Received: 5 October 2018

DOI: 10.1002/jobm.201800447
| Revised: 5 December 2018
| Accepted: 16 December 2018

RESEARCH PAPER

Isolation and characterization of high temperature tolerant

mutant from the cyanobacterium Anabaena doliolum

Yattapu P. Reddy | Ravindra K. Yadav | Keshawanand Tripathi |

Gerard Abraham

Centre for Conservation and Utilization of

BGA, ICAR-Indian Agricultural Research
Institute, New Delhi, India
Correspondence
Gerard Abraham, CCUBGA, ICAR-Indian
Agricultural Research Institute, New Delhi-
110012, India.
Email: abrahambga64@gmail.com
The mutant strain of the diazotrophic cyanobacterium Anabaena doliolum able to
tolerate high temperature was isolated by induced mutation techniques using ethyl
methane sulphonate. This mutant strain exhibited higher temperature tolerance than
the wild type. The wild type was able tolerate temperature up to 40 °C whereas the
mutant was able to grow at an elevated temperature of 48 °C. This mutant exhibited
higher growth rate, heterocyst frequency, and nitrogen fixation. Mutant strains
exhibited comparable levels of chlorophyll, phycocyanin, PS II activity, and O2
evolution as compared to unexposed control. Results also showed that the mutant
accumulated low levels of peroxides and lipid peroxidation products with enhanced
activity of antioxidant enzymes. The FAME analysis revealed quantitative and
qualitative changes in the profile of fatty acids in the mutant strain. Maximum number
of saturated fatty acids was observed in the mutant strain followed by control whereas
the wild type exposed to elevated temperature showed least diversity of fatty acids.
Enhanced level of antioxidant enzymes coupled with efficient modulation of fatty
acid profile could therefore enhance the mutant to resist the high temperature stress.
The results could be exploited further to decipher molecular mechanisms underlying
the temperature tolerance and enhancing the utility of A. doliolum as efficient
biofertilizer for rice paddy keeping in view of the future climatic change scenario.

KEYWORDS
antioxidant enzymes, biofertilizer, cyanobacterium, elevated temperature, fatty acids, mutant

