wayne State University,

5050 Anthony Wayne Drive, Detroit, MI 48202, USA

Corresponding author: Steven O. Salley

Department of Chemical Engineering and Materials Science

Wayne State University

5050 Anthony Wayne Drive

Detroit, Michigan 48202

Tel: (313) 577-5216

Fax: (313) 577-3810

Email: sssalley@wayne.edu

© 2011 Wiley Periodicals, Inc.

Received November 1, 2010; Revised February 25, 2011; Accepted March 25, 2011

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tAbstract

Microalgae are among the most promising of non-food based biomass fuel feedstock

alternatives. Algal biofuels production is challenged by limited oil content, growth rate, and

economical cultivation. To develop the optimum cultivation conditions for increasing biofuels

feedstock production, the effect of light source, light intensity, photoperiod, and nitrogen

starvation on the growth rate, cell density, and lipid content of Chlorella minutissima were studied.

The fatty acid content and composition of Chlorella minutissima were also investigated under the

above conditions. Fluorescent lights were more effective than red or white light-emitting diodes

(LEDs) for algal growth. Increasing light intensity resulted in more rapid algal growth, while

increasing the period of light also significantly increased biomass productivity. Our results

showed that the lipid and triacylglycerol (TAG) content were increased under N starvation

conditions. Thus, a two-phase strategy with an initial nutrient-sufficient reactor followed by a

nutrient deprivation strategy could likely balance the desire for rapid and high biomass generation

(124 mg/L) with a high oil content (50%) of Chlorella minutissima to maximize the total amount

of oil produced for biodiesel production. Moreover, methyl palmitate (C16:0), methyl oleate

(C18:1), methyl linoleate (C18:2), and methyl linolenate (C18:3) are the major components of

Chlorella minutissima derived FAME, and choice of light source, intensity, and N starvation

impacted the FAME composition of Chlorella minutissima. The optimized cultivation conditions

resulted in higher growth rate, cell density, and oil content, making Chlorella minutissima a

potentially suitable organism for biodiesel feedstock production.

Keywords: microalgae, Chlorella minutissima, algae cultivation, biodiesel production

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t1 Introduction

Developing biomass feedstocks for biofuel production is vital for our energy security and

national economy, as well as for our environment. Biodiesel is a renewable fuel defined as alkyl

monoesters of long chain fatty acids, which has been widely used in diesel engines especially in

Europe and the USA (Ma and Hanna 1999; Vasudevan and Briggs 2008). However, the cost and

availability of traditional feedstocks for biodiesel has become a critical concern. Algae are among

the most promising non-food-crop-based biomass feedstocks (Chisti 2007). Some green algae

species have been reported to have a high lipid content and potentially may serve as a viable

biodiesel oil source , such as Nannochloropsis sp. (Gouveia and Oliveira 2009; Rodolfi et al.

2009), Tetraselmis sueccica (Rodolfi et al. 2009), Chlorella sp. (Rodolfi et al. 2009), and

Scenedesmus sp. (Matsunaga et al. 2009; Rodolfi et al. 2009). However, at present there are a very

limited number of large-scale commercial operations that grow and harvest algae and these

mainly produce relatively high value products such as food supplements and none economically

produce algae with the potential for biofuel use. Several factors limit algal production of oil and

hydrocarbons for biofuels: existing algae strains do not possess both high oil content and a high

growth rate and they cannot be grown to high cell densities; the design of large scale production

systems (open ponds and photobioreactor systems) have not been fully optimized; and harvesting

of algae and extraction and processing of oil remain a challenge. Moreover, the key properties of

biodiesel are highly dependent on the fatty acid (FA) composition of the original triacylglycerol

(TAG) (Tang et al. 2008). A high oil-producing species with an optimized FA profile for

production of high quality biodiesel is desirable for a microalgal biodiesel which meets standards

for user acceptance.

Chlorella minutissima is an eukaryotic alga, with relative fast growth and easy cultivation,

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tand the levels of amino acids and polyunsaturated fatty acids in Chlorella minutissima are high,

which could be potentially useful in health foods and pharmaceuticals (Seto et al. 1984). There

have been few studies focused on biofuel feedstock production using Chlorella minutissima

(Bhatnagar et al. 2010). Moreover, Chlorella minutissima is an extremely high-CO2-tolerent alga,

which may be developed to use waste CO2 for algal cultivation (Papazi et al. 2008). Therefore,

one freshwater strain, Chlorella minutissima, was chosen in the present research to find the best

conditions of cultivation, including light source, light intensity, photoperiod, and nutrient, for high

growth rate and cell density, oil content, and suitable FA profile for biodiesel production.

