Introduction

Energy consumption in Indonesia is still focused

on the fossil fuel. The current fossil fuel consumption

in Indonesia reached 1.3 million barrels per day.

Meanwhile, the production only reached 900,000

barrels per day. Fossil fuel is a non-renewable

resource, it will run out eventually. The consumption

is mostly used for transportation, households, and

industries (Nuryadhyn, 2012).

Microalga is one of the prospective materials for

biofuels. Microalgae are photosynthetic organisms

capable of using CO2 and sunlight into carbohydrates

and biomass. Microalgae have various morphology

and size of cells. Unicellular microalgae can live and

form colonies. Most microalgae are phototrophic,

although some species are heterotrophic.

Carbohydrate content in microalgae is capable to be

converted into bioethanol through fermentation

process, while the lipid content is converted into

biodiesel through transesterification process (Chisti,

2007).

One of microalgae potentially to be developed as

biofuel source is Tetraselmis sp. which has high lipid

content. Lipid content in Tetraselmis sp. can reach 15-

23% of the dry weight (Chisti, 2007; Rodolfi et al,

2009). Nevertheless, the production of biomass from

the microalgae is currently not optimal yet. The

microalgal biomass and lipid content can not meet the

needs of biofuel consumption.

Biomass of microalgae can be increased by

optimizing photosynthetic process of microalgae

(Marchetti et al., 2012; Mercado, 2004). Blue light and

red light are known to be effectively used for

photosynthesis because chlorophyll absorbs light best

on the blue and red light. Photosynthesis will be more

efficient on both those lights (Taiz and Zeiger, 2002).

The blue light can regulate microalgae metabolism and

increase the productivity of its lipid and protein

content and decrease its carbohydrates, while the red

light is able to increase its cell growth up to 300% of

the ordinary white light (Marchetti et al., 2012;

Suyono et al., 2013).

Furthermore, in order to increase the lipid content

in microalgae, nitrogen starvation method is widely

used. Microalgae were grown under the conditions of

nitrogen deficiency directing its carbon metabolism

into lipid synthesis (Hu, 2004; Thompson, 1996).

ABSTRACT

Tetrselmis sp is a potential microalgae as source for biodiesel, however, its biomass and lipid content are not

optimal yet. It is reported that blue and red light as well as nitrogen starvation could overcome the problems.

Therefore, the research aim was to study the effect of blue and red light as well as nitrogen starvation to enhance

the biomass and lipid content. This study used microalgae Tetraselmis sp. grown in f/2 medium for 14 days under

red light and blue light. At 7th day, growth medium of microalgae replaced with f/2 medium without nitrogen and

f/2 medium with 50% nitrogen of the recipe. Standard f/2 medium and white light were used as a control. Number

of microalgae cells was counted everyday using a haemocytometer and dry weight was taken on day 1, 3, 5, 7 and

14. Chlorophyll content was calculated using Jeffrey and Humphrey's trichromatic equation taken on day 1, 3, 5,

7 and 14. Lipid content was calculated using Nile red staining method on day 7 and 14. A combination of red light

and nitrogen with an amount of half of the f/2 standard medium produced the highest cell number, while the

combination of blue light and the full nitrogen recipe (standard f/2 medium) produced the highest dry weight per

cell, accounted for 3.6 million cells/ mL and 251 pg /cell, respectively. Ratio of chlorophyll a/b was stable under

blue light treatment in all nitrogen levels of medium. The combination of blue light and f/2 medium without

nitrogen generated the highest lipid, accounted for 15.89%.

Keywords : Biodiesel, Lipid, Blue Light, Red Light, Nitrogen Starvation.

