Optimization of Growth of Two Microalgal Isolates For Biofuel Feedstock
Optimization of Growth of Two Microalgal Isolates For Biofuel Feedstock
Optimization of Growth of Two Microalgal Isolates For Biofuel Feedstock
info
BANGLADESH JOURNAL
Bangladesh J. Sci. Ind. Res. 51(3), 183-192, 2016 OF SCIENTIFIC AND
INDUSTRIAL RESEARCH
BCSIR
E-mail: [email protected]
Abstract
A research involving random isolation and characterization of naturally occurring microalgae in Bangladesh was carried out for assessing
their potential for biofuel feedstock and other uses. Among the isolates, one identified as a green alga Chlamydomonas noctigama and anoth-
er one as blue-green alga Nostoc spongiaeforme were grown in modified Chu-10D medium. The optimization of their growth was performed
following incubation of the isolates under different levels of media concentration, temperature, pH, light intensity and aeration. Both the
species showed optimum growth in terms of total chlorophyll at a temperature of 25oC. However, significant differences (at 5% level) in
growth were observed for the isolates under other conditions. The optimum growth of Chlamydomonas noctigama was observed for
the vitamin B1+B6, whereas there was no significant difference on growth of Nostoc spongiaeforme for any vitamin supplement. The
optimum pH for the growth of Chlamydomonas noctigama and Nostoc spongiaeforme were 6.5 and 7.5, light intensity 110 Em-2 s-1 and
70 Em-2 s-1, and media concentration 2x and 1x of normal concentration, respectively.
Keywords: Algae; Green alga; Blue-green alga; Chlamydomonas noctigama; Nostoc spongiaeforme; Optimization
Introduction
Algae are emerging to be one of the most promising long- ideal growth conditions for microalgal cultures are strain
term, sustainable sources of biomass and oils for fuel, food, specific and microalgae cultivation requires specific envi-
feed, and other co-products. Algae efficiently use CO2, and ronmental conditions including temperature ranges, light
are responsible for more than 40% of the global carbon fix- intensities, mixing conditions, nutrient composition, and gas
ation, with the majority of this productivity coming from exchange. It is known that microalgae respond with physio-
marine microalgae. Algae can produce biomass very rapidly, logical alterations to the environmental conditions where
with some species doubling in as few as 6 hrs, and many they grow (Schenk et al., 2008). This behavior can be
exhibiting two doublings per day (Hannon et al., 2010). All viewed as a biotechnological attribute that can be manipulat-
algae have the capacity to produce energy-rich oils, and a ed in order to control the algal biochemical composition and
number of microalgal species have been found to naturally growth, focusing on specific compounds and higher produc-
accumulate high oil levels in total dry biomass (Rodolfi et tivity. The need for clean and low-cost algae production
al., 2009). demands for investigations on algal physiological response
under different growth conditions.
Microalgae biofuels may provide a viable alternative to fos-
sil fuels; however, this technology must overcome a number A few thousand algal species are reported to occur in fresh
of hurdles before it can compete in the fuel market and be water and marine environment in Bangladesh (Ahmed et al.,
broadly deployed. These challenges include native strain 2008). However, the potential for these natural resources as
identification and improvement, both in terms of oil produc- biofuel feedstock and other probable uses are not assessed to
tivity and crop protection, nutrient and resource allocation
that extent. Despite of its importance related to biodiesel
and use, and the production of co-products to improve the
production as reported in many countries of the world, quite
economics of the entire system (Pankaj and Awasthi, 2015).
a few studies have been conducted in Bangladesh with a very
Algae are easy to grow and cultivate anywhere with less insignificant number of species such as Spirulina,
energy requirements and using very few of the nutrients. The Cholorococcum, Spirogyra sp. (Qudus and Halim, 2012)
thus requiring a comprehensive work involving isolation,
*Corresponding author. e-mail: [email protected]
184 Microalgal isolates of biofuel 51(3) 2016
characterization and optimization of their growth and selec- tics as described by Siddiqui et al. (2007) and Ahmed et al.
tion of algal strains as a potential feedstock. Therefore, the (2008).
present investigation was undertaken in order to evaluate in
vitro growth of some of the isolates under different condi- Characteristics of blue-green alga (Nostoc spongiaeforme)
tions and their potential in terms of biomass production was
Class: Cyanophyceae
assessed.
Order: Nostocales
Materials and methods Family: Nostocacae
Genus: Nostoc
Isolation and Identification Species: Nostoc spongiaeforme
Figs. 1. A-D. Nostoc spongiaeforme, A. Hormogonia of various length, B. Fully developed fila-
ments with low cyanophycin granules, C. Several developed filaments with inter-
calary heterocyst, D. Mature filaments with numerous granules
Ali, Tarin, Mondol, Chamon, Aziz and Rahman 185
Class: Chlorophyceae The liquid medium used in this study is not an absolute inor-
Order: Volvocales ganic medium but with two organic compounds, the EDTA
Family: Chlamydomonadaceae as a chelating agent and HEPES as a buffer.
