Bioresource Technology 123 (2012) 528–533
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Bioresource Technology
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Cultivation of green alga Botryococcus braunii in raceway, circular ponds
under outdoor conditions and its growth, hydrocarbon production
A. Ranga Rao 1, G.A. Ravishankar, R. Sarada ⇑
Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" B. braunii (LB-572 and N-836) strains
Cultivation of Botryococcus braunii in raceway and circular ponds.
"
"
"
"
were used for the current study.
Cultivated LB-572 and N-836 in both
raceway & circular ponds.
Evaluated biomass yield,
hydrocarbon content and fatty acid
profile.
Biomass and hydrocarbon content
were observed in various seasons.
Effect of NaHCO3 on biomass and
hydrocarbon production in N-836
were evaluated.
a r t i c l e
i n f o
Article history:
Received 16 April 2012
Received in revised form 6 July 2012
Accepted 7 July 2012
Available online 26 July 2012
Keywords:
Microalgae
Botryococcus braunii
Outdoor cultivation
Hydrocarbon
Fatty acids
a b s t r a c t
The present study focused on cultivation, seasonal variation in growth, hydrocarbon production, fatty
acids profiles of Botryococcus braunii (LB-572 and N-836) in raceway & circular ponds under outdoor conditions. After 18 days of cultivation the biomass yield and hydrocarbon contents were increased in both
raceway and circular ponds. The fat content was found to be around 24% (w/w) with palmitic and oleic
acids as prominent fatty acids. Hydrocarbons of C20–C30 carbon chain length were higher in raceway and
circular ponds. Maximum biomass yield (2 g L 1) and hydrocarbon content (28%) were observed in Nov–
Dec. In case of B. braunii (N-836) after 25 days of cultivation the biomass yield was 1 g L 1 and hydrocarbon content was 27%. Supplementation of 0.1% NaHCO3 in the medium resulted in biomass yield of
1.5 g L 1 and hydrocarbon content of 30% compared to control.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Botryococcus braunii is a green colonial fresh water micro alga
which produces hydrocarbons. It is recognized as one of the renewable resource for the production of hydrocarbons. Three races of B.
braunii have been documented, and they are differentiated on the
basis of the characteristic hydrocarbons they produce. The ‘A’ race
produces odd numbered C25 to C31, n-alkadienes and trienes. The
⇑ Corresponding author. Tel.: +91 821 2516501; fax: +91 821 2517233.
E-mail addresses: [email protected], [email protected] (R. Sarada).
Present address: Department of Applied Sciences and Mathematics, Arizona State
University, 7001 E. Williams Field Road, Mesa, AZ 85212, USA.
1
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.07.009
‘B’ race produces triterpenoid hydrocarbons known as botryococcenes and ‘L’ race produces lycopadiene, a C40 tetraterpene. Another
difference among the races is the colony color in the stationary
phase. Race ‘A’ and ‘B’ strains are known to produce exopolysaccharides up to 250 g m3, whereas ‘L’ race produced up to 1 kg m3
(Banerjee et al., 2002; Niehaus et al., 2011). However the amount
of exopolysaccharides production varies with the strains and the
culture conditions.
B. braunii is a promising renewable resource for the production
of hydrocarbons and it has been reported that on hydrocracking,
the distillate yields 67% gasoline, 15% aviation turbine fuel, 15%
diesel fuel and 3% residual oil (Hillen et al., 1982; Samori et al.,
2010). B. braunii is also known to produce large amounts of fatty
A. Ranga Rao et al. / Bioresource Technology 123 (2012) 528–533
acids. The quantity and composition of fatty acids varies with species and also among the races (Grice et al., 1998; Metzger et al.,
1990; Niitsu et al., 2011; Weiss et al., 2010). Being a photosynthetic
organism, it can reduce CO2 emissions by 1.5 105 tons y 1 per
8.4 103 ha of micro algal cultivation area would be necessary
(Sawayama et al., 1999; Tanoi et al., 2011). The alga B. braunii produces hydrocarbons in the range of 2–86% (on dry weight basis).