Growth and Lipid Content of Microalgae Tetraselmis sp Cuture Using Combination of

Red-Blue Light and Nitrogen Starvation as an Effort to Increase Biodiesel Production

Eko Agus Suyono and Thoriq Teja Samudra

Corresponding author

eko_suyono@ugm.ac.id

Faculty of Biology, Gadjah Mada University, JalanTeknika Selatan, Sekip Utara, Yogyakarta, Indonesia

Material and Methods

Materials

The materials used in this study were a thermometer,

culture bottles, aerator, erlenmeyer, hand

refractometer, pH meter, autoclave, magnetic stirrer

with hotplate, centrifuges, microscopes, and

haemacytometer.

Tetraselmis sp. isolate Ancol was obtained from

BBAP Gondol Bali, sea water, distilled water, alcohol,

and chemicals to make the culture medium: 1 mL

stock concentration of NaH2PO4 with 0.00565 g / L, 1

mL stock concentration of NaNO3 with 0.075 g / L, 1

mL of stock trace elements (Na2 EDTA 4.16 g / L,

FeCl36H2O 3:15 g / L, CuSO45H2O 0.01 g / L,

ZnSO47H2O 0.022 g / L, CoCl26H2O 0.01 g / L,

MnCl24H2O 0,18 g / L, Na2MoO42H2O 0.006 g / L),

and 1 ml of vitamin stock (Cyanocobalamin 0.0005 g

/ L, vitamin B1 0.1 g / L, Biotin 0.0005 g / L).

Method

Preparation of medium stock

Medium used for the study was f/2 (Guillard, 1975).

Nitrogen starvation medium 50% was using NaNO3

concentration as much as half of the recipe, while

nitrogen starvation medium 0% was no NaNO3.

Preparation of starter culture of Tetraselmis sp.

Starter culture was prepared by increasing the

number of microalgal cells before treatment.

Tetraselmis sp. was cultured in the bottle with 500 mL

f/2 medium. A total of 50 mL isolate Tetraselmis sp.

was inoculated into 150 mL of medium f/2 and then

incubated for one week with aeration and continuous

illumination. White light was used for the starter

culture.

Incubation of culture with various lights

50 mL starter cultures were taken and added with

200 mL f/2 medium. Then, they were incubated for

one week. The cultures were incubated and treated by

giving a different lights (blue and red) and white light

was a control. All those treatments were run for 7 days

with 18 hours lightning per day with 3 replications.

Nitrogen starvation treatment

After a week of treatments with red light and blue

light, then transfered into the f/2 medium with nitrogen

starvation. Nitrogen levels used were 100% (full

nitrogen recipe), 50% (half nitrogen recipe), and 0%

(without nitrogen) of standard f/2 medium.

Meassurements

a. Lipid The lipid was stained using a Nile red method (Doan

and Oppbard, 2011), then observed by a fluorescence

microscope. The lipid content was counted using cell

profiler software (Carpenter et al., 2006). Lipid levels

were observed on day 7 and 14.

b. Cell number The number of cell was counted with the direct

calculation method using a haemocytometer with the

following formula:

=

5 25 104

(Punchard, 2001)

c. Dry weight Dry weight was measured by inserting a 2 mL

microalgae sample in a centrifuge tube. The sample

was centrifuged at a speed of 3300 rpm. The

supernatant was discarded. The pellet and centrifuge

tube were put in the oven with a temperature 34C to get constant weight. The difference between the

weight of the centrifuge tube at begining and the end

was measured to get the dry weight of the microalgae.

The dry weight was measured on day 0, 1, 5, 7 and 14.

d. Chlorophyll a and b content Calculation of chlorophyll a and b was performed

on day 0, 1, 5, 7, and 14 using a spectrophotometer

with a wavelength of 630 nm, 647 nm, 664 nm, and

750 nm. The amount of chlorophyll a and b was

calculated using the following formula of Jeffrey and

Humphrey's Tricrhomatic Equation:

Ca = 11.85 (D664-D750) - 1.54 (D647-D750) - 0.08

(D630-D750)..........................................................(1)

Cb = 21.03 (D647-D750) - 5.43 (D664-D750) - 2.66

(D630-D750)..........................................................(2)

