Download Isolation and optimization of growth conditions for green alga

Isolation and optimization of growth conditions for green alga Botryococcus
Rashmi Tyagi and Arham Partap Jain
Applied Sciences & Humanities Department, ITM University, Sector 23-A, Palam Vihar,
Gurgaon (Haryana), India
[email protected]
Abstract
Life of conventional fuels is limited, necessitating switch on to renewable, environment
friendly, non-conventional fuel sources. Diesel-like hydrocarbon rich green alga Botryococcus
braunii was isolated from the freshwater Udaisagar Lake, Udaipur, Rajasthan, India. Various
growth conditions like temperature (27±1o C), light intensity (1.2 ± 0.2 klux) and light:dark cycle
(16:8) for the alga were optimized. For large-scale and economical production of biomass and
hydrocarbons, the growth of this green alga was studied in different nutrient media such as basal
medium, Chu-10, desmidiacean medium and Chu-13 medium. Among the different media tested,
Chu-13 medium was found to be the best for the growth of the alga, in which the alga grew
exponentially up to about two weeks and then was followed by the stationary phase.
In order to make the selected Chu-13 medium economic, concentration of various
components of the nutrient medium were varied, one at a time, and growth was measured. When
the concentration of the nitrogen source KNO3 was varied, maximum growth was observed in
control concentration of KNO3. In an attempt to substitute KNO3 by urea the growth was much
slower than the growth obtained in control; whereas up to 60% NaNO3 supported almost the
same growth as the control concentration of KNO3. Almost the same growth was observed with
up to 20% concentration of K2HPO4 to that of the control amount and its substitution by single
super phosphate. When MgSO4 concentration was varied in Chu-13 medium, almost same
growth was observed in B. braunii up to 60% concentration in comparison to the growth
obtained in control concentration. Unfortunately, to further enhance the growth, supplementation
of different types of soil extracts to Chu-13 medium did not enhance any growth, rather the
growth was slightly slower than the growth obtained in medium without any soil extract.
Thus, in the present study, the cost of commercial production of hydrocarbon rich B.
braunii can be reduced to some extent by substituting the K2HPO4 with much lower
concentration (20 %) of single super phosphate, KNO3 with 60 % NaNO3 and by reducing the
concentration of MgSO4 to 60 % in Chu-13 medium with almost equivalent growth as observed
in control medium. Further work of standardization of economic nutrient medium by reducing
the number of components of Chu-13 medium and supplementation of this modified medium
with treated sewage water, tap water and soil extract to obtain maximum growth and
hydrocarbon production is in progress.
Introduction:
The colonial green micro alga Botryococcus braunii (Dayanand et al., 2007, Pal et al., 1998,
Papa et al., 2008) is a rich source of hydrocarbons and lipids amounting to 30-70% of its dry
weight under different conditions of growth (Wolf, 1983; Yamaguchi et al., 1987). However, no
growth conditions have been defined which may be suitable for the large scale cultivation of the
alga for its use as an efficient hydrocarbon producer. For cultivation of B. braunii, a modified
improved Chu-10 medium has been developed (Rai et al., 1987), and the growth of the alga was
also found to be increased with the application of growth promoting substances (Ahmad, 1988).
In the present work we have isolated Botryococcus braunii from Udaisagar Lake, Udaipur,
Rajasthan and optimized the growth conditions.
Materials and Methods:
Botryococcus braunii is isolated from Udaisagar Lake, Udaipur, Rajasthan in Chu-13
nutrient medium (Chu, 1942, Yamaguchi, et al., 1987) by serial dilution followed by plating on
solidified nutrient medium. The individual colonies were isolated and inoculated into liquid
medium and incubated at 27 ± 1ºC under 1.2 ± 0.2 klux light intensity with 16:8 hrs light:dark
cycle. The purity of the culture was ensured by repeated plating and by regular observation under
microscope (Fritsch, F. E., 1935, Smith, G. M., 1920).