This variation in the content of hydrocarbon is due to the differences among different strains/races in the production of hydrocarbons and changes in cultural and physiological conditions (Barupal
et al., 2010; Samori et al., 2010). Dayananda et al. (2005) reported
optimization of media constituents for growth and hydrocarbon
production in B. braunii (SAG 30.81). In the changing energy scenario it is necessary to exploit the potential of this microalga as a
source of hydrocarbons. One of the important strategies may be
adaptation of the organism for outdoor cultivation. The present
study focused on the growth and hydrocarbon production of B.
braunii in both raceway and circular ponds under outdoor culture
conditions.
2. Methods
2.1. Micro algal strains
B. braunii strains were obtained from various culture collection
centers such as B. braunii (LB-572, ‘A’ race) from UTEX culture collection, USA and B. braunii (N-836, ‘B’ race) from National Institute
for Environmental Studies, Tsukuba, Japan respectively. The stock
cultures were maintained both in agar slants and liquid medium
of modified Chu 13 (Largeau et al., 1980).
2.2. Experimental design
2.2.1. Cultivation of B. braunii in raceway and circular ponds
Forty litres medium was inoculated with 25% (v/v) of B. braunii
culture and grown in raceway pond (Length 1.13 m; Width 0.6 m;
Depth 0.3 m) under outdoor conditions with 15 rpm agitation. The
culture volume was increased gradually to the pond capacity (80 L)
and then continued cultivation for a period of 18 days.
Forty litres medium was inoculated with 25% (v/v) of B. braunii
culture and grown in circular ponds (Diameter 1.21 m; Depth 0.
25 m) under outdoor conditions without agitation. The cultures
were mixed twice a day manually. One batch culture was run for
a period of 25 days and the yields were estimated.
2.2.2. Effect of NaHCO3 on B. braunii (N-836)
A set of 500 mL Erlenmeyer conical flasks were taken and
200 mL of modified Chu 13 medium was distributed and sodium
bicarbonate was added in the range of 0.05–0.1% to the flasks.
Two weeks old culture of B. braunii (N-836) grown in modified
Chu 13 was used as inoculum at 25% (v/v). The culture flasks were
incubated for 25 days at 26 ± 1 °C temperature under 20 lmol photons m 2 s 1 light intensity and 16:8 h light dark cycle. All the
experiments were carried out in triplicates.
529
content in the pooled extract was estimated by reading absorbance
at 645 and 661.5 nm using spectrophotometer and quantified by
the method of Lichtenthaler (1987).
2.3.3. Carbohydrate estimation
A known quantity of cell free (spent) medium was taken and
analyzed for total carbohydrate using phenol–sulfuric acid method
(Dubois et al., 1956).
2.3.4. Hydrocarbon extraction
The dry biomass was homogenized in mortar and pestle with nhexane for 15 min and centrifuged. The extraction process was repeated twice and supernatant was transferred to pre-weighed
glass vial and evaporated under the stream of nitrogen to complete
dryness. The quantity of residue was measured gravimetrically
(Sawayama et al., 1992) and hydrocarbon content was expressed
as percent of dry weight.
2.3.5. Hydrocarbon analysis
Hydrocarbon extract was purified by column chromatography
on silica gel. The hydrocarbon sample was analyzed using ELITE 5 capillary column. The conditions used were as per Dayananda
et al. (2005). The initial temperature of oven was at 130 °C for
5 min which was increased to 200 °C at the rate of 8 °C per minute.
After maintaining at 200 °C for 2 min, the temperature was increased to 280 °C at the rate of 5 °C/min and maintained for
15 min. The injector port and the detector temperatures were
240 °C and 250 °C respectively. Hydrocarbons were grouped into
three categories as less than C20, higher than C30 and in between
C20 and C30 with reference to their elution with that of the retention times of the internal standard.
