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International Journal of Marine Science 2013, Vol.3, No.17, 135-144
http://ijms.sophiapublisher.com
136
as membrane components, storage products, metabolites
and sources of energy under some special production
conditions (Deng et al., 2009).
For biodiesel product, the economic feasibility of
microalgal mass culture have to be taken into
consideration, the searching of microalgal species
with high lipid content and high cell growth is a great
importance (Lv et al., 2010). However, there are two
categories of microalgae that used for lipid production
such as: 1) high lipid content but low growth rate, for
example
Botryococcus braunii
with lipid content of
50% but had low biomass productivity of 28 mg/L/day
(Dayananda et al., 2007); 2) high growth rate but low
lipid content, such as,
Chlorella vulgaris
(Griffiths
and Harrison, 2009).
Biodiesel consists of fatty acid methyl esters, which
commonly are derived from triacyglycerols (TAGs) by
transesterification with a short chain alcohol such as
methanol, with glycerol as a by product (Chen et al.,
2010). Most microalgal species produce largae amounts
of TAG under stress condition, e.g, under nutrient
depletion and temperature stressed (Chen et al., 2010).
Enhancement lipid production in cell at various
cultivation condition such as nitrogen deprivation and
phosphate limitation (Rodolfi et al., 2009; Phadwal
and Singh, 2003; Cheng et al., 2010), light intensity
and temperature stressed (Tsovenis et al., 2003;
Norman et al., 1985), iron supplementation and silicon
deficiency (Liu et al., 2008; Griffiths and Harrison,
2009) and different CO
2
concentration (Chiu et al.,
2009; Ho et al., 2010) had been tested. Among them,
nutrient deprivation has been the most studied aspect.
Cheng et al (2010) reported that nitrogen starved cell
of
Dunaliella tertiolecta
had accumulated significant
amounts of neutral lipids by day 3 and reached
maximum lipid content per OD unit by day 4 of
culture. Rodolfi et al (2009) found that N-deprived
could stimulate the lipid accumulation with low
biomass productivity. Nitrogen starvation created an
environmental stresses for microalgae to increase a
lipid production as a consequence of inhibiting cell
division (Sukenik and Livne, 1991). It showed that
lipid accumulation was commonly correlated by
nitrogen limited growth rates and due to overall the
low lipid production (Lv et al., 2010).
Most previous study have investigated separately the
effect of those factors to cell growth or lipid production
of various microalgal species, they had hardly been
investigated simultaneously and comprehensively
between cultivation condition, especially for three
different species (
Dunaliella tertiolecta, Scenedesmus
sp.
and
Nannochloropsis
sp.) which are promising
species as a lipid production from microalgae.
Noteworthy that chlorophyll play an essential role for
capturing CO
2
and solar energy to generate the
metabolic flux for not only cell growth but also lipid
accumulation of microalgae photosynthesis (Cohen et
al., 1988; Li et al., 2008). Therefore, to get better
understanding of the relationship between cell growth
and lipid accumulation, the measurement chlorophyll
a (Chl
a
) of cell during the cultivation process is
important to be done.
In this study, the batch culture of three species
microalgae (
Dunaliella tertiolecta, Scenedesmus
sp
.
and
Nannochloropsis
sp.) were carried out. The
variation methods of lipid measurement from cultured
species were applied, including gravimetric method of
Bligh and Dyer (1959), Nile Red staining method
(Cooksey et al., 1987; Elsey et al., 2007; Chen et al.,
2009; Chen et al., 2011) and FTIR method (Dean et
al., 2010; Giordano et al., 2001). The effects of cultivation
conditions including nutrient and temperature
condition on cell growth, Chl
a
content and lipid
content of
Dunaliella tertiolecta, Scenedesmus
sp
.
and
Nannochloropsis
sp. were thoroughly investigated.
The influence of culture conditions on the cell growth,
chl
a
content and lipid production were further discussed.
Finally, the different method of lipid analysis were
compared and discussed thoroughly.
2 Materials and Methods
2.1 Microalgae and culture medium
Three microalgal species were used in this study,
specifically
Dunaliella tertiolecta, Scenedesmus
sp
.
and
Nannochloropsis
sp. (all species from culture
collection of Algae, Algal Physiology Laboratory,
Biological Science, Monash University). All microalgae
are eukaryotic photosynthesis microorganisms that
grow rapidly as a consequence of their simple
structure (Li et al., 2008).
Dunaliella tertiolecta
and
Nannochloropsis
sp. are marine microalgal were
cultured in PhK medium, consisting of 2L of pasteurized
artificial seawater which has the following composition
(per liter): 22 g NaCl, 5.0 g MgSO
4
.7H
2
O, 0.6 g KCl,