2. Materials and methods

2.1 Algal cultures

Chlorella minutissima (UTEX 2219) was obtained from the University of Texas at Austin

(UTEX). It is a green unicellular alga and has a cell size of about 2 ~ 5 µm. The cells were

cultured in Modified Bold 3N medium, consisting of 850 mL of DI H2O, 6 mL of P-IV metal

solution (0.75 g/L of Na2EDTA·2H2O (Sigma ED255), 0.097 g/L of FeCl3·6H2O (Sigma 1513),

0.041 g/L of MnCl2·4H2O (Baker 2540), 0.005 g/L of ZnCl2 (Sigma Z-0152), 0.002 g/L of

CoCl2·6H2O (Sigma C-3169), and 0.004 g/L of Na2MoO4·2H2O (J.T. Baker 3764)), 30 mL of 10

g/400 mL of NaNO3 (Fisher BP360-500), 10 mL of 1 g/400 mL of CaCl2·2H2O (Sigma

C-3881), 10 mL of 3 g/400 mL of MgSO4·7H2O (Sigma 230391), 10 mL of 3 g/ 400 mL of

K2HPO4 (Sigma P 3786), 10 mL of 7 g/400 mL of KH2PO4 (Sigma P 0662), 10 mL of 1

g/400mL of NaCl (Fisher S271-500), 40 mL of soil-water (1 teaspoon of garden soil (SCHULTZ

enriched garden soil, St. Louis, Missouri) was added in 200 mL of DI water), 1 mL of vitamin

B12 (0.1 mM of Vitamin B12 (cyanocobalamin, (Sigma V-6629) was added to 200 mL of 50 mM

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tHEPES buffer (Sigma H-3375) and the pH was adjusted to 7.8), 1 mL of biotin vitamin solution

(0.1 mM of biotin (Sigma V-4639) was added in 200 mL of 50mM HEPES buffer (Sigma

H-3375) and the pH was adjusted to 7.8), and 1 mL of thiamine vitamin solution (6.5 mM of

thiamine (Sigma T-1270) was added in 50 mL of 50mM HEPES buffer (Sigma H-3375) and the

pH was adjusted to 7.8). The total pH of Modified Bold 3N medium is 6.2. The components of

N-depleted medium were the same as the above Modified Bold 3N medium except excluding

NaNO3 and soil-water. In other experiments, it was found that elimination of vitamins from

Modified Bold 3N medium did not affect growth of Chlorella minutissima (data not shown).

Algae cultures were set up in 650 mL culture flasks (4.45×10.16×14.38 cm3, Greiner

Bio-One Gmbh, Germany) with a feed gas flow rate of 60 mL/min bubbled air/CO2 mixture

through an aquafizz 1 inch air stone (Petsolutions, Beavercreek, OH). The culture flask was

illuminated with external lights that were automatically turned on/off to simulate a circadian

cycle. Images of culture flask have been showed elsewhere (Tang et al. 2010). The temperature

of the reactor was maintained at 23 oC. The pH was not controlled. Three different light sources,

including a total of 20 strips of white light-emitting diodes (LEDs are supplied in strips, with 24

LEDs per 9 inch strip (0.8 watts), Super Bright LEDs, Inc. St. Louis, Missouri), 20 strips of red

LEDs, and one fluorescent light (one 12 inch 8 watts, Phillips), with the same initial light

intensity (100 µE/ (m2

s)) were evaluated for their effect on algal growth rate. Moreover, the

initial light intensities of 100, 200, 350 and 400 µE/(m2

s) were attained using one, two, three,

and four of fluorescent lights, respectively. Photoperiods of 15 hr light: 9 hr dark, 12 hr light: 12

hr dark, and 24 hr light: 0 hr dark was investigated with the initial light intensity of 200 µE/ (m2

s).

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t2.2 Growth analysis

Algal growth was measured by daily changes in both optical density at 680 nm with a

spectrophotometer (Evolution 60, Thermo Scientific, Waltham, MA) and cell numbers counted

under an OLYMPUS BX51 microscope (Optical Analysis Corp., Nashua, NH) with a

haemacytometer (Hausser Scientific, Horsham, PA). One unit of OD680 corresponded to 4 × 107

cells/mL. Determining the dry weight of Chlorella minutissima is by drying the algae for 2 hr in

an oven at 110°C. The temperature and pH were recorded every day using an Oakton pH 110

advanced portable pH meter (Euthech Instrument, Singapore).

The light intensity was measured at eighteen fixed spots on the side of the flask opposite

the light source using a light meter (LI-1400, LI-COR Inc., USA), and the average of eighteen

readings of light intensity was obtained at the same time daily.

The inorganic nitrogen content was quantified in the forms of nitrate using a portable

spectrophotometer (HACH DR 2800, Loveland, Colorado). Briefly, 5 mL algae culture was

collected, and then was centrifuged and the supernatant was transferred to another clean tube. A

one mL sample was analyzed for nitrogen content using reaction kit Nitrate (0-30 mg/L NO3-N,

Hach Lange GmbH).

