Chapter-4
Isolation and screening of microalgae for carbon sequestration and its lipid content
Abstract:
Samples collected from Gujarat coast and from polluted habitat, were enriched for
isolation and screening of potential microalgae for carbon sequestration and its lipid
content. Most of the unicellular eukaryotic microalgae have higher lipid and biomass
productivity. Out of the studied strains, Monoraphidium minutum and Chlorella
variabilis were selected because of their higher lipid, biomass productivity and CO2
tolerance.
1. Introduction:
Isolation and screening of microalgae for higher lipid productivity is important
criterion for biodiesel production and CO2 sequestration (Dunahay, 1998). The tolerance
of CO2 is one of the important characteristics which should be evaluated while selecting
the microalgal strain in different media as well as in varied pH (Griffiths et al., 2009;
Wang et al., 2008). The present chapter deals with the selection of promising microalgal
strains from the coastal environment.
2. Materials and methods:
2.1. Isolation of microalgal samples:
The collected microalgal samples from described assessed sites in Chapter- 3
(Table-1) were inoculated for enrichment. Each individual sample distributed and
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poured in different autoclaved media (BG11, modified ZM, ASN-3, BBM, ACB and F2) under aseptic conditions and allowed for 7 – 10 days for enrichment. After
enrichment, all the samples were taken for serial dilution (10-1, 10-2, 10-3) and, streaking
were done on solid CSMCRI-agarose/ agar containing media plates under aseptic
conditions. Isolated colonies which were observed after 5-20 days were picked and
transferred in autoclaved fresh respective medium (10 ml.) as well as plates in sterile
laminar flow chamber (Rippka, 1988; Anderson, 2005). Some of the microalgae like
Monoraphidium and Navicula isolated by single cell isolation technique (Anderson,
2005). Purity of the culture was examined under optical microscope with different
magnifications (10X, 40X and 100X).
The media composition for BG 11 (Blue green 11; Stainer et al., 1971), modified
ZM (Zarrouk’s media; Zarrouk, 1966), ACB (Algae culture broth, Himedia), F/2 media
(Guillard, 1975) and BBM (Bold basal media; Kantz and Bold, 1969) were used. All
media were prepared in distilled water and autoclaved.
One liter of Zarrouk’s medium consists of (part A) NaHCO3 (16.80 g/l) and K2HPO4
(0.50 g/l); (part B) NaNO3 (2.50 g/l), K2SO4 (1.00 g/l), NaCl (1.00 g/l), MgSO4·7H2O
(0.20 g/l), EDTA-Na2·2H2O (0.08 g/l), CaCl2·2H2O (0.04 g/l), and FeSO4·2H2O
(0.01 g/l); trace elements mixture A5 (part C 10 mL/l): 1.00 ml, trace elements mixture
B6 (part D 1.0 ml/l): 1.00 mL; part C (mg/L): H3BO3 2.86, MnCl2·4H2O 1.810 g,
ZnSO4·7H2O 0.222 MoO3·0.015, and CuSO4·5H2O 0.074 (the used amount is 10 mL/l);
part D mg/L: NH4VO3 22.9, NiSO4·7H2O 47.8, NaWO2 17.9, Ti2(SO4)3·6H2O, and
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Co(NO3)2·6H2O 4.4 (the amount used was 1.0 mL/l). The pH of the media was
maintained at 10.00.02 (Zarrouk, 1966).
BG-11 media composition was having NaNO3 (1.5 g/l), K2HPO4 (0.04 g/l),
MgSO4.7H2O (0.075 g/l), CaCl2.2H2O (0.036 g/l), FeC6H5O7NH4OH (0.006 g/l),
C6H8O7 (0.006 g/l), C10H14N2Na2O8 (0.001 g/l), Na2CO3 (0.02 g/l). In addition to this
trace metal mix (A5) was used 1ml/l. The trace metal mix solution was prepared
separately with composition of H3BO3 (2.86 g/l), MnCl2.4H2O (1.81 g/l), ZnSO4.7H2O
(0.222 g/l), NaMoO4.2H2O (0.39 g/l), CuSO4.5H2O (0.079 g/l), Co(NO3)2.6H2O (49.4
g/l) in distilled water. The pH of the final medium was 7.1 ± 0.2 (Stainer et al., 1971).
The detailed composition of the of autoclaved F/2 media in distilled water was as
follows: NaCl
(29.23 g/l), KCl (1.105 g/l), MgSO4·7H2O (11.09 g/l), Tris-base
(1.21 g/l) , CaCl2 2H2O (1.83 g/l), NaHCO3 (0.25 g/l), and a 3.0 mL trace metal solution
that was composed of 281.3 mg NaNO3, 21.2 mg NaH2PO4·H2O, 16.35 mg Na2·EDTA,
11.8 mg
FeCl3·6H2O,
675 MnCl2·4H2O,
37.5 CoCl2·6H2O,
37.5 ZnSO4·7H2O,
22.5 Na2MoO4, 0.375 mg vitamin B1, and 0.188 of biotin (Guillard, 1975).
The algae culture broth was prepared in sterile distilled water and, final composition
of the media consist of NH4Cl (0.05 g/L), CaCl2 (0.058 g/L), K2HPO4 (0.25 g/L), FeCl3
(0.003g/L),, MgSO4 (0.513 g/L), NaNO3 (1g/L). The final pH of the medium was
adjusted up to 7.00.02.