1 | INTRODUCTION exhibit considerable ability to tolerate a number of

Increased anthropogenic activities and rapid industrialization
has created a lot of pressure on the earth's atmosphere and
resulted in an increase in the level of green house gases which
trap heat and enhance the temperature leading to the
phenomenon “global Warming.” According to Mckenzie
et al. [1] the earth is warming and the global mean temperature
change over the 21st century is likely to be about fivefold
greater than in the past century. An increase in the
temperature can lead to adverse impact on various organisms
including the cyanobacteria. In general, the cyanobacteria
J Basic Microbiol. 2019;1–9.
abiotic stress factors [2–5]. Elevated temperature leads to
inhibition of growth and photosynthesis, production of
reactive oxygen species and damage of RNA and protein [6–
9]. Mishra et al. [10] observed that the cyanobacterium
Anabaena doliolum exposed to temperature stress encounter
oxidative stress conditions due to the production of reactive
oxygen species. Stimulation in the level of antioxidant
enzymes in cyanobacteria exposed to elevated temperature
stress has been reported in cyanobacteria [11]. Oxidative
stress conditions have been reported to lead to changes
in the fatty acid composition of the membranes [12].
www.jbm-journal.com © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim | 1
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Tasaka et al. [13] and Allakhverdiev et al. [14] demonstrated
that an increase in the unsaturation of fatty acids in membrane
lipids enhances the tolerance to salt stress in the mutant
cyanobacterium Synechocystis sp. PCC 6803. Inductionn of
temperature tolerance in these organisms could be better
understood through random mutagenesis. However, most of
these studies have been conducted by exposing the organism
to high temperature and comparing the response with respect
to the control. A number of studies have been carried out in
cyanobacteria to enhance their efficiency to tolerate
environmental stress [15–17].
The cyanobacteria are distributed in terrestrial, freshwa-
ter, and marine ecosystems and these organisms exhibit
remarkable capacity to tolerate various environmental
extremes [18]. Nitrogen fixing cyanobacteria are used as
biofertilizer for rice cultivation and sensitivity of the
cyanobacteria to elevated temperature may limit their
exploitation as biofertilizer for rice paddy. Therefore,
cyanobacterial strains adapted to grow and fix nitrogen under
high temperature can be selected for developing biofertilizer
considering the anticipated increase in temperature due to
climate change. This will help in developing a sound
cyanobacterial biofertilizer technology and it may help
reducing the dependence of the chemical nitrogen fertilizers
to a certain extent and also to minimize the global climatic
change. Therefore, in the present study, efforts were made to
develop temperature tolerant mutants from the cyanobacte-
rium Anabaena doliolum through random mutagenesis.
2 | MATERIALS AND METHODS
2.1 | Experimental organism and culture
conditions
The experimental organism Anabaena doliolum was provided
by Prof. A.K. Rai, Department of Botany, Banaras Hindu
University, Varanasi, Uttar Pradesh, India. A. doliolum and
the cyanobacterium was routinely maintained in BG-11
medium without added nitrogen [19]. The pH of the medium
was adjusted to 7.5 and the cultures were routinely maintained
in a culture room at 30 °C illuminated with white fluorescent
tubes emitting 72 μmol photon m−2 s−1 PAR (photosyntheti-
cally active radiation). Cultures were shaken manually at least
two to three times a day. Experiments have been conducted in
triplicate with triplicate samples for each experiment.
2.2 | Mutagenesis, screening, and selection of
mutants
For the mutagenesis, freshly prepared filter-sterilized solution
(50 mM sodium phosphate buffer; pH 7.5) of potent chemical
mutagen Ethyl Methane Sulphonate (Sigma, USA) was used.
Exponentially growing filaments of A. doliolum were
ET AL.
harvested by centrifugation at 10,000 rpm for 5 min and
were washed with 50 mM sodium phosphate buffer (pH 7.5).
The washed filaments were resuspended in 500 µL of 0.1 M
phosphate buffer (pH 7.0) and exposed to different concen-
trations (0.05–0.5%) of the mutagen for 5 min in the dark at
room temperature. Mutagen was finally removed from the
cultures by washing the cells with sterile sodium thiosulphate
(10% w/v) for 5 min. Cells were washed with phosphate
buffer (0.1 M), centrifuged at 1250 g for 5 min, and the pellet
was resuspended in BG11 medium and kept for 24 h in dark.
The treated cultures were further plated on agar plates.
Cultures were treated with LD50 concentration of EMS and
subsequently exposed to high temperature ranging from 40 to
50 °C for screening. Colonies developed at high temperature
of 48 °C was used for developing batch culture of the mutant
cyanobacterium. These mutant cultures were again plated on
agar plates containing minimal medium and sub cultured
several times. The mutant thus isolated were sub cultured
several times in BG11 medium and observed for the genetic
stability for at least five generations. The stable mutant thus
obtained was used for further experiments. Mutant was grown
and maintained at a temperature of 48 °C and a comparison
was made between the wild type (control) and wild type
exposed to 40 °C.
2.3 | Measurement of growth
Growth was determined in terms of dry weight. A known
volume of homogenized cyanobacterial suspension was
filtered through Whatman No. 42 filter paper and oven dried
at 60 °C and cooled in a desiccator until constant weight was
achieved. The difference in initial and final weights was
recorded as dry weight [20].
2.4 | Estimation of pigments
Total chlorophyll content was determined by cold extraction
method developed by McKinney [21]. The phycocyanin
content was estimated according to the method of Bennett and
Bogorad [22].
2.5 | Estimation of PS II activity and
photosynthetic oxygen evolution
Photosynthetic O2 evolution was measured using polaro-
graphic oxygen electrode enclosed in air tight reaction vessel
and connected to an oxygen analyzer (Digital oxygen system,
model 10, Rank Brother, UK). A known amount of A.
doliolum culture (4.5 mL) was transferred into the reaction
vessel. Corresponding temperature of the vessel was
maintained by circulating water in the jacket chamber around
the reaction vessel. Polarizing voltage was set at 0.6 volt.
Light was provided using a projector lamp at photon fluence
REDDY ET AL..
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rate of 95 μmol m−2 s−1 on the surface of reaction vessel. O2

evolution was recorded for 3 min and expressed as
μ mol mg chl−1 h−1. Photosystem II (PS II) activity was
measured in terms of O2 evolution in the presence of p-
benzoquinone (PBQ, 2 mM). Photosynthetic electron trans-
port activities were measured according to the methods
described in Rai and Raizada [23] and Lien [24].