2.3 Lipid analysis

2.3.1 Lipid extraction

The lipid extraction method was based on the methods of Bligh & Dyer (Bligh and Dyer

1959). The algae suspension was centrifuged at 5000 rpm (Eppendorf centrifuge 5804R, Germany)

for 10 min; the upper layer of medium was removed and the concentrated algae was obtained, and

subsequently frozen at -20 °C for 24 hr. After freezing, the about 4 g of algae was disrupted with

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tglass beads (Sigma, St. Louis) in a vortex mixer (Scientific Industries Inc, Bohemia, NY) for 10

min. The lipid was then extracted with 4 mL of chloroform: methanol (2:1), and then the extracted

lipid was centrifuged at 5000 rpm (Eppendorf centrifuge 5804R, Germany) for 10 min to separate

into three layers. The upper layer (methanol layer including water) was removed and the

chloroform layer including lipid was collected. The residues were subjected to repeated extraction

three times. All extracts obtained were then mixed together to form the crude oil extract from

which the chloroform was evaporated to yield the resultant algae oil, and finally weighed to give

the total extracted organic content (TEO). The TEO content was evaluated by its weight relative

to the weight of dry algal biomass. The definition of TEO content is the same as the total lipid

content quantified gravimetrically by others (Bhatnagar et al. 2010; Gouveia and Oliveira 2009;

Liu et al. 2008).

2.3.2 FAME analysis

The FAME was prepared from the resultant algae oil. The sodium methylate catalyst

(CH3ONa, 0.1 M/MEOH, 1.5 mL), and tetrahydrofuran solubility improver (5 mL, EMD

chemical Inc. Gibbstown, NJ) were added to the algal oil and heated at 110 °C for 5 hours in a

reactor (General purpose vessel 4744, Parr Instrument Company, Moline, IL). After the reaction

was completed, the products were cooled to room temperature, and solvent was evaporated. The

content and composition of FAME was further analyzed via GC-MS.

The FAME sample was dissolved in 1 mL of heptane, then a 100 L sample was mixed

with 100 L of ethyl arachidate (~ 10 mg/mL, Nu-Chek Prep, Inc., Elysian, Minnesota), which

was used as an internal standard. Finally, 900 L of heptane was added to the vial. The prepared

sample was analyzed using a PerkinElmer Clarus 500 GC-mass spectrometer (GC-MS) with a

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tsplit automatic injector and a Rtx-WAX (Restek, Bellefonte, PA) column (length: 60 meters; ID:

0.25 mm, coating: 0.25 µm). Details of the procedure have been described elsewhere (Tang et al.

2008). The column was held at 120 ºC for 1 minute and ramped to 240 ºC at 20 ºC/min, and it was

then held at 240 ºC for 13 minutes. The transfer line between GC and MS was kept at 240 ºC. The

FAME contents were determined by comparing their peak areas with that of the internal standard

(EEC20:0). The total FAME content was evaluated by its weight relative to the weight of dry algal

biomass.

2.3.3 FFA, TAG, DG, and MG analysis

A high performance liquid chromatographic (HPLC) method was used to determine the

free fatty acid (FFA), TAG, diglyceride (DG), and monoglyceride (MG) content in the resultant

algae oil (Foglia and Jones 1997). The HPLC analysis was conducted using a PerkinElmer Series

200 with an Altech 3300 Evaporative Light Scattering Detector (ELSD) and a Perkin Elmer

Brownlee Validated Cyano column (250×4.6mm, 5µm) with guard column (7.5×4.6mm, 5µm) as

the stationary phase and a flow rate of 1.0 mL/min. Mobile phase solvents were hexane with 0.4%

acetic acid (Phase A) and methyl t-butyl ether with 0.4% acetic acid (Phase B). A detailed elution

scheme is given in Table 1. The column temperature was set to 25 °C and the injection volume was

20 µL.

3. Results

3.1 Effect of light sources

Chlorella minutissima flask cultivations were performed with 100 µE/(m2

s) initial light

intensity of red LEDs, white LEDs, and fluorescent lights under a photoperiod of 24 hr light, 4%

CO2, temperature of 23 ºC, and pH of between 6.4 and 7.0. The fastest growth of Chlorella

minutissima was observed with florescent light with the cell concentration reaching 2.3×108

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tcells/mL after 13-day cultivation, followed by white LEDs (1.7×10

8 cells/mL) and red LEDs

(1.7×108 cells/mL). Moreover, the corresponding light intensity with red LEDs, white LEDs, and

fluorescent lights was found to be decreased with the increasing cell density as a function of time

(Fig. 1). The varying light intensity corresponding to cell density is consistent with three types of

light sources. The higher biomass content (dry weight) with fluorescent light was greater than

with the white and red LEDs (Table 2).