2.3.6. Fatty acid analysis
The lipids were extracted with chloroform–methanol (2:1, v/v)
and quantified gravimetrically. The lipid sample was dissolved in
benzene and 5% methanolic hydrogen chloride (95 mL chilled
methanol + 5 mL of acetyl chloride). The mixture was refluxed for
2 h and then 5% sodium chloride solution was added and the fatty
acid methyl esters (FAME) were extracted with hexane. The hexane
layer was washed with 2% potassium bicarbonate solution and
dried over anhydrous sodium sulphate (Christie, 1982). FAME were
analyzed by GC–MS (PerkinElmer, Turbomass Gold, Mass spectrometer) equipped with FID using SPB-1 (poly(dimethysiloxane))
capillary column (30 m 0.32 mm ID 0.25 lm film thickness)
with a temperature programming 150–280 °C at a rate of
5 °C min 1. The FAME were identified by comparing their fragmentation pattern with authentic standards (Sigma) and also with NIST
library.
2.4. Statistical analysis
Results were expressed as the mean ± SD of three replicates.
Difference between the groups were statistically analyzed by using
one-way ANOVA.
2.3. Analytical methods
3. Results and discussion
2.3.1. Biomass estimation
The cultures were harvested by centrifugation at 5000 rpm and
the cells were washed with distilled water. The pellet was freeze
dried. The dry weight of algal biomass was determined gravimetrically and growth was expressed in terms of dry weight (g L 1).
2.3.2. Chlorophyll estimation
A known volume of B. braunii culture was centrifuged and the
residue was extracted with methanol repeatedly. The chlorophyll
3.1. Growth and hydrocarbon production of B. braunii (LB-572) in
raceway and circular ponds under outdoor conditions
B. braunii (LB-572) was able to grow in raceway and circular
ponds under outdoor conditions. The biomass yields were observed at different time intervals in raceway and circular ponds.
Marginally higher chlorophyll (26 lg mL 1) and biomass
(1.8 g L 1) contents were achieved in raceway pond at the end of
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A. Ranga Rao et al. / Bioresource Technology 123 (2012) 528–533
the 18th day as shown in Fig. 1A and B. Variation in pH (7.5–9.5)
was observed during the growth of alga.
As B. braunii (LB-572) strain is known to produce hydrocarbons
and polysaccharides, these were estimated during cultivation of
alga. Hydrocarbon content in B. braunii was found to be higher in
raceway pond (Fig. 1C). After 18 days of cultivation, hydrocarbon
content was found to be 24% and 19% in raceway and circular
ponds respectively. Maximum hydrocarbon content was observed
in raceway pond. However the hydrocarbon profile as analyzed
by GC indicated that relative proportion of hydrocarbons of less
than C20, between C20–C30 and higher than C30 was not changed
much between the alga grown in raceway pond and that grown
in circular pond (Table 1).
Fig. 1D shows that the carbohydrate content in the medium as
analyzed in terms of total sugars increased with the growth of the
alga. The maximum carbohydrate content was observed in race-
Table 1
Hydrocarbon profile of B. braunii (LB-572) in raceway and circular ponds under
outdoor conditions as analyzed by GC.
B. braunii
(Race ‘A’)
Less than
C20 (%)
Between
C20–C30 (%)
Higher than
C30 (%)
Raceway pond
Circular pond
11.20 ± 1.54
28.23 ± 3.38
60.39 ± 4.26
51.45 ± 2.13
28.41 ± 3.19
20.32 ± 2.81
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
Table 2
Fatty acid profile of B. braunii (LB-572) in raceway and circular ponds under outdoor
conditions as analyzed by GC.
Fatty acid
Raceway pond (%)
Circular pond (%)
16:0
16:1
18:0
18:1
18:2
22:0
22:1
24:0
16.52 ± 0.15
15.45 ± 0.07
8.21 ± 0.01
34.23 ± 0.13
12.26 ± 0.11
3.87 ± 0.05
5.27 ± 0.09
1.69 ± 0.01
22.13 ± 0.04
12.5 ± 0.08
5.19 ± 0.11
28.35 ± 0.07
16.24 ± 0.10
2.31 ± 0.09
8.42 ± 0.06
Trace
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
way pond at the end of the 18 days of cultivation. The total fat content of the alga was found in the range of 20–24% (w/w). The fatty
acid profile is shown in Table 2, which indicated the presence of
C16:0, C16:1, C18:0, C18:1, C18:2 and C22:0 fatty acids in both the ponds
with variation in their relative proportion. Palmitic and oleic acids
were the major fatty acids in both raceway and circular ponds.