Genus: Chlamydomonas
Species: Chlamydomonas noctigama Sterilization
Figs. 2. A-D. Chlamydomonas noctigama, A-B. Early stage of mature non-granular cells,
developing cells and zoospores, C-D. Mature cells and granular zoospores and
with one distinct pyronoid in each cell
After identification, isolated microalgae were used as inocu- Stock cultures were maintained in 60 ml liquid medium
lants for algal monoculture. Identification of species fol- incubated standing in an average 25C growth room under
lowed by the genus was done with the 4, 8 and 15 days old continuous light (ca 40 Em-2s-1.). Subcultures to fresh
monoculture. In vitro algal culture was done in modified medium were made after about three months. Stocks for
Chu-10D culture medium (Sinclair and Whitton, 1977) incu- experimental purposes were maintained at 32C under con-
bated in the controlled growth room, at 23 to 28C tempera- tinuous light of average 71 Em-2s-1..
ture in an average light flux of 71 Em-2s-1.
186 Microalgal isolates of biofuel 51(3) 2016
Table I. Composition of modified Chu-10D medium (mg l-1 of salts) used in the present study with Chu-10D of Sinclair
and Whitton (1977)
Estimation of growth
Table II. Composition of modified Chu-10D medium
(mg l-1 of elements) of Aziz and Whitton (1987) Total chlorophyll and optical density (O.D.) have been used
used in the present study and comparison with to estimate growth. During early stage of growth, the whole
Chu-10D of Sinclair and Whitton (1977) contents of each flask were used for either total chlorophyll
or O.D.
Elements Modified Chu-10D medium Chu-10D
N (only for GA) 6.83 6.83 Chlorophyll a (for blue-green alga) was estimated by follow-
P 1.78 1.78
ing the procedure based on the recommendations of Marker
K 2.24 2.24
et al. (1980).
Na ca 40.0* 6.69
Ca 9.78 9.78
Chlorophyll a and b can be calculated by following formu-
Mg 2.47 2.47
lae according to APHA (American Public Health
S 3.25 3.25
Association), 1985.
Fe 0.5 0.5
Si - 1.44
By measuring the optical density at 750 nm growth was esti-
Cl 17.26 0.016
mated using a spectrophotometer as described by Rodolfi et
Mn 0.012 0.012
al., 2009. During early stage of growth, the whole contents
Mo 0.0028 0.0028
Zn 0.013 0.013 of each flask were used for O.D.
Cu 0.005 0.005
Optimization of growth conditions
Co 0.002 0.002
B 0.125 0.125
The need for clean and low-cost algae production demands
*Na concentration increased from 6.69 to 40.0 mg l-1 due to
for investigations on algal physiological response under dif-
the addition of 1M NaOH solution (ca 1.48 ml l-1) during
buffering with HEPES to pH 7.2 ferent growth conditions. Experiments were carried out in
batch culture under continuous light. In the growth room
Ali, Tarin, Mondol, Chamon, Aziz and Rahman 187
The growth of both GA and BGA was observed at four dif- Results and discussion
ferent temperature (i.e. 20, 25, 30 and 35 C) to find out opti-
mum temperature for respective algal growth in the medium. The effect of different growth conditions, namely pH, light
intensity, temperature, nutrient concentration in growth
Light intensity medium, vitamins and air-bubbles supply were evaluated to
optimize the conditions of growth as well as the production
Five light intensities (i.e. 30, 50, 70, 90 and 110 Em-2s-1.) of biomass for selected microalgal samples (Chlamydomonas
had been used to find out the optimum light intensity for the noctigama and Nostoc spongiaeforme). The standard devia-
growth of selected algal strains. tion was done to measure the sample fluctuation, analysis of
variance (ANOVA) of the data was computed to determine
Vitamin supplements the F-value and the test of significance was computed by
Duncan's Multiple Range Test (DMRT), using IBM SPSS
Four vitamin solutions, individually (i.e. B1, B6, B7, B12) Statistics V.22.
and in six combinations (i.e. B1+B6, B1+B7, B1+B12,
B7+B12, B1+B7+B12 and B1+B6+B7+B12) had been used pH
to find out the effect of vitamin for two algal growths in the
The pH can affect the growth of microorganisms and each
medium.
species has a definite pH growth range and pH growth opti-
Fig. 3. Effect of pH on the growth as total chlorophyll Fig. 4. Effect of pH on the growth as chlorophyll
(chl a and b) of Chlamydomonas noctigama (chl a) of Nostoc spongiaeforme
188 Microalgal isolates of biofuel 51(3) 2016
mum. Extreme pH conditions influence photosynthesis, the activity of Nostoc punctiforme as 7.6 which was also
growth and nutrient assimilation in algae (Gensemer et al., very close to the findings of this research. Lang et al. (2008)
1993). The effect of different pH values on growth of found optimum growth of Nostoc sp. at pH 7.0.