Chlorophyll a, b (mg/dm3) = ((C)(Va))/((Vc))......(3)

Where:

Ca = absorbance of chlorophyll a

Cb = absorbance of chlorophyll b

C = value of (1) or (2)

Vc = culture samples used (litre)

Va = aceton used as diluting agent (ml)

(Jeffrey and Humphrey, 1975)

Results and Discussion

It could be seen from Figure 1 that the microalgae

cell growth was fastest under treatment of red light

irradiation, followed by blue and white light

treatments. Microalgae growth under red light was

faster than other treatments because the energy and

wavelength of the red light were optimum for its

photosynthesis.

Basically, photosynthesis had 2 stages of its excited

chlorophyll. Those stages were higher excited stage

when chlorophyll absorbed the high energy light (blue

light) and lower excited stage when the excited

chlorophyll absorbed the low energy light (red light).

In higher excited stage, chlorophyll was unstable and

released energy in the form of heat, then the energy

was going to be lower. On this condition, the energy

was in the lower excited stage (Taiz and Zeiger, 2002).

In the lower excited stage, red light was used

chlorophylls for photosynthesis, so that it made the

microalgae photosynthesis was faster. Therefore, the

microalgae growth under the red light was faster than

white and blue light.

Red light which has optimal energy for

photosynthesis caused more rapid growth of

microalgae cells despite medium conditions of

nitrogen deficiency. Microalgae growing in the

amount of nitrogen 50% (a half of normal f/2 medium)

was faster than others. This showed that the treatment

gave a more optimal energy and nutrient for the

growth process.

On day 14, the number of cells of microalgae under

a combination of blue light treatment and nitrogen

starvation was low. It was assumed that by the absence

of nitrogen in the medium, the microalgae in this

treatment tend to store energy in the form of lipids.

In Figure 2, the ratio of chlorophyll a/b in blue light

treatments was more stable than others. This meant

that the content of chlorophyll a and chlorophyll b in

the blue light treatment was not changed very much

during the period of cultivation. The blue light

treatments did not give much effect on the ratio of

chlorophyll a and b.

It could be seen in Figure 3 that the highest dry

weight was produced by treatment of red light in

standard f/2 medium with full of nitrogen recipe (RN

100%), while the the highest dry weight per cell was

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 1 5 7 14

Rat

io o

f C

hlo

rop

hy

ll a

/b

Days

N 100% N 50% N 0%(a)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 1 5 7 14

Rat

io o

f C

hlo

rop

hy

ll a

/b

Days

N 100% N 50% N 0%(b)

Figure 2. Ratio of Chlorophyll a/b under (a) white light, (b) red light, (c) blue light;

full nitrogen recipe (N100%),half nitrogen recipe (N50%), without

nitrogen (N0%)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 1 5 7 14

Rat

io o

f C

hlo

rop

hy

ll a

/b

Days

N 100% N 50% N 0%(c)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Cel

l N

um

ber

(C

ell.

ml-

1)

x1

06

Days

Figure 1. Cell growth of Tetraselmis sp under various lights and nitrogen starvation; Red light with full nitrogen recipe (_______), Red

light with half nitrogen recipe ( ), Red light without nitrogen ( ), White light with full nitrogen recipe

(hhhhhhhh), White light with half nitrogen recipe (________), White light without nitrogen ( ), Blue light with full

nitrogen recipe ( ), Blue light with full nitrogen recipe (_ ), Blue light without nitrogen ( )

produced by combination of blue light and standard f/2

medium with full of nitrogen recipe (BN 100%).

Eventhough the number of cells was high in the

treatment of red light and standard f/2 medium (RN

100%), however, the dry weight per cell was the

lowest among other treatments. This suggested that

cell division of microalgae under the treatment was not

offset by significant weight gain cell. At the red light

treatment, microalgae cells were divided rapidly.