3.2. Seasonal variation on growth, biomass yield and hydrocarbon
production of B. braunii (LB-572) in raceway pond
The seasonal variation on growth, biomass yield and hydrocarbon production of B. braunii in raceway and circular ponds were
evaluated. During Jan–Dec period the hydrocarbon content was
estimated in raceway pond. May onwards due to seasonal continuous rains the outdoor culture got diluted frequently which resulted in lower biomass yields. As shown in Fig. 2 the biomass
yields were lower during rainy season (Jun–Aug). Maximum biomass (2 g L 1) was obtained during winter (Oct–Dec). Hydrocarbon
production correlated with biomass yields and maximum hydrocarbon content 28% (w/w) was observed during winter season
(Nov–Dec). The hydrocarbon profile was analyzed by GC data
(Table 3). During Nov–Dec month higher than C30 hydrocarbons
increased compared to other seasons.
Fig. 1. Biomass yield (A), chlorophyll (B), hydrocarbon (C) and carbohydrate (D) of
B. braunii (LB-572) in raceway and circular ponds under outdoor conditions. Data
represents mean ± SD of three replicates. Data recorded for 18 day old culture.
Fig. 2. Biomass yield and hydrocarbon production of B. braunii (LB-572) in raceway
pond under outdoor conditions during different months in a year. Data represents
mean ± SD of three replicates. Data recorded for 18 day old culture.
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A. Ranga Rao et al. / Bioresource Technology 123 (2012) 528–533
Table 3
Hydrocarbon profile of B. braunii (LB-572) in raceway pond during different seasons in
a year as analyzed by GC.
Season
Table 4
Hydrocarbon profile of B. braunii (N-836) in raceway and circular ponds as analyzed
by GC.
Max temp
(°C)
Less than C20
(%)
Between C20–C30
(%)
Higher than C30
(%)
Time in
(days)
Winter
Oct
Nov
Dec
Jan
Feb
25 ± 1
27 ± 1
29 ± 1
28 ± 1
31 ± 1
10.00 ± 0.98
11.31 ± 1.65
10.25 ± 1.23
17.48 ± 2.31
15.12 ± 3.21
33.36 ± 2.76
22.40 ± 3.45
21.13 ± 2.01
33.10 ± 3.65
32.72 ± 3.01
56.24 ± 5.23
66.22 ± 5.81
68.22 ± 4.96
49.32 ± 5.67
52.34 ± 4.31
Summer
Mar
Apr
May
32 ± 1
31 ± 1
29 ± 1
13.22 ± 0.95
13.79 ± 1.53
11.25 ± 2.45
37.18 ± 4.25
31.18 ± 2.86
41.51 ± 3.09
49.60 ± 4.98
55.03 ± 5.96
47.24 ± 3.78
Rainy
Jun
Jul
Aug
Sep
24 ± 1
23 ± 1
23 ± 1
25 ± 1
14.20 ± 1.97
18.20 ± 2.35
15.90 ± 1.14
10.25 ± 0.76
51.38 ± 4.32
52.08 ± 3.92
59.55 ± 4.41
34.06 ± 2.38
34.41 ± 4.25
29.83 ± 3.48
24.55 ± 2.05
55.59 ± 6.72
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
3.3. Growth and hydrocarbon production in B. braunii (N-836) culture
under outdoor conditions
Since B. braunii (LB-572) was found to be of ‘A’ race producing
saturated higher hydrocarbons, N-836 which is ‘B’ race was selected for outdoor cultivation to compare the hydrocarbon yields
in A & B races and their adaptability to outdoor conditions. Growth
and hydrocarbon yields of B. braunii (N-836) grown in both circular
and raceway ponds are shown in Fig. 3A–B. The biomass yield and
Less than C20 (%)
Between C20–C30
(%)
Higher than C30
(%)
Raceway
5
10
15
20
25
25.92 ± 2.65
19.34 ± 2.54
12.22 ± 1.98
10.46 ± 1.06
9.34 ± 2.38
54.32 ± 3.24
42.80 ± 3.98
45.24 ± 2.16
43.28 ± 3.76
38.62 ± 3.98
19.76 ± 2.06
37.86 ± 3.75
42.54 ± 5.11
46.22 ± 3.83
52.03 ± 4.65
Circular
5
10
15
20
25
22.81 ± 1.23
19.25 ± 3.02
15.03 ± 2.87
12.61 ± 1.53
6.78 ± 0.54
55.06 ± 2.87
48.93 ± 2.91
49.79 ± 3.20
47.02 ± 4.52
46.06 ± 2.10
21.69 ± 2.50
32.41 ± 2.17
35.13 ± 4.28
40.19 ± 5.13
47.16 ± 4.09
Data represents mean ± SD of three replicates. Data recorded on 25 day of old
culture.