There was no significant difference in total FAME (TFAME) content (~ 10%) with three

types of light sources (Table 2). Chlorella minutissima methyl esters were predominantly methyl

linolenate (C18:3), methyl palmitate (C16:0), methyl linoleate (C18:2), and methyl oleate (C18:1),

with minor fractions of methyl palmitoleate (C16:1), methyl hexadecadienoate (C16:2), and

methyl hexadecatrienoate (C16:3). The total unsaturated FAME content didn’t change with

different light source, and it constituted ~ 82% of the known total FAME fraction (Table 2).

However, for methyl linolenate (C18:3) and methyl linoleate (C18:2), there were significant

differences with florescence light, white LEDs, and red LEDs as light source: methyl linolenate

(C18:3) composition with red LEDs was significantly decreased from ~ 30% with florescence

light and white LEDs to ~ 20%; while methyl linoleate (C18:2) composition was increased.

3.2 Effect of light intensity

Fluorescent light with 100, 200, 350, and 400 µE/(m2s) initial light intensities were utilized

for Chlorella minutissima cultivation with a photoperiod of 24 hr light, 4% CO2, and temperature

of 23 ºC. The results of the cell density and nitrate concentration as a function of time are shown

in Fig. 2. A significantly higher cell grow rate was observed with increasing light intensity of 100,

200 and 350 µE/ (m2

s), while no apparent difference in cell grow rate was found with light

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tintensity of between 350 and 400 µE/ (m

2s). The nitrate concentration declined continuously with

cultivation time, and was zero when the cell density reached a maximum value, indicating that

growth may have been nitrate limited. The biomass content of Chlorella minutissima increased

with increasing light intensity (Table 3) consistent with the growth shown in Figure 2.

The total FAME content by weight relative to the weight of dry algal biomass was not

significantly different for any light intensity. However, the fraction of saturated FAME with 200,

350, and 400 µE/ (m2s) of initial light intensity was higher than 100 µE/ (m

2s) (~31% vs. ~22%),

which was related to the increasing methyl palmitate (C16:0) at the higher light intensity.

3.3 Effect of photoperiod

Figure 3 shows the growth of Chlorella minutissima as a function of illumination time with

three different photoperiods of 12 hr light: 12 hr dark, 15 hr light: 9hr dark, and 24 hr light: 0 hr

dark. Each algal culture was illuminated by fluorescent light with a light intensity of 200 µE/(m2

s), 4% CO2, temperature of 23 ºC, and pH of between 6.4 and 7.0 in culture flasks. For a given

total time of illumination, the photoperiods of 12 hr light: 12 hr dark and 15 hr light: 9hr dark

achieved a higher cell density as compared to the photoperiod of 24 hr light: 0 hr dark. However,

the total biomass productivity with continuous light was higher than with the other photoperiods

(Table 4). Table 4 also shows that the photoperiod had no significant influence on TFAME content

(~10%) and composition of Chlorella minutissima (~77% unsaturated FAME).

3.4 Effect of N starvation

Starting from a culture grown in nutrient-sufficient medium which achieved an

exponential growth phase after 4-day cultivation (1.2×108 cells/mL), and N-deprivation

cultivation was achieved by centrifuging the culture and replacing the medium with the same

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tvolume of N-depleted medium. As shown in Fig. 4, the cells of Chlorella minutissima still

exhibited exponential growth under N-depleted medium and obtained a cell density of 2.5 ×108

cells/mL after 5 additional days of cultivation. The biomass content increased from 560 mg/L

(before N-deprivation cultivation) to 1240 mg/L (after N-deprivation cultivation) (Table 5).

The TEO content determined by gravimetric method was 35.3% before N-depleted

medium cultivation (1.2×108 cells/mL of cell concentration at the exponential growth phase),

while the TEO content was up to 50% after N-deprived medium cultivation (Table 5).

N-deprivation also led to a significant increase of the TFAME content from ~ 6% to 18%.

Moreover, methyl linolenate (C18:3) composition was significant decreased from ~27% to 17%,

while methyl oleate (C18:1) composition was increased from 21% to 32%; however, the total

unsaturated composition had no significant change under N-deprivation.

Table 6 shows FFA, TAG, DG, and MG content (% dry weight biomass) determined by

HPLC analysis. The total oil content including FFA, TAG, DG, and MG content was significantly

increased from around 12% to 20% in N-deprived medium. TAG and MG content were found to

be increased from ~ 1% to 9% and ~ 2% to 8%, respectively; while the FFA content was decreased

from 9% to 2% in the N-deprived medium. The TFAME content ( ~ 18%) determined by GC-MS

and total FFA, TAG, DG and MG content (~ 20%) determined by HPLC are much less than the

TEO content (50%) determined by gravimetric Bligh & Dyer method. This is likely because the

total extracted lipids include fatty acids, triglycerides, chlorophyll, and the other components.