Microalgae under red light treatment used all the

energy produced from photosynthesis for the cell

division process (Domozych et al., 1981; Lee, 2008;

Da Silva et al., 2009). Therefore, the total dry weight

of microalgae under red light treatment was the

highest. On the other hand, a dry weight per cell of

treatment of microalgae under blue light was highest

(Figure 4.). Presumably, the each cell under blue light

treatment was significantly heavier. This suggested

that the combination of blue light that had high energy

and a standard f/2 medium with full nitrogen recipe

was able to increase dry weight per cell of microalgae

better than other treatments.

It could be assumed that by changing the metabolic

pathway could shift the carbohydrate production into

lipids (Nelson and Cox, 2006). Moreover, the lipid

production was influenced by the type of lights used.

Blue light was used to increase the lipid content. This

was because blue light had high energy, so it

stimulated lipid production of microalgae (Suh and

Lee, 2003). Synthesis of lipids was also influenced by

the level of nitrogen in the growth medium (Nelson

and Cox, 2006). It coud be seen from the Figure 5 that

the all nitrogen starvation treatments increased lipid

levels.

In the growth medium with nitrogen deficiency,

microalgae were growing slowly. This was because

microalgae took more nitrogen from chlorophyll

synthesis process. This reduced the photosynthesis

rate of microalgae since the chlorophyll was the main

element of photosynthesis and the nitrogen played an

important role in the synthesis of chlorophyll (Da

Silva et al, 2009). Without any nitrogen in the medium, microalgae experienced chlorosis. Some studies suggested that nitrogen deficiency conditions

caused a decline in the rate of photosynthesis in

microalgae (Kolber et al., 1988; Herzig and

Falkowski, 1989; Berges et al., 1996; Young and

Beardall, 2003).

Figure 3. Dry weight of microalgae cells (a) under white light (b) cells under red light (c) under blue light; full nitrogen recipe (N100%), half nitrogen recipe (N50%), without nitrogen (N0%)

0

1

2

3

4

5

0 1 5 7 14

Dry

wei

ght

(g.

mL

)x1

0

Day

N 100% N 50% N 0%

(c)

0

2

4

6

8

10

0 1 5 7 14

Dry

wei

ght

per

cel

l (p

g.se

l)

x10

Day

N 100% N 50% N 0%(b)

0

1

2

3

4

5

0 1 5 7 14

Dry

wei

ght

(g.

mL

)x1

0

Day

N 100% N 50% N 0%

(a)

0

2

4

6

8

10

0 1 5 7 14

Dry

wei

ght

per

cel

l (p

g.se

l)

x10

Day

N 100% N 50% N 0%

(a)

0

2

4

6

8

10

0 1 5 7 14

Dry

wei

ght

Per

cel

l (p

g.se

l)

x10

Day

N 100% N 50% N 0%

0

1

2

3

4

5

0 1 5 7 14

Dry

wei

ght

(g.

mL

)x1

0

Day

N 100% N 50% N 0%

(b)

(c)

Figure 4. Dry weight per cell of microalgae (a) under white light (b) under red light (c) under blue light; full nitrogen recipe (N100%), half nitrogen recipe (N50%), without nitrogen (N0%)

Moreover, the reduced photosynthesis rate caused

cell glucose became less, whereas glucose cell was

needed for microalgae growth. Less glucose content affected the synthesis of acetyl co-A, which owned the

precursor of lipids. Microalgae altered its metabolism

into lipid synthesis that served as food reserves when

the condition was seized (Nelson and Cox, 2006; Moat

et al., 2002).

Eventhough the combination of blue light and

nitrogen starvation did not give a big change of the

microalgae chlorophyll a/b ratio, but it accelarated the

lipid synthesis. Furthermore, the microalgae stored the

lipid in the form of droplets that were in the vicinity of

chlorophyll. (Lee, 2008; Geider et al., 1998).

