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EFFECT OF DIFFERENT CULTURE MEDIA FOR THE GROWTH AND OIL YIELD IN
SELECTED MARINE MICROALGAE

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EFFECT OF DIFFERENT CULTURE MEDIA FOR THE
GROWTH AND OIL YIELD IN SELECTED MARINE
MICROALGAE

PRABA. T1, AJAN. C1, CITARASU.T1, SELVARAJ. T2, ALBIN DHAS 1.S, GOPAL. P1, MICHAEL
BABU, M*1

1
Centre for Marine Science and Technology, Manonmanium Sundaranar University,
Rajakkamangalam, Tamil Nadu, India.
2
Departamento de Biología, Universidad Técnica Particular de Loja, UTPL, Loja, Ecuador ,
South America.

ABSTRACT
This study reveals the effect of five different culture media on biomass and oil production of five
marine microalgae, Tetraselmis sp, Chaetoceros sp, Chlorella sp, Nannochloropsis sp and Dunaliella
sp which were harvested by Flocculation method using Aluminium sulphate. The Conway, MN+, and
ASN-III medium resulted in a significantly higher growth (P<0.05) when compared to the Guillard and
Chu 10 medium. The results suggest that the ASN-III medium is the best growth medium for Chlorella
sp, Nannochloropsis sp and Chaetoceros sp than the other species studied. The maximum oil yield
was obtained in the Nannochloropsis sp that was grown in Conway medium. The oil yield in relation
with species and culture media are significant (P < 0.05).

Keywords : Microalgae, Outdoor Culture, Growth, Flocculation, Oil production.

INTRODUCTION
Micro algae are photosynthetic microorganism that convert sunlight, water and carbon dioxide
in to algal biomass. Many microalgae are exceedingly rich in oil (Chisti, 2007; Banerjee et al.,
2002). Oil content of some microalgae exceeds 80% of the dry weight of algae biomass (Chisti,
2007; Banerjee et al., 2002). Agricultural oil crops, such as soybean and palm are widely being
used to produce oil, however they produce oils in amounts that are miniscule (e.g less than 5%
of total biomass basis) compared with microalgae. Unlike other oil crops, they grow extremely
rapidly (Chisti, 2007), which is significantly quicker than other oil crops.

J. Aqua Trop. Vol. 31, No. (3-4) 2016, Pages 165-177

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166 Praba. T, Ajan. C, Citarasu.T, Selvaraj. T, Albin Dhas.S, Gopal. P, Michael Babu, M

Micro algal oil production that requires light, carbon dioxide, water, inorganic nutrients, then
mainly nitrates, phosphates, iron and trace elements. Sea water supplemented with commercial
nitrate and phosphate fertilizers, and a few other essential micro nutrients, are commonly used
for growing marine microalgae (Molina Grima et al., 1999).
The elements required for the growth of green algae are N, P, K, Mg, Ca, S, Fe, Cu, MN+, and
Zn and these elements are added in the form of salts (Kaplan et al., 1986). There have been
extensive studies on the growth of Chlorella in culture media with different concentration of
different carbon sources (Jayasankar and Vasala, 2008; Sostaric et al., 2009). Variation in the
elemental composition of Chlorella under different conditions and different stages of growth has
been reported (Oh-Hama and Miyachi, 1988; Harrison et al., 1990).
Certain nutrients in appropriate quantities are needed in culture media for the algae to
multiply. As the algae belonging to different classes and family, the requirements of nutrients
may naturally vary. Another factor to be noted is that among the culture media, the
constituents also vary. All media possess nitrogen, phosphorous and carbonate as major
nutrients and lack trace metals, vitamins and other mineral nutrients. Some algae require
trace metals and minor nutrients for better growth. Isochrysis sp needs minor nutrients like
cobalamine B12 and thiamine B1 rapid growth and multiplication (Provasoli et al., 1958; Kain,
and Folk., 1960).
Microalgae are photosynthetic eukaryotic organisms which can produce high-added-value
compounds such as hydrocarbons, pigments, carbohydrates, proteins and lipids (Chisti, 2007;
Banerjee et al., 2002; Tran et al., 2009; Hidalgo et al., 2013). These microorganisms can
accumulate important quantities of lipids (Hidalgo et al., 2013; Balat and Balat, 2010) and
additionally, they present a fast growth and high productivity compared to agricultural crops
(Chisti, 2007; Hidalgo et al., 2013; Mata et al., 2010).
Oil yield (%) is based on the amount of lipid accumulated in the algal cells. The increase or
decrease in the high lipid content may be due to the depletion and addition of nutrient from the
media which enhances the lipid accumulation in the algal cells during the final week of the study
(Brown and Zeiler, 1993)
The growth of B. braunii LB 572 and SAG 30.81 in four media—Bold basal medium (BBM),
Bold basal with ammonium carbonate (BBMa), BG11 and modified Chu 13 medium has been
evaluated (Dayananda et al., 2007). The investigators found the highest biomass production in
BG11 medium and they also produced high hydrocarbon (oil) yields in BG11 (Dayananda et
al., 2007). However, CHU 13 medium was better for B. braunii growth than either BG11 or
BBM media (Cheng and Timilsina, 2011). Another study compared the growth of B. braunii in
four autotrophic media (CHU13, Z8, BBM, and BG11) at 25°C and found that the highest
biomass was produced in BG11 medium (Ambati et al., 2010). Reports on other microalgae
(for example Ankistrodesmus alcatus) have shown better growth in BBM compared to BG11