hydrocarbon content was estimated at five day intervals. After
25 days of cultivation in outdoor pond maximum biomass yield
of 1 g L 1 with 27% hydrocarbon (w/w) content was obtained.
The hydrocarbon profile as analyzed by GC indicated that major
proportion of hydrocarbons after 25 days of outdoor cultivation
were of higher than C30 chain length (Table 4).
3.4. Effect of NaHCO3 on growth and hydrocarbon production in B.
braunii strain (N-836)
B. braunii (N-836) was evaluated for growth and metabolite production at different levels of NaHCO3. It was found that 0.1% (w/v)
NaHCO3 favored rapid growth resulting in increased biomass accumulation and hydrocarbon production at the end of the experimental period. B. braunii strain (N-836) was able to grow at all
the concentrations of NaHCO3 (0.01%, 0.020%, 0.050% and 0.1%)
tested. The biomass yields were analyzed after 25 days. The biomass yields were found to increase with increasing concentrations
of NaHCO3 and maximum biomass was achieved at 0.1% NaHCO3
concentration (Fig. 3C).
Hydrocarbon content in B. braunii was found to be similar to
growth pattern as shown in Fig. 3C. Hydrocarbon content varied
in the range of 16–30% at different NaHCO3 levels and maximum
hydrocarbon content was found at 0.1% NaHCO3. The hydrocarbon
profile as analyzed by GC, indicated that C30 category hydrocarbon
level increased up to NaHCO3 concentration of 0.05% while C20
category hydrocarbons decreased (Table 5).
The unicellular photosynthetic micro alga B. braunii is a member of the chlorophyceae which produces hydrocarbons. To date
only a limited number of micro algae such as Dunaliella (high
salinity), Spirulina (high alkalinity), and Chlorella (high nutrient)
have been maintained as monocultures and successfully cultivated in open raceway ponds for using commercially. These
micro algae were mass cultured in custom made raceway and
circular ponds for using as a source of biomass and biomolecules.
Raceway ponds are most widely utilized at the industrial level of
algal biomass production in many countries like Israel, United
Table 5
Effect of NaHCO3 on hydrocarbon profile in B. braunii (N-836) as analyzed by GC.
Fig. 3. Biomass yield (A) and hydrocarbon content (B) of B. braunii (N-836) in
raceway and circular ponds under outdoor conditions (C). Effect of NaHCO3 on
biomass and hydrocarbon content. Data represents mean ± SD of three replicates.
Data recorded for 25 day old culture.
NaHCO3 (%)
Less than C20 (%)
Between C20–C24 (%)
Higher than C30 (%)
Control
0.025
0.05
0.1
24.23 ± 2.81
14.64 ± 0.98
11.38 ± 2.33
9.76 ± 1.27
29.44 ± 3.29
34.10 ± 2.11
22.40 ± 2.14
22.97 ± 2.37
46.32 ± 5.32
51.26 ± 4.87
66.22 ± 5.76
67.27 ± 4.30
Data represents mean ± SD of three replicates. Data recorded for 25 day old culture.
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States of America, China, Japan, Taiwan and Indonesia. The present study attempted to cultivate B. braunii in open raceway and
circular ponds. In our earlier studies on B. braunii sp. under indoor (controlled) conditions we reported maximum biomass
yield of 2.0 and 2.8 g L 1 and hydrocarbon content of 46% and
33% on dry weight in B. braunii SAG 30.81 and LB-572 respectively under 16:8 h light and dark cycle with 1.2 ± 0.2 klux light
intensity at 25 ± 1 °C temperature (Dayananda et al., 2005, 2007).