4 Discussion

Algal production systems included open pond/raceway systems (outdoor cultivation), and

photobioreactor (PBR) systems (indoor cultivation). Open ponds are the less expensive than PBR

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tsystem, but the disadvantage of open ponds system is ease of contamination, low productivity,

high harvesting cost, and large volume of water loss (Hu et al. 2006; Shen et al. 2009; Xu et al.

2009). A PBR is a closed system that reduces the chances of contamination, allows precise

environmental control, operates as a continuous process for harvesting and producing of algae

biomass to improve efficiency; however, PBRs have a higher capital cost than open pond systems

as well as a likely high energy cost associated with cooling (Pulz 2001). The main factor affecting

microalgae cell growth in photoautotrophic cultivation is light. Algal growth is limited by

insufficient light, but also may be inhibited by over exposure to light. Our results show that red

LEDs, white LEDs, and fluorescent lights are efficient for Chlorella minutissima photosynthesis

but with a higher growth rate with fluorescent light than with white and red LEDs and comparable

intensity. This observation is contrary to a previous study where fluorescent lights, red LEDs,

and white LEDs had similar growth rates with Dunaliella tertiolecta (Tang et al. 2010). Moreover,

our findings indicated that a higher light intensity (350 µE/(m2

s) was more efficient to improve

algae Chlorella minutissima biomass density than lower light intensity (100 ~ 200 µE/(m2

s)), and

the biomass density could reach up to 1000 mg/L dry mass after 7 days of cultivation, but a light

intensity between 350 and 400 µE/(m2

s) could not further significantly increase growth rate. An

earlier study showed that the density of Chlorella minutissima was 386 mg/L dry mass with ~23

µE/(m2s) light intensity and 5% CO2 concentration after 4 days of cultivation, while the biomass

density was 66 mg/L dry mass with ~ 3 µE/(m2s) light intensity (Seto et al. 1984). Bhatnagar et al.

(Bhatnagar et al. 2010) found that the biomass density was 73 mg/L dry mass under ambient CO2

concentration in standard BG 11 medium under phototrophic conditions (30 µE/(m2

s) light

intensity in 6/18 light/dark cycle) after 10 days of growth, while 379 mg/L dry mass under

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tmixotrophic condition (30 µE/(m

2s) light intensity and BG 11 medium supplemented with

glucose), and 140 mg/L under heterotrophic condition (BG 11 medium supplemented with

glucose in dark). In our conditions, the best biomass productivity of Chlorella minutissima was

~143 mg/L/day under continuous illumination with 350 µE/(m2s) fluorescent light combined with

4% CO2, and temperature of 23 ºC, and the biomass productivity was significantly increased as

compared with Vazhappilly and Chen’s results (~89.4 mg/L/day) (Vazhappilly and Chen 1998).

The dry biomass productivity (143 mg/L/day) is higher compared to 90 mg/L/day (dry biomass)

reported for both Neochloris oleoabundans and Nannochloropsis sp. in an outdoor Raceways

Pond (Gouveia and Oliveira 2009), a maximum biomass productivity of 47 mg/L/day with 11%

(w/w) of lipid content (da Silva et al. 2009) for Neochloris oleoabundans in an open race pond

(375 L) (da Silva et al. 2009), which were used as a suitable feedstocks for biodiesel production.

Moreover, Jiang et al. compared the cultivation of Dunaliella salina between outdoors and in

PBR and found that cultivation of algae in the PBR ( 24 mg/L/day of biomass productivity) was

more effective than that outdoors (20 mg/L/day) at the end of the logarithmic phase (Jiang and

Zhu 2010). Since our results are for a PBR system, they may not be directly transferable to

outdoor cultivation conditions.

Light penetration is dependent on the geometry of the reactor and the cell density of the

culture. Our results showed the effective light intensity decreased to zero at the vessel wall

opposite the light source as the cell concentration increased (Fig. 1). Light limitation was one of

the factors which could decrease algal photosynthetic rate in the log phase. While light

determined the growth rate, nitrate consumption appears to limit overall yield (Fig. 2). The

exponential growth period (log phase) was shorter under higher light intensity, i.e., at 350 and 400

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tµE/(m

2s), as compare to lower light intensity such as (100 and 200 µE/(m

2s) ). However, it is

worth noting that algae cells in N-depleted medium still had a higher growth rate when the

nitrogen starvation-stage culture process began from algae cells which were in the beginning of

log growth phase (Fig. 4). It is possible that nitrogen was accumulated within the algal cells

during the exponential phase period, and then was consumed during the algae growth in the

N-depleted medium (Lourenco et al. 1998).