A time course study of B. braunii growth was carried out by using two week old culture
as inoculum at 5 % in Erlenmeyer flasks of 250 ml capacity, containing 100 ml medium for a
period of 3 weeks. The culture flasks were incubated at 27 ± 1ºC under 1.2 ± 0.2 klux light
intensity with 16:8 hrs light and dark cycle. Cultures were harvested and chlorophyll was
estimated at different time intervals. All the experiments were carried out in triplicates.
Concentration of various components of the nutrient medium Chu-13 were varied, one at
a time, and growth was measured by chlorophyll estimation in 90 % methanol using
spectrophotometer at different time intervals. A known volume of culture was centrifuged (8000
rpm) for 10 min and the pellet was treated with the same volume of 90 % methanol and kept in
water bath at 60ºC for 30 min. Absorbance of the supernatant was measured at 652 and 665 nm
and chlorophyll (a + b) was estimated using Lichtenthaler equations (Lichtenthaler, 1987).
Results and Discussion:
In order to select the best nutrient medium, growth of isolated B. braunii was checked in
various nutrient media, Chu-13, Chu-10, Basal medium and Desmidiacean medium; and Chu-13
was found to support best growth (Fig. 1). The growth curve shows that the alga grew
exponentially up to about two weeks and it was followed by the stationary phase.
Chlorophyll (ug/ml) x 10-1
100
10
1
0
5
10
15
20
Time (Days)
Fig. 1: Growth curve of B. braunii in Chu-13 medium.
Growth of test organism was compared in continuous light, light:dark cycles of 16:8,
12:12 and 8:16 hrs by incubating the culture at 27±1 oC temperature and 1.2 ± 0.2 klux light
intensity. Highest growth was found in 16:8 hr light-dark cycle. In order to make the selected
Chu-13 medium economic, growth was further checked by varying the composition of the
nutrient medium. When the concentration of the nitrogen source KNO3 (Rs. 607/kg) was varied
in Chu-13 medium, maximum growth was observed in control concentration of KNO3 (0.4 g/L)
and reduction of KNO3 concentration considerably retarded the growth of the test organism (Fig.
2).
9
KNO3
Cholorophyll Conc. (g/ml)
8
120 %
100 %
80 %
60 %
40 %
20 %
10 %
7
6
5
4
3
2
1
0
0
6
12
Time (Days)
18
Fig. 2: Growth of B. braunii in Chu-13 medium with different concentrations of KNO3 (100 % =
0.4 g/L). Growth was measured on 6th, 12th and 18th days.
Then, efforts were made to substitute the KNO3 with cheaper source of nitrogen urea (Rs.
290/kg scientific grade and Rs. 8.70/kg commercial grade), but the growth was much slower than
that of the control (0.4 g/L KNO3) (Fig. 3). Observation with commercial and scientific grade
urea was almost the same; results shown here are for commercial grade of urea.
Urea
Cholorophyll Conc. (g/ml)
8
Control
120 %
100 %
80 %
60 %
40 %
20 %
10 %
6
4
2
0
0
6
12
18
Time (Days)
Fig. 3: Growth of B. braunii in Chu-13 medium with different concentrations of urea (100 % =
0.14 g/L) on 6th, 12th and 18th days (Control: 0.4 g/L KNO3).
Once it became apparent that urea cannot economically replace KNO3, another source of
nitrogen, NaNO3 (Rs. 364/kg) was tested in Chu-13 medium, and it was found that up to 60%
NaNO3 was able to support almost same growth as the control concentration of KNO3 (Fig. 4).
Thus, KNO3 can be substituted for by NaNO3 (60 %).
NaNO3
Cholorophyll Conc. (g/ml)
8
Control
120 %
100 %
80 %
60 %
40 %
20 %
10 %
6
4
2
0
0
6
12
Time (Days)
18
Fig. 4: Growth of B. braunii in Chu-13 medium with different concentrations of NaNO3 (100 %
= 0.34 g/L) on 6th, 12th and 18th days.