2.2. Mass propagation and culture maintenance:
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Culture volume of each isolated monoalgal culture successively increased by adding
fresh autoclaved medium under laminar flow conditions. Isolated colonies of microalgae
identified morphologically by using standard monographs (Desikachary, 1959) under
40X and 100X magnification of Carl Zeiss Microscope. The isolated cultures were also
maintained as mother culture as well as on solid agar containing media plates and slant
test tubes.
2.3. Screening of microalgal strains:
Isolated microalgal strains were checked for their growth in different media (BG11,
ZM, ACB, BBM) with some selective range of pH 3.0-10) to know their optimum
growth. Growth was monitored at OD540, dry weight (oven dry weight at 80ºC) and pH.
All the cultures were harvested in stationary phase by centrifugation at 8000 rpm. The
promising microalgae of higher biomass productivity were grown at 1000 ml volumetric
scale. After harvesting and drying of microalgal biomass, total lipid extracted, separated
and weighed gravimetrically. Microalgae having higher biomass productivity and lipid
content (lipid productivity) were selected for further experiments.
The total lipid was extracted from freeze-dried biomass using Chloroform: Methanol
in 2:1 ratio (Bligh and Dyer, 1959). The extracted total lipid for each sample was
weighed gravimetrically after removal of solvent and, %lipid calculated (Bligh and
Dyer, 1959). All sample analyzed in triplicates. The resistance of microalgal strains
towards CO2 was checked by inhibition of growth in sterile CO2 incubator under
different CO2%. The 100 ml of each selected microalgal culture having OD540 of 0.25
was inoculated in 250 ml flask containing favorable media. All culture flasks transferred
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in CO2 incubator at 50µM photons/m2/sec. light and 30ºC temp. for incubation wherein
mouth of the flasks kept open for direct exposure of CO2. The light source was provided
externally and light transmitted through glass door of CO2 incubator. The OD540 and pH
were checked daily.
3. Results and discussions:
The
Oscillatoria
spp.,
Phormidium
spp.,
Lyngbya
sp.
Chlorella spp., Monoraphidium sp., Navicula sp., Chroococcus sp., Spirulina sp.,
Synechococcus sp., Synechocystis sp. and Scenedesmus sp. were isolated and identified
as shown in Table 1. Out of total 17 strains, unicellular microalgae were chosen for the
study. The most of the strains were alkalophilic but some of them were able to tolerate
acidic conditions eg. Chlorcoccum sp., Synechoccoccus sp., and Synechocystis sp.
showed growth even when pH was acidic (6.80, 4.0 and 3.0) in BBM and BG11, which
showed the resistance of these strains.
The highest OD540 was observed in
Monoraphidium minutm and Chlorella variabilis in ZM as shown in Table 2.
The Monoraphidum minutum, Synechococcus sp., Chlorococcum sp. and Chlorella
sp. had higher biomass productivity (Table 3). Monoaraphidium minutum showed
maximum biomass productivity (24.75 mg/L/day) at pH 8.0 in ZM. Monoraphidium
minutum also showed comparable biomass productivity (23.83 mg/L/day) at pH 10.0 in
the same ZM. The second most promising strain was Chlorella variabilis with 23.57
mg/L/day and 24.75 mg/L/day at pH 10.0 and pH 8.0 in ZM, respectively. Both M.
minutum and C. variabilis were also having good growth in ACB after ZM as shown in
Table 2 & Table 3. These both strains had CO2 tolerance of 2%. The total lipid% of the
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four promising microalgae was estimated. The lipid% of Monoraphidum minutum,
Synechococcus sp, Chlorococcum sp. and Chlorella sp. were 20.93±1.12, 14.60±0.43,
14.07±0.58 and 15.3±0.55, respectively.
4. Conclusion:
Out of the studied strains, Monoraphidium minutum and Chlorella variabilis were
selected because of their higher lipid, biomass productivity and CO2 tolerance. Chlorella
variabilis was also selected because of its ongoing successful trial of mass cultivation in
open ponds by CSIR-CSMCRI team.
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Table 1. Isolated microalgal strains from different sites.
Major region
Vasad
Ankleshwar
Sub-site Code
MAHI
A-DP
A-CP2
A-AML2
Isolated strains
Chlorella,
Lyngbya
Chlorella
Scenedesmus
Chlorella
A-AML
Oscillatoria,
Phormidium
Su- IE
Chroococcus, Scenedesmus, Monoraphidium
Surat- Hazira
Su-CS
Somanath
Madhavpur
Bet-Dwarka
Som-CS
M-CS
D-CS
Navicula
Synechococcus, Spirulina
Phormidium
Synechocystis, Chlorococcum, Phormidium
122
Table 2. OD540 of the selected microalgal strains on 18th day in different media and pH.