The major factors affecting lipid accumulation are nutrient starvation, salinity,

growth-medium pH, temperature, and light intensity, followed by growth phase and/or aging of

the culture (Hu et al. 2008). Our results indicated that nitrogen starvation was an effective method

to improve TEO (total lipid) content and TAG content in the algae Chlorella minutissima. The

light source, light intensity, and photoperiod had no significant effect on TEO content and FAME

content. These results are in agreement with other studies, which reported that the lipid content of

Nannochloropsis sp., Neochloris oleoabundans, Scenedesmus obliqus, and Chlorella vulgaris

was increased from 29 to 60 %, 16 to 34 %, 22 to 55 %, and 25 to 50 % under N starvation,

respectively (Gouveia et al. 2009; Li et al. 2008; Rodolfi et al. 2009; Widjaja et al. 2009). Rodolfi

et al. (Rodolfi et al. 2009) also reported that nitrogen starved cells can accumulate up to 85% lipid

in its biomass while the typical content of cultures in the exponential phase was only about 5%.

However, the increased oil content of the algae may not lead to increased overall productivity of

oil because higher levels of oil in the cells are often more than offset by lower rates of cell growth

(Sheehan et al. 1998). Nitrogen level in the medium has an inverse influence on microalgal

growth and lipid storage (Li et al. 2008; Widjaja et al. 2009). A two-stage strategy with an initial

nutrient-sufficient reactor followed by a nutrient deprivation reactor can make biomass density of

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tChlorella minutissima to increase up to 1240 mg/L with 50% of total lipid content under

N-depleted condition, which was achieved at 400 µE/(m2

s) light intensity of continuous

illumination and 4% CO2 after 9- day cultivation in our study. Bhatnagar et al. (Bhatnagar et al.

2010) reported that the biomass of Chlorella minutissima was 73 mg/L, 379 mg/L, and 140 mg/L

under phototrophic, mixotrophic, and heterotrophic conditions, respectively, while the

corresponding lipid contents was 3.9%, 56.4%, and 16.5%. Therefore, the two-phase strategy with

an initial nutrient-sufficient reactor followed by a nutrient deprivation strategy may balance the

need for rapid and high biomass generation with high oil content.

GC-MS analysis demonstrates that FA profile from Chlorella minutissima mainly contains

the long chain and un-branched fatty acids with C16 to C18 carbons, thus, Chlorella minutissima

could be considered as a potential organism for biodiesel production. Among the unsaturated fatty

acids of Chlorella minutissima, special attention should be taken in relation to the about 30% of

linolenic methyl ester (18:3), which may result in the poor stability of fuel. Moreover, an

important factor affecting the FA profile of algae is light intensity. Earlier study showed that a

higher light intensity can increase TAG accumulation and saturated FA, while mainly polar lipids

(phospholipids and glycolipids), structurally and functionally associated with cell membranes, are

formed under lower light intensity (Hu et al. 2008). This is consistent with our results that the

lower light intensity (100 µE/ (m2s)) led to increases in the unsaturated FA as compared with the

higher light intensity. Seto et al. (Seto et al. 1984) also reported that light intensity affected the

composition of FA of Chlorella minutissima. However, the FA composition didn’t significantly

change with 200 to 400 µE/ (m2

s) of light intensity in our study. Interestingly, our results

indicated the light source and N-starvation also influenced the FA profile of Chlorella minutissima:

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tlinolenic acid (C18:3) composition of Chlorella minutissima was significantly decreased with red

LEDs as light source and under N-starvation conditions; but the total unsaturated FA didn’t

significantly change. It should be noted that the profile of FFA, TAG, DG, and MG was

significantly changed in N- starvation condition: The N-deprivation increases the TAG

accumulation, while FFA is formed in the nutrient-sufficient medium.

5 Conclusions

This study has demonstrated that maximum biomass productivity of Chlorella

minutissima can reach to ~143 mg/L/day under fluorescent light with 350 µE/ (m2

s) light

intensity combined with continuous light, 4% CO2, and temperature of 23 ºC. Chlorella

minutissima under N-deficient culture medium showed a great increase in lipid content and TAG

content, and Chlorella minutissima presents adequate desirable fatty acid profile. Therefore,

Chlorella minutissima may be suitable as raw material for biodiesel production

Acknowledgements

Financial support from National Institute of Food and Agriculture for this research is

gratefully acknowledged.

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AcceptedPrepri n

tList of Figure Captions:

Figure 1 Cell densities of Chlorella minutissima with red light-emitting diodes (LEDs), white

LEDs, and fluorescent lights as light source and corresponding light intensity as a

function of illumination time. The average of duplicate cultures is shown. Cultures

were illuminated under a photoperiod of 24 hr light with 100 µE/(m2s) initial light

intensity, 4% CO2, and temperature of 23 ºC.