Journal of Aquaculture in the Tropics

 Effect of Different Culture Media for the Growth and Oil Yield in Selected Marine Microalgae 167

medium (Kalita et al., 2011). However, there are no reports comparing the growth of B. braunii
in BG11 medium to the growth in JM medium despite the fact that both media have been used
for culture (Rani et al., 2011; Li et al., 2013).
The present study has been conducted to isolate and identify the microalgae and to find out the
suitability of the commercial media for growth and oil yield.

MATERIALS AND METHOD

(i) Isolation of microalgae:

Marine algal sample were collected from west coast area of Rajakkamangalam. A mono species
of algae were isolated by serial dilution method. All species were identified by external morphology
using microscope and microalgal identification keys included, The structure and reproduction of
algae (Fritsch, 1935); Introduction to Marine Plankton (Abijit Mitra et al., 2004) and Identifying
Marine Phytoplankton (Carmelo, 1997).

(ii) Stock culture maintenance of microalgae:

One litre of filtered and sterilized seawater after cooling was added with stock solution of Conway
medium and considered as working solution, which was used to maintain the stock culture. Ten
to twenty percentage of the inoculums in the growing phase were transferred into 1culture flask
and placed in front of the light (1000 lux). In 4-5 days, the culture reached log phase. Then in
8-10 days, the maximum exponential phase was achieved.

(iii) Growth of isolated microalgae in outdoor mass culture:

In outdoor cultures, the isolated micro algae culture was cultivated in five different media. After
inoculation, all the five isolated algae were cultured at 38±2ºc under 3±0.2 k lux light with 12-
12 hours light dark cycle. The purity of the culture was ensured by regular microscopic
observations.

In open culture system, 500 L FRP tank was chosen for the biomass cultivation, inoculum of
100ml/L medium was initially used. The tank was kept in a good light source. Purity of the
culture was checked daily from day zero to day of harvest26 (Huang et al., 2010).

The growth of the isolated algae in all the media were determined by visible cell count using
Neubauer’s chamber.

Algal growth was checked at 24hrs interval, starting from day zero to the day of harvest. A drop
of well mixed culture was placed on the counting slide graticule, and the viable cells were
counted using microscope at 40X level.

Journal of Aquaculture in the Tropics

168 Praba. T, Ajan. C, Citarasu.T, Selvaraj. T, Albin Dhas.S, Gopal. P, Michael Babu, M

Total cell count =  Total No. of squares  10000

(iv) Preparation of media:

The media such as Conway, Guillard’s, Chu 10, MN+, and ASN-III medium were prepared as per
standard published methodologies.
The Conway media was as described by Laing (1991). The Guilard’s media was by Smith et al.
(1993). The Chu 10 media was by Stein (1973). The MN+ media was according to Rippka (1979).
The ASN-III media was also by Rippka (1988).