It was also reported that the hydrocarbon content varied in the
range of 14–28% at different CO2 levels and the fat content in
the range of 25–30% in different species of B. braunii (Ranga
Rao et al., 2007a).
Various algae are reported to accumulate high levels of secondary metabolites under various stress conditions (Banerjee et al.,
2002; Ranga Rao et al., 2010). Fang et al., 2004 reported palmitic
acid and oleic acids as major components in the Botryococcus sp.
Ashok Kumar and Rengasamy (2012) reported that oleic, linolenic
and palmitic fatty acids were the major fatty acids in B. braunii
Kutz (AP-103). B braunii (B. mahabali) was scaled up in open raceway ponds in batch mode and the biomass yields were found to be
2 g L 1 (w/w) (Dayananda et al., 2010).
Present results showed that both the B. braunii strains (LB-572
and N-836) were successfully grown under outdoor conditions
although they differed in biomass yields and hydrocarbon content.
Interestingly these strains differed in the type of hydrocarbons
they produce. B. braunii (LB-572) belong to ‘A’ race producing
saturated hydrocarbons and alkadienes while B. braunii (N-836)
belongs to race ‘B’, which produces triterpenoid hydrocarbons
known as botryococcenes (Dayananda et al., 2006). The yields in
terms of biomass and hydrocarbon are found to be more with
‘A’ race compared to ‘B’ race. The B. braunii (LB-572) culture also
exhibited seasonal variations in growth and hydrocarbon profiles
(Fig. 3 and Table 3). Surprisingly growth and hydrocarbon yields
in both the strains were considerably less under outdoor conditions when compared to that obtained under controlled conditions (Sakamoto et al., 2012). This may be possibly due to the
exposure of the culture to different light intensities and temperatures during the day night cycles unlike in controlled cultures.
Hu and Richmond (1996) also suggested that optimum biomass
concentration was difficult to achieve in raceway ponds at high
and low irradiances of day/night. Thus, it is practically impossible
to operate at optimum cell concentration for the whole range of
irradiance, which changes throughout the day. Moreover Guterman et al. (1989) suggested that the biosyntheses of outdoor
micro algal cultures lag behind photosynthesis, which responds
to rapidly changing irradiance in the day. Although the biomass
yields were improved by bicarbonate addition, occasional CO2
bubbling and mode of cultivation (Yaming et al., 2011), increasing
the metabolite production under outdoor condition is a challenging aspect. Since hydrocarbons are accumulated in the intercellular spaces of the cells the shear forces under outdoor conditions
might be disrupting the colony morphology thereby the accumulation sites or the cultures are amenable to hydrocarbon degrading micro flora under outdoor conditions. Selective adaptation of
B. braunii to extreme conditions would be an alternative as monoculture of algae is usually achieved by maintaining an extreme
culture environment, such as high salinity, high alkalinity and
high nutritional status (Lee, 1986). Further Ranga Rao et al.
(2007b) reported that the biomass yields increased with increasing concentration of sodium chloride and maximum biomass yield
was achieved in 17 mM and 34 mM NaCl and the hydrocarbon
content varied in the range of 12–28% in different salinities and
maximum hydrocarbon content was observed in 51 mM and
68 mM NaCl. Detailed studies are necessary on B. braunii to make
its outdoor cultivation a commercial viability for obtaining high
content of hydrocarbons.
4. Conclusion
Botryococcus is known for hydrocarbon production and
throughout the world efforts are continuing to improve its cultivation methodologies. In this context, the present study was focused
on cultivation of B. braunii in both raceway and circular ponds under outdoor conditions for studying its growth and hydrocarbon
production. Our findings suggest that this organism can be
exploited for mass cultivation, biomass and hydrocarbon production under outdoor culture conditions. The study shows the adaptability of B. braunii to outdoor conditions and the most challenging
aspect is how to improve its hydrocarbon content in outdoor
conditions.
Acknowledgements
The authors thank Department of Biotechnology, Government
of India, New Delhi, for their financial support. The award of Senior
Research Fellowship to Dr. ARR by the Indian Council of Medical
Research, New Delhi is gratefully acknowledged.
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