Figure 2 Cell density of Chlorella minutissima with light intensity of 100, 200, 350, and 400

µE/(m2

s) and corresponding nitrate concentration in medium as a function of

illumination time. Cultures were illuminated by fluorescent light with a photoperiod

of 24 hr light, 4% CO2, and temperature of 23 ºC.

Figure 3 Cell density of Chlorella minutissima with different photoperiod as a function of

illumination time. Cultures were illuminated by fluorescent light with a light

intensity of 200 µE/(m2s), 4% CO2, and temperature of 23 ºC.

Figure 4 Growth curve of Chlorella minutissima with/without nitrogen starvation as a function

of illumination time. Cultures were illuminated by a photoperiod of 24 hr fluorescent

light with a light intensity of 400 µE/(m2s), 4% CO2, and temperature of 23 ºC.

AcceptedPrepri n

tList of Table Captions:

Table 1 Gradient condition of the HPLC method.

Table 2 Summary of fatty acid (FA) composition, biomass content, total extracted organic

content (TEO), and total fatty acid methyl ester (FAME) content of Chlorella

minutissima cultured with florescence light, white LEDs, and red LEDs after 14 days of

cultivation.

Table 3 Summary of FA composition, biomass content, TEO content, and total FAME content of

Chlorella minutissima with four levels of light intensity: 100, 200, 350, and 400 µE/ (m2

s) after 7 days of cultivation.

Table 4 Summary of FA composition, biomass content and productivity, TEO content, and total

FAME content of Chlorella minutissima with different photoperiod of 9 hr light: 15 hr

dark, 12 hr light: 12 hr dark, and 24 hr light: 0 hr dark, after 14 days of cultivation.

Table 5 Summary of FA composition, biomass content, TEO content, and total FAME content of

Chlorella minutissima with before and after nitrogen starvation cultivation.

Table 6 FFA, TAGs, DG, and MG content (% dry weight biomass) determined by HPLC

analysis.

AcceptedPrepri n

tTable 1

Step Time (min) A (%) B (%)

Equilibrium 15 100 0

1 5 100 0

2 15 20 80

3 17 20 80

17.1 17.1 100 0

5 27 100 10

AcceptedPrepri n

tT

able

2

Fat

ty a

cid

co

mp

osi

tio

n a (

%)

Flu

ore

scen

tW

hit

e L

ED

R

ed L

ED

C16:0

18.4

± 0

.4

18.2

3 ±

0.2

1

18.6

3 ±

0.9

3

C16:1

2.8

7 ±

0.2

5

1.4

3 ±

0.0

6

1.2

± 0

.26

C16:2

1.9

3 ±

0.2

1

0.7

7 ±

0.0

6

1.5

3 ±

0.2

1

C16:3

7.8

± 0

.26

10.9

± 0

.17

10.9

± 0

.17

C18:0

00

0

C18:1

18.9

7 ±

0.7

4

20.1

3 ±

0.1

5

21.9

33 ±

0.3

8

C18:2

19.6

3 ±

0.2

5

19.3

3 ±

0.1

5

27.3

7 ±

0.4

2

C18:3

30.3

7 ±

0.5

5

29.2

± 0

.1

19.5

3 ±

0.4

9

SF

A18.4

3 ±

0.4

18.2

3 ±

0.2

1

18.6

3 ±

0.9

3

UF

A81.5

7 ±

0.4

81.7

7 ±

0.2

1

81.3

7 ±

0.9

3

Bio

mas

s co

nte

nt

(dry

wei

gh

t, g

/L)

0.9

30

.85

0.8

7

Ex

trac

ted

org

anic

co

nte

nt b

(%

) 3

8.9

36

.73

4.4

TF

AM

E c

on

ten

t c(%

)9

.41

± 0

.47

1

1.3

5 ±

1.9

9

.99

± 0

.61

a P

erce

nta

ge

calc

ula

ted

bas

ed o

n t

he

tota

l k

no

wn

fat

ty a

cid

s.

b E

xtr

acte

d o

rgan

ic/

dry

cel

l w

eig

h ×

10

0%

c T

ota

l fa

tty

aci

d m

eth

yl

este

r (F

AM

E)/

dry

cel

l w

eig

ht

×1

00

%

AcceptedPrepri n

tT

able

3

Fat

ty a

cid

co

mp

osi

tio

n (

%)