(v) Culture and harvest:

The five marine microalgae Tetraselmis sp, Chaetoceros sp, Chlorella sp, Nannochloropsis sp
and Dunaliella sp. were stock cultured and outdoor mass cultured using five standard culture
media individually. The algal cell’s were harvested at late log phase by following the method of
Michael Babu et al. (2013)

(vi) Extraction of algal oil (Michael Babu et al. (2013))

The algal oil was extracted by The method described by Michael Babu et al. in 2013.

(vii) Statistical analysis:

The data obtained in the present study are expressed as mean SD and were analyzed using
two way ANOVA at 5% significant level.

RESULT

1. Isolation and identification of microalgae:

To know the growth, the enriched samples were serially diluted. The isolated samples were
singled out using Conway medium by spread plate and broth culture techniques. The isolated
species were identified as Tetraselmis sp, Chaetoceros sp, Chlorella sp, Nannochloropsis sp
and Dunaliella sp. Isolated microalgae were grown outdoor.

2. Growth of algae in different standard culture media:

In outdoor culture, Tetraselmis sp showed the highest growth in MN+ media with the cell count
385×104 cells/ml. This was higher than Conway, Guillard’s, Chu 10, and ASN-III media. Whereas
Chaetoceros sp, Chlorella sp and Nannochloropsis sp showed the maximum biomass in ASN-
III media with the cell count 380, 715 and 785×104 cell/ml respectively. The best growth of
Dunaliella sp was obtained in Conway media with the cell count 985 ×104 cells/ml (fig 1- 5).

Journal of Aquaculture in the Tropics

Effect of Different Culture Media for the Growth and Oil Yield in Selected Marine Microalgae 169

Figure 1. Growth of Tetraselmis sp in different growth media

Figure 2. Growth of Chaetoceros sp in different growth media

Figure 3. Growth of Chlorella sp in different growth media

Journal of Aquaculture in the Tropics

170 Praba. T, Ajan. C, Citarasu.T, Selvaraj. T, Albin Dhas.S, Gopal. P, Michael Babu, M

Figure 4. Growth of Nannochloropsis sp in different growth media

Figure 5. Growth of Dunaliella sp in different growth media

3. Oil yield in different standard culture media:

Tetraselmis sp, Chaetoceros sp, Chlorella sp, Nannochloropsis sp and Dunaliella sp were grown
in five different standard culture media, and the maximum oil yield (ml/100g) varied based on the
species. In Tetraselmis sp, the maximum oil yield was obtained in ASN-III medium and MN+
medium which yielded 20.30 ml/100g each and followed by 18.20, 16.52 and 13.00 ml/100g in
Conway, Guillard’s and Chu 10 media respectively.
In Chaetoceros sp, the maximum oil yield of 30.20 ml/100g was obtained in MN+ medium and
followed by 28.52, 27.15, 24.21 and 18.60 ml/100g in ASN-III, Conway, Guillard, and Chu 10
media respectively; in Chlorella sp, the maximum oil yield 25.64 ml/100g was obtained in ASN-
III medium and followed by 18.52, 18.00, 17.41, and 11.82 ml/100g in Conway, Guillard, MN+ and
Chu 10 media respectively; in Nannochloropsis sp, the maximum oil yield of 32.45 ml/100g

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 Effect of Different Culture Media for the Growth and Oil Yield in Selected Marine Microalgae 171

was obtained in Conway medium and followed by 30.25, 29.22, 28.25, and 26.15 ml/100g in,
MN+, ASN- III, Chu 10 and Guillard, media respectively; in Dunaliella sp, the maximum oil yield
22.00ml/100g was obtained in ASN-III medium and Conway medium and followed by 19.60,
18.35, and 15.44ml/100g, in MN+, Chu 10 and Guillard media respectively (Table 1).