The combination of blue light and medium without

nitrogen produced a low dry weight. This was due to

the cell metabolism was shifted to produce more lipid

as food reserves. Microalgae which live in conditions

of nitrogen deficiency stimulated to produce more

lipid due to the poor environmental conditions (Nelson

and Cox, 2006; Taiz and Zeiger, 2002; Takagi et al.,

2000; Da Silva et al., 2009; Lee, 2008).

Conclusion

In conclusion, a combination of red light and

nitrogen with an amount of half of the f/2 standard

medium produced the highest cell number, while the

combination of blue light and the full nitrogen recipe

(standard f/2 medium) produced the highest dry

weight per cell, accounted for 3.6 million cells/ mL

and 251 pg /cell, respectively. Ratio of chlorophyll a/b

was stable under blue light treatment in all nitrogen

levels of medium. The combination of blue light and

f/2 medium without nitrogen generated the highest

lipid, accounted for 15.89%. Therefore, the

combination of blue light and nitrogen starvation was

the prospective method to increase the lipid in the

microalgae as a source of biodiesel.

Acknowledgement

The experiments were carried out in the Laboratory

of Biotechnology, Faculty of Biology, Gadjah Mada

University, Indonesia, as a part of the National Priority

Research Project. The project was financially

supported by the Institute for Research and

Community Services, Gadjah Mada University and

Directorate General of Higher Education, Ministry of

National Eduacation and Culture, Republic of

Indonesia. The authors appreciate all the concerned

agencies and people very much.

References

Berges, J.A., Charlebois D.O., Mauzerall D.C.,

Falkowski P.G., Differential effects of N

limitation on photosynthetic efficiency of

Figure 5. Lipid Content of Tetraselmis sp sp under various lights and nitrogen levels on day 7 and 14; Red light with full

nitrogen recipe (RN100%), Red light with half nitrogen recipe (RN50%), Red light without nitrogen (RN0%), White

light with full nitrogen recipe (WN100%), White light with half nitrogen recipe (WN50%), White light without

nitrogen (WN0%), Blue light with full nitrogen recipe (BN100%), Blue light with full nitrogen recipe (BN50%),

Blue light without nitrogen (BN0%)

0%

2%

4%

6%

8%

10%

12%

14%

16%

18%

RN 100% RN 50% RN 0% WN 100% WN 50% WN 0% BN 100% BN 50% BN 0%

Lip

id C

on

ten

t (%

)

Treatment

day 7 day 14

photosystems I and II in microalga. Plant

Physiol. 110: 689696. (1996) Carpenter, A.E., Jones T.R., Lamprecht M.R., Clarke

C., Kang I.H., Friman O., Guertin D.A., Chang

J.H., Lindquist R.A., Moffat J., Golland P.,

and Sabatini D.M., CellProfiler: image

analysis software for identifying and

quantifying cell phenotypes. Genome Biol.

7:R100 (2006)

Chisti, Y., Biodiesel from microalgae. Biotechnology

Advances. 25, 294306 (2007) Da Silva, A., Lourenco S.O., and Chaloub R.M.,

Effects of nitrogen starvation on the

photosynthetic physiology of a tropical marine

microalga Rhodomonas sp. (Cryptophyceae).

Aq Bot. 91, 291297 (2009) Doan, T.T.Y., and Obbard J. P., Improved Nile Red

staining of Nannochloropsis sp. J Appl Phycol

23, 895901 (2011) Domozych, D. S., Stewart K. D., and Mattox K. R.,

Development of the cell wall in Tetraselmis:

Role of the Golgi apparatus and extracellular

wall assembly, J. Cell Sci. 52, 35171 (1981) Geider, R., MacIntyre H.L., Graciano L.M., McKay

R.M.L., Response of the photosynthetic

apparatus of Dunaliella tertiolecta

(Chlorophyceae) to nitrogen and phosphorus

limitation, Eur. J. Phycol. 33, 315332 (1998) Guillard, R.R.L., Culture of phytoplankton for feeding

marine invertebrates. pp 26-60. In Smith W.L.

and Chanley M.H (Eds.) Culture of Marine

Invertebrate Animals. Plenum Press, New

York, USA (1975)

Herzig, R., Falkowski P.G., Nitrogen limitation in

Isocrysis galbana (Haptophyceae). I.