10

0µ

E/m

2s

20

0µ

E/m

2s

35

0µ

E/m

2s

40

0µ

E/m

2s

C16:0

21.8

± 0

.75

30.1

± 1

.42

30.8

± 1

.78

29.7

± 1

.25

C16:1

2.9

± 0

.12

1.5

± 0

.21

1.3

± 0

.32

1.3

± 0

.35

C16:2

1.2

± 0

.15

0.3

± 0

.12

0.3

± 0

.15

0.4

± 0

.21

C16:3

8.3

± 0

.1

6.6

± 0

.51

6.1

± 0

.38

6.4

± 0

.51

C18:0

0.3

± 0

.06

1.2

± 0

.1

1.3

± 0

.1

1.3

C18:1

14.8

± 0

.46

13.5

± 1

.01

16 ±

0.6

1

17.5

± 0

.7

C18:2

11.9

± 0

.21

7.2

± 0

.5

7.8

± 0

.26

8.4

± 0

.20

C18:3

38.8

± 1

.36

39.6

± 0

.99

36.5

± 0

.66

35 ±

0.8

5

SF

A22.1

± 0

.80

31.3

± 1

.36

32.1

± 1

.88

31 ±

1.2

5

UF

A77.9

± 0

.85

68.7

± 1

.42

67.9

± 1

.88

69 ±

1.2

5

Bio

mas

s co

nte

nt(

dry

wei

gh

t, g

/L)

0.4

90

.74

11

.03

Ex

trac

ted

org

anic

co

nte

nt

(%)

35

.73

7.1

32

.33

0.3

TF

AM

E c

on

ten

t (%

) 1

1.2

6 ±

0.7

4

10

.23

± 0

.37

1

0.7

1 ±

0.5

9

8.8

1 ±

0.0

8

AcceptedPrepri n

tT

able

4

Fat

ty a

cid

co

mp

osi

tio

n (

%)

12

hr

15

hr

24

hr

C16:0

23.4

± 0

.61

24.8

± 0

.38

27.3

± 0

.25

C16:1

1.8

± 0

.06

1.6

± 0

.06

1.1

± 0

.06

C16:2

2.7

± 0

.06

1.9

± 0

.06

0.8

± 0

.06

C16:3

9.3

± 0

.12

9.4

± 0

.10

7.4

± 0

.12

C18:0

0.6

± 0

.06

0.7

± 0

.06

1.7

± 0

.06

C18:1

14.9

± 0

.4

15.7

± 0

.15

21.4

± 0

.21

C18:2

16.5

± 0

.12

16.8

± 0

.10

15.6

± 0

.10

C18:3

30.9

± 0

.46

29.1

± 0

.21

24.7

± 0

.1

SF

A24 ±

0.6

4

25.5

± 0

.36

29 ±

0.2

6

UF

A76 ±

0.7

2

74.5

± 0

.36

71 ±

0.2

6

Bio

mas

s co

nte

nt

(dry

wei

gh

t, g

/L)

1.1

1.3

11

.59

Bio

mas

s p

rod

uct

ivit

y (

g/L

/day

)

0

.07

90

.09

40

.11

4

Ex

trac

ted

org

anic

co

nte

nt

(%)

37

.83

8.5

39

.6

TF

AM

E/D

W (

%)

8.3

2 ±

0.6

8

10.4

4 ±

1.2

3

10.2

6 ±

0.6

5

24

AcceptedPrepri n

t T

able

5

Fat

ty a

cid

co

mp

osi

tio

n

Bef

ore

N-d

epri

vat

ion

A

fter

N-d

epri

vat

ion

C16:0

25.9

7 ±

0.8

5

28.2

± 0

.1

C16:1

2.2

± 0

.26

2.0

3 ±

0.1

2

C16:2

12.4

7 ±

0.3

1

4 ±

0.1

C16:3

02.5

7 ±

0.0

6

C18:0

00

C18:1

21.0

3 ±

0.5

9

32.6

3 ±

0.0

6

C18:2

11 ±

1.1

41

11.9

7 ±

0.1

5

C18:3

27.3

3 ±

1.1

5

18.6

3 ±

0.1

SF

A25.9

7 ±

0.8

5

28.2

± 0

.1

UF

A74.0

3 ±

0.8

5

71.8

± 0

.1

Bio

mas

s co

nte

nt

( d

ry w

eig

ht,

g/L

) 0

.56

1.2

4

Ex

trac

ted

org

anic

co

nte

nt

(%)

35

.35

0

TF

AM

E/D

W(%

)6

.22

± 0

.55

1

8.0

5 ±

0.9

0

25

AcceptedPrepri n

tTable 6

% Normal Starvation

FFA/DW 9.38% 2.16%

TG/DW 0.88% 9.47%

DG/DW 0.50% 1.08%

MG/DW 1.56% 7.67%

Total/DW 12.31% 20.38%

AcceptedPrepri n

t

Figure 1

27

AcceptedPrepri n

t

Figure 2

28

AcceptedPrepri n

t

Figure 3

29

AcceptedPrepri n

t

Figure 4

30