Name of the
strain/genus
BG11
ZM
ACB
BBM
Initial
pH
7.1
6.0
3.0
10.0
8.0
6.0
3.0
8.0
6.0
3.0
6.8
4.0
Chlorococcum
sp.
Mean
0.82
0.51
0.40
0.46
0.55
0.65
0.29
0.71
0.99
NG
1.52
0.75
SD
0.00
0.02
0.01
0.06
0.00
0.04
0.00
0.10
0.05
0.00
0.01
0.43
Synechococcus
sp.
Mean
0.95
0.84
0.43
0.68
1.71
0.42
0.41
0.65
0.44
NG
0.40
NG
SD
0.03
0.03
0.00
0.06
0.03
0.01
0.01
0.00
0.00
0.00
0.00
Synechocysits
sp.
Mean
0.88
0.79
0.68
0.45
0.30
0.50
0.00
0.48
0.57
NG
SD
0.03
0.02
0.00
0.01
0.00
0.01
0.00
0.03
0.01
0.00
Chlorella
Variabilis
Mean
0.44
0.30
NG
1.79
1.88
0.63
0.00
0.92
1.21
SD
0.02
0.00
0.00
0.05
0.08
0.01
0.00
0.03
0.01
0.00
0.01
0.01
Monoraphidium
minutum
Mean
0.92
0.43
NG
1.81
1.89
0.70
0.00
1.74
1.22
NG
0.72
NG
SD
0.01
0.04
0.00
0.02
0.05
0.00
0.00
0.01
0.03
0.00
0.00
0.00
Chroococccus
sp.
Mean
0.42
0.44
NG
1.08
1.37
0.95
0.31
1.22
0.42
NG
0.45
NG
SD
Mean
0.01
0.99
0.04
0.64
0.00
0.29
0.02
0.71
0.05
0.69
0.00
0.53
0.02
NG
0.01
0.89
0.01
0.96
0.00
NG
0.00
0.73
0.00
NG
SD
0.01
0.00
0.00
0.02
0.01
0.01
0.00
0.01
0.02
0.01
0.03
0.00
Scenedesmus
sp.
0.00
NG
0.30
0.00
1.10
NG
0.00
NG
Italic value in the table shows optimum growth. NG shows that no growth was observed.
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Table 3. Biomass productivity (mg/L/day) of some of the selected microalgal strains.
BG11
ZM
ACB
BBM
Name of the strain/genus
Initial pH
7.1
6
3
10
8
6
3
8
6
3
6.8
4
Mean
10.80
6.72
5.27
6.06
7.24
8.56
3.82
9.35
13.04
NG
20.01
9.88
SD
0.01
0.26
0.13
0.79
0.01
0.53
0.09
1.32
0.66
-
0.13
0.66
Mean
12.51
11.06
5.66
8.95
22.52
5.53
5.40
8.56
5.79
NG
5.27
NG
SD
0.40
0.40
0.04
0.79
0.40
0.13
0.13
0.04
0.03
-
0.03
-
Mean
11.59
10.40
8.95
5.93
3.95
6.58
0.01
6.32
7.51
NG
3.95
NG
SD
0.40
0.26
0.01
0.13
0.05
0.13
NG
0.40
0.13
-
0.01
-
Mean
5.79
3.95
NG
23.57
24.75
8.30
NG
12.11
15.93
NG
14.48
NG
SD
0.26
0.01
0.01
0.66
1.05
0.13
0.01
0.40
0.13
-
0.20
-
Mean
12.11
5.66
NG
23.83
24.89
9.22
NG
22.91
16.06
NG
9.48
NG
SD
0.13
0.53
0.00
0.26
0.66
0.00
NG
0.13
0.40
-
0.00
-
Mean
5.53
5.79
NG
14.22
18.04
12.51
4.08
16.06
5.53
NG
5.93
NG
SD
0.13
0.53
0.00
0.26
0.66
0.00
0.26
0.13
0.04
-
0.10
-
Mean
13.04
8.43
3.82
9.37
9.16
7.03
NG
11.31
12.64
NG
9.66
NG
SD
0.13
0.00
0.00
0.26
0.13
0.13
0.00
0.13
0.26
-
0.40
-
Growth inhibition
by minimum CO2
(%)
2
Chlorococcum sp.
1
Synechococcus sp.
Synechocystis
sp.
1
2
Chlorella variabilis
Monoraphidium
minutum
Chroococccus sp.
Scenedesmus
sp.
2
0.5
0.5
Italic value in the table shows optimum biomass productivity. NG shows that no growth was observed.
124
Fig. 1. Total lipid content of four promising microalgae at 18th day.
25.00
Lipid (%)
20.00
15.00
10.00
5.00
0.00
Monoraphidium minutumChlorella variabilis
Synechococcus
Chlorococcum
125
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