2.6 | Estimation of heterocyst frequency and

nitrogenase activity
The number of heterocysts per hundred vegetative cells is
referred as heterocyst frequency. For the estimation of
nitrogenase, acetylene reduction assay was performed
according to Stewart et al. [25]. The nitrogenase activity
was expressed in terms of nmol C2H4 mg chlorophyl l −1 h−1.
FIGURE 1 Growth (dry weight) of the wild type cyanobacterium
A. doliolum at 30 and 40 °C and mutant (48 °C) in response to
2.7 | Determination of lipid peroxidation and elevated temperature
peroxide content
Lipid peroxidation was assessed by measuring the total heterocyst frequency of the mutant cyanobacterium did not
thiobarbituric acid reactive substances and it is expressed as change significantly in response to exposure to elevated
equivalent of malondialdehyde (MDA) with minor modifi- temperature. As compared to control, 34.1% increase in the
cations as suggested by Cakmak and Horst [26]. Total heterocyst frequency was observed in the wild type
peroxide content was estimated according to the protocol cyanobacterium. The mutant cyanobacterium, on the other
given by Sagisaka [27]. hand, showed a marginal increase of 9% in the heterocyst
frequency with respect to the unexposed (control) wild cells.
It was also observed that the mutant cyanobacterium exhibit
2.8 | Estimation of antioxidant enzymes better nitrogen fixation potential as compared to the wild type.
Superoxide dismutase activity was assayed by monitoring the In the wild type exposed to 40 °C, the nitrogenase activity was
inhibition in the reduction of nitroblue tetrazolium (NBT) found to decrease by 74% whereas in the mutant strains able to
according to the method of Giannopolitis and Ries [28]. grow at 48 °C the inhibition was 39% (Fig. 2).
Ascorbate peroxidase activity was determined according to
Nakano and Asada [29]. The catalase was assayed by
measuring the disappearance of H2O2 according to Aebi [30].
Glutathione reductase activity was estimated according to
Tietze [31].