Photosynthetic energy conversion and growth

efficiencies, J. Phycol. 25, 462471 (1989) Hu, Q., Environmental Effects on Cell Composition.

In: Richmond, Amos ed. Handbook of

Microalgal Culture: Biotechnology and

Applied Phycology. Blackwell Science: India.

P. 86 (2004)

Jeffrey, S.W., Humphrey., New spectrophotometric

equation for determining chlorophyll a, b, c1

and c2. Biochem. Physiol. Pflanz., 167, 194-

204 (1975)

Kolber, Z., Zher J., Falkowski P.G., Effects of growth

irradiance and nitrogen limitation on

photosynthetic energy conversion in

photosystem II. Plant Physiol. 88, 923929

(1988) Lee, R. E., Phycology. 4th edition. Cambridge

University Press, (2008)

Marchetti, J., Bougaran G., Jauffrais T., Lefebvre S.,

Rouxel C., Saint-Jean B., Lukomska E.,

Robert R., and Cadoret J.P., Effects of blue

light on the biochemical composition and

photosynthetic activity of Isochrysis sp. (T-

iso). J. Appl. Phycol (2012)

Mercado, J. M., Snchez-Saavedra M. P., Correa-

Reyes G., Lubin L., Montero O., Figueroa F.

L., Blue light effect on growth, light

absorption characteristics and photosynthesis

of ve benthic diatom strains. Aq Bot. 78, 265277 (2004)

Moat, A.G., Foster J.W., Spector M. P., Microbial

physiology 4th ed. Wiley-liss inc. New York

(2002) Nelson, D.L., Cox M.M., Lehninger Principles of

Biochemistry 4th ed. John Wiley and sons inc.

New York (2006)

Nuryadhyn, A. Konsumsi BBM 1,3 Juta Barel Per

Hari. BANGKA POS. 15 Maret September

2012. (2012).

Punchard, N. A., Haemocytometer Instruction Sheet

(for improved Neubauer Haemocytometer).

University of East London. London. UK.

(2001) Rodolfi, L., Zittelli, G.C., Bassi, N., Padovani, G.,

Biondi, N., Bonini, G., Tredici, M.R.,

Microalgae for oil: strain selection, induction

of lipid synthesis and outdoor mass cultivation

in a low-cost photobioreactor. Biotechnol.

Bioeng, 102 (1), 100112 (2009) Suh, I.S. and Lee S.B., A light distribution model for

an internally radiating photobioreactor.

Biotechnol. Bioeng. 82, 180189 (2003) Suyono, E.A., Nuhamunada, M., Samudra, T.T.

Pengaruh pemberian cahaya merah dan biru

terhadap kandungan pertumbuhan Tetraselmis

sp sebagai sumber biofuel. Seminar Nasional Bioteknologi,Universitas Gadjah Mada

(2013) Taiz, L., and Zeiger E., Plant Physiology 3th ed.

Sinauer Associates. Sunderland. England

(2002) Takagi, M., Watanabe K., Yamaberi K., Yoshida T.,

Limited feeding of potassium nitrate for

intracellular lipid and triglyceride

accumulation of Nannochloris sp. UTEX

LB1999. Appl Microbiol Biotechnol 54, 112-

117 (2000)

Thompson, Jr. G.A. 1996. Lipids and membrane

function in green algae. Biochem. Biophys.

Acta, 13(02), 17-45 (1996)

Young, E.B., Beardall J., Photosynthetic function in

Dunaliella tetiolecta (Chlorophyta) during a

nitrogen starvation and recovery cycle, J.

Phycol. 39, 897905 (2003)