It was observed that up to 20% reduction in the concentration of K2HPO4 (Rs. 963/kg) to
that of the control amount (0.08 g/L) supported almost the same growth (Fig. 5). Thus,
concentration of K2HPO4 can be effectively reduced to 20 %.
9
K2HPO4
Cholorophyll Conc. (g/ml)
8
120 %
100 %
80 %
60 %
40 %
20 %
10 %
7
6
5
4
3
2
1
0
0
6
12
Time (Days)
18
Fig. 5: Growth of B. braunii in Chu-13 medium with different concentrations of K2HPO4 (100 %
= 0.08 g/L).
Fortunately, when K2HPO4 was replaced by economic source of phosphorus, single super
phosphate (SSP) (Rs. 6.50/kg), almost equivalent growth was observed in B. braunii cultures
over the control without K2HPO4 up to 20% concentration. Thus, K2HPO4 can efficiently be
substituted by much lower concentration of SSP (20-40 %) (Fig. 6).
SSP
Control
120 %
100 %
80 %
60 %
40 %
20 %
10 %
Cholorophyll Conc. (g/ml)
8
6
4
2
0
0
6
12
18
Time (Days)
Fig. 6: Growth pattern of B. braunii in Chu-13 medium with different concentrations of SSP
(100 % = 0.07 g/L) on 6th, 12th and 18th days.
In the experiment when MgSO4 (Rs. 274/kg) concentration was varied in Chu-13
medium, almost same growth was observed in B. braunii up to 60% concentration of MgSO4 in
comparison to the growth obtained in control concentration (Fig. 7).
9
MgSO4
Cholorophyll Conc. (g/ml)
8
120 %
100 %
80 %
60 %
40 %
20 %
10 %
7
6
5
4
3
2
1
0
0
6
12
Time (Days)
18
Fig. 7: Growth pattern of B. braunii in Chu-13 medium with different concentrations of MgSO4
(100 % = 0.2 g/L) on 6th, 12th and 18th days.
In an attempt to further enhance the growth; Chu-13 medium was supplemented with
different amounts of soil extracts obtained from various places. Unfortunately, all the types of
soil extracts did not enhance any growth, rather the growth was slightly slower than the growth
obtained in medium without any soil extract. Results of the soil extract from a garden in Vasant
Kunj, New Delhi area are shown in Fig. 8.
9
Soil
Control
6 g/L
4.8 g/L
3.6 g/L
2.4 g/L
1.2 g/L
Cholorophyll Conc. (g/ml)
8
7
6
5
4
3
2
1
0
0
6
12
Time (Days)
18
Fig. 8: Growth pattern of B. braunii in Chu-13 medium with different amounts of soil extracts on
6th, 12th and 18th days.
With further understanding of the cultural conditions, the alga can be exploited for
outdoor cultivation. In the present study done so far, the cost of commercial production of
hydrocarbon rich Botryococcus braunii can be reduced to some extent. KNO3 in Chu-13 medium
can be economically substituted by 60% concentration of NaNO3. Concentration of K2HPO4 in
Chu-13 nutrient medium can be reduced to 20% without much affecting the growth. K2HPO4 can
efficiently be substituted by 20% concentration of the more economic source of phosphorus,
single super phosphate with almost equivalent growth as found in control amount of K2HPO4 in
Chu-13 nutrient medium. Concentration of MgSO4 can be effectively reduced to 60 % in Chu-13
medium.
References:
Ahmad, M. R. (1988) Physiological studies on Botryococcus. I Effect of growth promoting
substances on the growth of green alga Botryococcus braunii. Crypto. Algo. 9(1): 127-131.
Chu, S. P. (1942) The influence of the mineral composition of the medium on the growth of
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This study was performed in ITM University by RT and APJ. RT thanks DST (Department of
Science and Technology), N. Delhi for providing financial assistance.