3 | RESULTS

3.1 | Growth, heterocyst frequency, and

nitrogenase activity
An inverse relationship was observed in the growth of the
cyanobacterium with exposure to temperature (Fig. 1). More
than 24.9% inhibition in the growth was observed when the
cyanobacterium was exposed to elevated temperature of
40 °C, while exposure to 45 °C inhibited the growth
completely. However, the mutant strains of the cyanobacte-
rium A. doliolum were able to grow at 48 °C and produced
significant quantity of biomass. Elevated temperature FIGURE 2 Heterocyst frequency and nitrogenase activity of wild
resulted in increase in the heterocyst frequency of the wild type A. doliolum and mutant in response to elevated temperature
type cyanobacterium (Fig. 2). On the other hand, the (Values are means of three independent observations)
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3.2 | Pigment content and photosynthetic
activity
Chlorophyll, phycocyanin content, gross photosynthesis, and
PS II electron transport chain activity by the wild type A.
doliolum and its high temperature tolerant mutant strain are
presented in Table 1. Wild type exposed to elevated
temperature showed decrease in the chlorophyll and
phycocyanin content as compared to the mutant strains
which showed marginal reduction in the pigment content. In
the present experiment, the wild type strain unexposed to high
temperature exhibited the highest O2 evolution and PS II
activity, followed by the mutant strain and the wild type
exposed to elevated temperature. PS II activity of the wild
type and mutant cyanobacterium was found to be reduced by
31 and 16%, respectively (Table 1). Similarly, gross
photosynthesis (O2 evolution) decreased by 19.6 and 7.8%.
The mutant strain was able to maintain comparable levels of
PS II activity and O2 evolution as the wild type strains
exposed to elevated temperature.
3.3 | Lipid peroxidation and antioxidant
enzyme activity
The extent of oxidative damage in relation to exposure to
temperature in the wild type and mutant cyanobacteria was
studied by estimating the H2O2 and MDA content and the
activity of antioxidants. Significant increase in the
accumulation of H2O2 was observed in the wild type
cyanobacterium exposed to elevated temperature (p > 0.01,
Fig. 3). However, the mutants accumulated comparatively
less peroxides and products of lipid peroxidation as
compared to the wild type. This observation is supported
by the increase in the activity of antioxidants by the mutant.
Although, the SOD, APX, CAT, and GR activities increased
in response to temperature in the wild type cyanobacteria,
the mutant strain exhibited further enhancement in the
antioxidant enzyme activity (p > 0.01, Fig. 4a and b).
Exposing the cyanobacterium (both wild type and mutant)
to elevated temperature possibly resulted in enhanced
generation of ROS and corresponding increase in the SOD
activity. The CAT, APX, and GR activities also showed a
similar trend.
TABLE 1 Effect of elevated temperature on the pigment content, oxygen evolution and PS II activity of the wild type cyanobacterium A. doliolum
and mutant
Temperature Chlorophyll (µg mg−1 Phycocyanin (µg mg−1
(°C) dry weight) dry weight)
30 4.61 ± 0.02 7.05 ± 0.83
40 2.62 ± 0.01 4.92 ± 0.28
48 3.01 ± 0.01 6.01 ± 1.76
Values are means of three replicates (±SD).
ET AL.
3.4 | Fatty acid (FAME) profile
Fatty acids play an important role in relation to thermo
tolerance in cyanobacteria. The FAME profile of the wild
type and mutant cyanobacteria was analyzed to assess the
quantitative as well as qualitative changes in the fatty acid
profile (Fig. 5 and Table 2). Unsaturated fatty acids (35%)
constitute the major fatty acids in the wild type cyanobacteria,
followed by saturated fatty acids (23.2%), aromatic fatty acids
(21.3%), and higher alkanes (20.5%). However, in the wild
type exposed to elevated temperature, aromatic fatty acids
(91.7%) constituted the major fatty acids, followed by alkanes
(4.3%), unsaturated fatty acids (2.5%), and saturated fatty
acids (2.6%). On the other hand, the mutant showed marked
variation in fatty acid composition than the wild type control
and treatment. Unsaturated fatty acids (47.9%) constituted the
major fatty acids in the mutant followed by alkanes (31.3%),
saturated fatty acids (14.5%), and aromatic fatty acids (6.3%).
Maximum number of saturated fatty acids was observed in
mutant strain, followed by control. However, the wild type
exposed to elevated temperature showed the least diversity
of fatty acids. From the results, it appears that in the mutant of
A. doliolum the fatty acid biosynthesis was shifted from short
chain to long chain fatty acids as compared to control.
4 | DISCUSSION
In the present study, we have employed one of the most
commonly used chemical mutagenesis techniques to enhance
the thermotolerance of the cyanobacterium A. doliolum.
The mutagenesis resulted in stable alterations in this strain
and the mutant cyanobacterium was able to tolerate relatively
high temperature up to 48 °C. However, the wild type was
able to tolerate temperature up to 40 °C. Increase in dry
weight accumulation indicates that the mutant is tolerant to
high temperature. Bisen and Shanthy [15] observed increase
in the growth parameters of the herbicide resistant mutants of
the filamentous, diazotrophic cyanobacterium Anabaena
doliolum. The mutant cyanobacterium exhibited better
pigment profile in terms of high chlorophyll and phycocyanin
content. A wide range of pigments have been observed in the
cyanobacteria [32]. Better pigmentation especially the
chlorophyll probably helps the mutant cyanobacteria to
Oxygen evolution (µmol mg PS II activity (µmol mg
protein−1 h−1) protein−1 h−1)
153 ± 0.31 121.14 ± 0.18
123 ± 0.25 83.21 ± 0.12
141 ± 0.37 104.37 ± 0.16
REDDY ET AL..
FIGURE 3 Cellular malondialdehyde (MDA) and peroxide
(H2O2) content of the wild type cyanobacterium A. doliolum and
mutant in response to elevated temperature
capture the light energy more efficiently than the wild type in
the process of photosynthesis. Chlorophyll plays an important
role in the harvesting of light to chemical energy during the
photosynthetic electron transport [33]. Phycocyanin content
of the mutant cyanobacterium was found to increase. Increase
in the phycocyanin content in the mutant may help in efficient
channeling of energy to the photosystem as compared to the
wild type. Genetic manipulation of Synechocystis 6803
resulted in the generation of a number of strains with modified
phycobilisomes [34]. Increase in the phycocyanin content of
the mutant cyanobacterium Synechococcus elongates has also
been observed by Suzuki et al. [35]. Photosynthesis is one of
the most essential processes in cyanobacteria which convert
the solar energy to energy rich compounds [36,37]. The
distribution of absorbed light energy between the two
photosystems is dynamically balanced and regulated. Energy
transfer from LHCII to the core antenna complex of PSII is
affected at elevated temperatures [38]. Decrease in PS II
activity and O2 evolution in the wild type could be due to its
limited capacity to consume energy equivalents as observed
by Suzuki et al. [35]. However, the temperature tolerant
mutant maintained comparable levels of PS II activity and O2
evolution suggesting efficient transfer of energy from the
antenna pigments to the reaction center.
The number of heterocysts in the wild type increased due
to elevated temperature. Increase in the number of heterocysts
in the symbiotic cyanobacteria in response to osmotic stress
conditions was observed Rai et al. [39]. The mutant
cyanobacterium exhibited better nitrogen fixation potential
as compared to the wild type. Low levels of nitrogenase
activity in the wild type cyanobacteria could be attributed to
probable inactivation of the nitrogenase enzyme due to
| 5
FIGURE 4 The antioxidant enzyme activity (SOD, CAT, APX,
and GR) of the wild type cyanobacterium A. doliolum and mutant in
response to elevated temperature (a and b)
elevated temperature and consequent damage of the hetero-
cysts. Tel-Or and Melhamed-Harel [40] reported inhibition of
nitrogenase activity due to disturbed electron transport
activity imposed by osmotic stress. Osmotic stress conditions
have been reported to inhibit the assemblage of the
component proteins of the nitrogenase enzyme [41]. Stress
conditions have been reported to induce heterocyst specific
damage in cyanobacteria making the nitrogenase enzyme
sensitive to external oxygen [42]. Heterocysts are found to be
more susceptible to osmotic stress conditions [43]. Further,
the increase in the number of heterocysts in the wild type
exposed to elevated temperature also does not reflect the rate
of nitrogen fixation. This may be due to physiologically
inactive heterocysts devoid of active nitrogen fixing potential.
Significant increase in the accumulation of H2O2 was
observed in the wild type cyanobacterium. The mutants
accumulated comparatively less peroxides and products of
lipid peroxidation as compared to the wild type. The
thylakoid membranes of cyanobacteria are highly suscep-
tible to damage to lipid peroxidation [44]. Increase in
H2O2 and MDA level has been reported due to exposure to
elevated temperature leading to lipid peroxidation [10,45].
Ali et al. [46] reported lipid peroxidation and membrane
damage due to the oxidation of fatty acids by ROS. Hyper
activity of the antioxidant enzymes in relation to stress
conditions have been reported [47]. Scandalios [48]
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| REDDY ET AL.