Table 1. Extraction of Algal oil in different growth media

Name of the Oil yield (ml/100g)

algal species
Conway Guilard’s Chu 10 MN+ ASN-III
Tetraselmis sp, 18.20+0.42 16.52+0.16 13.00+0.18 20.30+0.45 20.30+0.45
Chaetoceros sp 27.15+0.16 24.21+0.62 18.60+0.24 30.20+0.22 28.52+0.52
Chlorella sp, 18.52+0.18 18.00+0.47 11.82+0.36 17.41+0.10 25.64+0.16
Nannochloropsis sp, 32.45+0.32 26.15+0.14 29.22+0.16 30.25+0.18 28.25+0.22
Dunaliella sp 22.00+0.20 15.44+0.66 18.35+0.48 19.60+0.42 22.00+0.10

(Variation due to species and media is significant (P< 0.05))

Table 2: ANOVA for oil production due to species variation and culture media

Source of Variation SS df MS F P-value F crit

Species 528.5139 4 132.1285 19.3724 5.62E-06 3.006917

Media 160.0049 4 40.00123 5.864896 0.004206 3.006917

Error 109.1272 16 6.82045

Total 797.646 24

The two way ANOVA showed that the oil production is significantly (p<0.05) influenced by the
species variation and type of culture media (Table 2).

DISCUSSIONS
In the present study, the samples were collected from Rajakkamangalam Sea, near Kanyakumari,
Tamil Nadu, India and enriched with Conway medium. Then the species were isolated and
identified by appropriate techniques. The growth of five microalgal species were standardized
using different culture media such as Conway, Guillard’s, Chu 10, MN+ and ASN-III. The isolated
samples were further cultivated by outdoor culture techniques.
In order to study the efficiency of culture media on growth of Tetraselmis sp, different culture
media such as Conway, Guillard, Chu 10, MN + and ASN-III were prepared and used for

Journal of Aquaculture in the Tropics

172 Praba. T, Ajan. C, Citarasu.T, Selvaraj. T, Albin Dhas.S, Gopal. P, Michael Babu, M

culturing the microalgae. Different culture media tested in this study had different effects on
growth on microalgae. Among the five culture media tested for this study, Chu 10 media was
recorded as less efficient in encouraging the growth of algae when compared with other
culture media. The culture media also played an important role in completing the duration of
growth cycle.
In the present study, the Chaetoceros sp, Chlorella sp and Nannochloropsis sp showed the
maximum biomass in ASN-III and the best growth of Dunaliella sp was obtained in Conway
media. The cell count of Skeletonema costatum was found to be maximum in exponential
phase in Conway medium (600×104 cells/ml), followed by Miquel’s medium (500×104cells/ml),
TMRL Medium (470×104 cells/ml) and TMRL medium with urea (450×104 cells/ml) which clearly
proved that the Convay medium was the most suitable medium for getting more algal biomass
in low volume of area which could be useful in hatcheries for raising mass cultures to use as live
feed (Mayavan and Thangavel, 2014). Blue Green algae exhibited relatively better growth in ASN
III N+ media (Nagle et al., 2010).

In order to know the influence of different culture media on multiplication of algal cells in exponential
phase, the increase in the cell count per day was calculated in the experimental algal species.
Among the five species experimented for the daily increase of cells, the Tetraselmis sp and
Dunaliella sp had the maximum multiplication rate.

Certain nutrients in appropriate quantities are needed in culture media for selected algae to
multiply. As the algae belonging to different classes and family, the requirements of nutrients
may naturally vary. Another factor to be recanted is that among the culture media, the
constituents also vary. All media posses the major nutrients like nitrogen, phosphorous and
carbonate and lack in trace metals, vitamins and other mineral nutrients. But some algae
may require these trace metals and minor nutrients for better growth. Minor nutrients like
Cobalamine B12 and Thiamine B1 are needed for the rapid growth and multiplication of Isochrysis.
This finding also confirmed our result obtained in this study (Provasoli et al., 1958; Kain, and
Folk, 1960).

The microalgae were when grown in Guillard medium, the maximum cell multiplication was
observed in Chlorella sp and followed by Dunaliella sp, Nannochloropsis sp, Tetraselmis sp,
Chaetoceros sp. The result obtained from this study showed that the Guillard’s media has
positive effect only in Chlorella sp, Chaetoceros sp, and Dunaliella sp, but did not support the
growth of Nannochloropsis sp and Tetraselmis sp.