Chlamydomonas noctigama (as total chlorophyll) and
Temperature
Nostoc spongiaeforme (as chlorophyll a) was presented in
Fig. 3 and 4, respectively. The result was statistically signif- The effect of different temperature on growth of
icant at 5% level. This means pH had significant influence Chlamydomonas noctigama and Nostoc spongiaeforme as
on the growth of both algal species. O.D. (750 nm) was statistically different from each other.
Fig. 5. Effect of temperature on the growth as Fig. 6. Effect of temperature on the growth as
O.D. of Chlamydomonas noctigama O.D. of Nostoc spongiaeforme
The highest growth (4.052 gml-1.) and the lowest growth The highest and lowest growth as O.D. (750 nm) was found
(3.116 gml-1.) as total chlorophyll (chlorophyll a and b) per at the temperature of 25C and 35C, respectively for both of
ml of Chlamydomonas noctigama were found at pH 6.5 and the isolates.
pH 7.0, respectively. The highest growth was slightly differ-
ent from the growth at pH 7.0, 5.5, 6.0 and 8.0, whereas the From the result of the study the optimum temperature
lowest growth was slightly different from the growth at pH Chlamydomonas noctigama is 25C which is similar to that
5.5 and 7.5. of Chlamydomonas reinhardtii found by Akimoto et al.
(1997). Harris (2009) reported that the usual laboratory
The optimum growth of Chlamydomonas noctigama in this species of Chlamydomonas grow well in the range of 20-
research was determined at pH 6.5 which is near about the 25C. Pocock et al. (2004) used 29C as optimum tempera-
pH condition of the habitat from which the species was iso- ture for the culture of Chlamydomonas noctigama in Bold's
lated. Skjanes et al. (2008) also found optimum pH for the basal medium. The result varies might be due to the change
growth of Chlamydomonas noctigama collected from fresh in medium.
water as 6.5.
The growth increases with the increase in temperature up to
The highest growth (19.019) and the lowest growth (9.951) its optimum and then declines with the increase in tempera-
as chlorophyll a per ml of Nostoc were found at pH 7.5 and ture. The optimum growth condition for the selected blue-
pH 6.5, respectively. The highest and the lowest growth were green alga was achieved at temperature of 25C which is
statistically different from all other growth. similar to the finding of Li-Juan et al. (2011) on Nostoc com-
mune. Spencer et al. (2011) found optimum growth of
The optimum pH range for Nostoc sp. was reported by
Rodriguez et al. (1986) as 6.4-8.2 which was almost similar Nostoc spongiaeforme at 26C on BG-11 medium.
to this research. Granhall (1975) reported optimum pH for
Ali, Tarin, Mondol, Chamon, Aziz and Rahman 189
Fig. 7. Effect of light intensity on the growth Fig. 8. Effect of light intensity on the growth as
as O.D. of Chlamydomonas noctigama O.D. of Nostoc spongiaeforme
Fig. 9. Effect of vitamins on the growth as O.D. of Fig. 10. Effect of vitamins on the growth as O.D. of
Chlamydomonas noctigama Nostoc spongiaeforme
190 Microalgal isolates of biofuel 51(3) 2016
The result was statistically significant for Chlamydomonas statistically significant. But the result was statistically signif-
noctigama and not statistically significant for Nostoc spon- icant for both of them at 48 hr and 72 hr.
giaeforme at 5% level as the growth had no statistical differ-
ence to each other and also to the control. The highest growth (0.346) and the lowest growth (0.315) as
O.D. (750 nm) of Chlamydomonas noctigama were found at
The result showed that the highest growth (0.226) and the 2x and 1.5x, respectively after 48 hr. whereas the highest
lowest growth (0.171) of Chlamydomonas noctigama were growth (0.788) and the lowest growth (0.671) as O.D. were
found at the vitamin B1+B6 and B1, respectively. The high- found at 2x and 1x, respectively after 72 hr. On the other
est growth was slightly different from the growth at B7, B12, hand, the highest growth (0.347) and the lowest growth
B1+B7, B1+B12, B7+B12 and B1+B6+B7+B12. (0.319) as O.D. (750 nm) of Nostoc spongiaeforme were
found at 1x and 2x, respectively after 48 hr. The highest
Croft et al. (2006) found that many algal species require growth (0.536) and the lowest growth (0.515) as O.D. were
exogenous cobalamin (vitamin B12), thiamine (vitamin B1) found at 1x and 1.5x, respectively after 72 hr. Miller et al.
or biotin (vitamin B7) for growth. In this study, it had been (1999) found that the increased nutrient concentration in
found that the addition of vitamin B1+B6 in the media had a medium resulted in better growth and higher biomass of blue
positive effect on the growth of Chlamydomonas noctigama. green algae.
McVeigh and Brown (1954) found stimulating effect on the
growth of Chlamydomonas noctigama after including vita-
min B12 with the medium.
Media concentration
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