FIGURE 5 Effect of elevated temperature on the FAME profile of the wild type (control), wild type exposed to elevated temperature
(treatment) and mutant cyanobacterium A. doliolum. Fatty acids comprised of saturated (SFA), monounsaturated (MUFA), diunsaturated
(DUFA), aromatic fatty acids and alkanes (A–C)

reported that a balance between SOD, CAT, and APX
activities is important in determining the intracellular
levels of ROS. Compared to CAT and POX, the APX
is considered to be specific to H2O2. Further, CAT is
involved in the conversion of H2O2 into oxygen and
water, while APX plays an active role in the ascorbate
glutathione cycle [49]. Enhanced activity of GR helped the
cyanobacterium Anabaena fertilissima to maintain the
optimal redox activities and overcome the salinity
stress [50].
The FAME profile of the wild type cyanobacteria exposed
to elevated temperature and the mutant cyanobacteria
revealed quantitative as well as qualitative changes. Unsatu-
rated fatty acids constituted 35 and 47.9%, respectively, in
the wild type and mutant cyanobacteria. Furthermore,
significant quantitative and qualitative changes in the fatty
acid profile of wild type and mutant cyanobacteria were
observed in response to elevated temperature. From the
results, it appears that in the mutant of Anabaena doliolum the
fatty acid biosynthesis was shifted from short chain to long
chain fatty acids as compared to control. Lipids and fatty acids
play an important role in the tolerance of cyanobacterial cells
to various environmental stresses such as desiccation, salt
induced damage, and low temperature [12]. The wild type A.
doliolum produced higher quantity of saturated fatty acids as
compared to the mutant and wild type exposed to elevated
temperature. These results are in agreement with the
observations of Patel et al. [51] who observed substantially
higher amount of saturated fatty acids in a wild type
Synechocystis strain. Decreased levels of unsaturated fatty
acids in their membrane lipids reduced the osmotic stress
tolerance in cyanobacteria [52]. Stress-induced lipid accu-
mulation was reported to be an immediate consequence in
cyanobacteria exposed to high temperature [53]. High
temperature results in excessive fluidity of membrane lipids
and alterations in hydrogen and electrostatic bonds between
polar groups of proteins which in turn leads to modification of
their structure leading to ion leakage [54]. Changes in
REDDY ET AL..
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TABLE 2 Fatty acids (%) identified by GC-MS in the wild type CONFLICTS OF INTEREST
cyanobacterium A. doliolum and mutant in response to elevated
The authors declare that there is no conflict of interest.
temperature
Common Fatty
Name acid Control Treatment Mutant ORCID
Lauric C12:0 0.23 – 0.10
Gerard Abraham http://orcid.org/0000-0001-6543-4174
Mirystic C14:0 – 0.03 0.45
Pentadecylic C15:0 0.95 – 0.30
Palmitic C16:0 16.39 – 5.90
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How to cite this article: Reddy YP, Yadav RK,
Tripathi K, Abraham G. Isolation and
characterization of high temperature tolerant mutant
from the cyanobacterium Anabaena doliolum.
J Basic Microbiol. 2019;1–9.
https://doi.org/10.1002/jobm.201800447