In Chu 10 media, the maximum cell multiplication was observed in Tetraselmis sp, followed by
Dunaliella sp, Nannochloropsis sp, Tetraselmis sp, Chaetoceros sp, but Chlorella had inferior
growth. Hence Chu 10 media is not advisable to culture Nannochloropsis sp, Chaetoceros sp
and Chlorella sp.

Journal of Aquaculture in the Tropics

 Effect of Different Culture Media for the Growth and Oil Yield in Selected Marine Microalgae 173

The modified Chu-10 medium qualified best for growth, pigment complex and morphology (Rekha
Sharma et al., 2011). Similar observations were also reported by other works; i.e. nutritional
studies with variations in the amounts of essential elements in the solution may show that
modifications of Chu 10 will result in a faster rate and greater amount of growth of many of the
algae (Gerald et al., 1950). Potassium dihydrogen orthophosphate was the source of phosphate
in modified Chu-10 medium, it may be responsible for the rapid growth of the alga under
experiment, as has earlier been reported in Selenastrum sp (Turpin, 1986), where other nutrients
were limiting. It is often assumed that the limiting nutrients are nitrogen and phosphorus (elements
that comprise higher percentages in the cellular composition).
The growth of microalgae not only depends on the temperature, light and nutrient availability, but
also has a direct impact on the available carbon in the culture medium. In modified Chu-10
medium carbon source was Sodium carbonate, higher chlorophyll content in this medium showed
that Sodium carbonate support the growth of C. vulgaris. Carbon to chlorophyll ratio is a sensitive
indicator of the physiological state of microalgae (Jayasankar and Vasala, 2008). Sodium
carbonate and sodium silicate provides alkaline buffering in the medium.
In the case of MN+ media, the maximum cell count per day in exponential phase was observed
in Tetraselmis sp and Dunaliella sp, which was followed by Nannochloropsis sp, Chaetoceros
sp and Chlorella sp. From this observation of this experiment, the MN+ media has been the best
media for the culture of all the experimented species.
MN + and Conway media had silicate as one of the constituents to help the growth of brown
algae. The diatom belongs to this group require silicate for the formation of siliceous cell
wall also varies based on the concentration of silicate in their media and increase the cell
division. According to Ketchum and Redfield (1938), the nutrient requirement of algae may
be absolute, normal, minimum or optimum. Although many media have been devised for
culturing different algae, the experiment proves that no single medium can be said to be the
best one. A clear understanding of the nutritional requirements of members of various classes
of microalgae is therefore a prerequisite for determining the techniques of culturing and
maintaining the algae for a long period apart from judging the trace metal profile of the
source seawater at the study site.
The varied level of trace metals have varied effect on microalgae during division of cells and that
addition of chelating agent like EDTA increase the availability of trace metals to the algae
(Schreiber, 1927). In the present study, MN+, Conway, Guilard’s contained EDTA and effected
cell multiplication among all the culture media studied. Cells are also involved in mechanism of
adaptation such as to substitute one metal for another, to shift alternative pathway and to
develop resistant forms in unfavorable trace metal conditions (Hunter, 1948). As a result, the
species vary widely in their metal tolerance and requirements also briefly limiting the rate of
photosynthesis.

Journal of Aquaculture in the Tropics

174 Praba. T, Ajan. C, Citarasu.T, Selvaraj. T, Albin Dhas.S, Gopal. P, Michael Babu, M

The five algae when grown in five different standard culture media, the maximum growth and oil
production varied between species to species and media to media. The algal species which has
the maximum growth and oil yield in one media couldn’t yield the maximum growth and oil in
other media and from this study it has been concluded that each microalgae has its own choice
for the culture media for the maximum growth and oil yield. The selection of culture media is
therefore very important for culturing microalgae for the oil production.

ACKNOWLEDGEMENT
One of the authors T. Praba greatly acknowledges the UGC for funding through BSR fellowship
to carry out this study. The funding support offered through UGC SAP is also acknowledged.

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*Corresponding Author:
Michael Babu M. – Centre for Marine Science and Technology, Manonmanium Sundaranar
University, Rajakkamangalam, Tamil Nadu, India

Received : 06.04.2016

Accepted : 08.06.2016

Journal of Aquaculture in the Tropics

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