World J Microbiol Biotechnol (2008) 24:139–141
DOI 10.1007/s11274-007-9439-y
BRIEF COMMUNICATION
n-Alkanes variability in the diazotrophic cyanobacterium
Anabaena cylindrica in response to NaCl stress
Pratiksha Bhadauriya Æ Radha Gupta Æ
Surendra Singh Æ Prakash Singh Bisen
Received: 18 March 2007 / Accepted: 9 May 2007 / Published online: 10 June 2007

Springer Science+Business Media B.V. 2007
Abstract n-Alkanes pattern in response to NaCl stress
has been studied in the cyanobacterium Anabaena cylind-
rica. Saturated hydrocarbons were separated and identified
by gas chromatography-mass spectrometry (GC-MS) using
serially coupled capillary column. Light chain n-alkanes in
the range of C9–C17 (43%) and heavy chain n-alkanes in
range of C17–C23 (34%) and C23–C31 (23%) were identified
as the major components of total hydrocarbons in the NaCl
adapted cells of A. cylindrica. In contrast, NaCl-untreated
cells of A. cylindrica had dominance of only long chain n-
alkanes in the range of C23–C31 comprising about 94% of
its total n-alkanes. The persistence of high level (43%) of
short chain n-alkanes (C9–C17) in NaCl adapted cells of A.
cylindrica as compared to its negligible level (0.2%) in
NaCl untreated counterpart clearly indicates that NaCl
stress causes the A. cylindrica to shift towards the synthesis
of short chain n-alkanes.
Keywords Anabaena cylindrica
Salinity-stress

Diazotrophic
n-Alkanes
Filamentous
Introduction
Cyanobacteria are Gram-negative photoautotrophic prok-
aryotes having plant type oxygenic photosynthesis (Stewart
1980). The cosmopolitan distribution of cyanobacteria
P. Bhadauriya
R. Gupta
P. S. Bisen (&)
Department of Biotechnology, Madhav Institute of Technology
& Science, Gwalior 474005, India
e-mail: psbisen@gmail.com
S. Singh
School of Studies in Microbiology, Jiwaji University, Gwalior
474011, India
indicates that they can cope with wide spectrum of global
environmental stresses such as heat, cold, dessication,
salinity etc. (Fay 1992; Tandeau de Marsac and Houmard
1993). They have developed a number of mechanisms by
which they defend themselves against environmental
stresses. Biologically active compounds isolated so far
indicate that cyanobacteria are a rich source of potentially
useful natural products. Over 40 different Nostocales spe-
cies, the majority of which are Anabaena and Nostoc spp.
produce natural products (secondary metabolites) having
antifungal, antimicrobial and anti HIV activities (Burja
2001). GC-MS analysis of volatile components of Spiru-
lina platensis indicates the presence of hydrocarbons hep-
tadecane and tetradecane, which also have antimicrobial
activity (Ozdemir et al. 2004). Nicotiana tobacum cells
showed n-alkanes variability in response to UV-B stress
and also shift towards the synthesis of short chain n-alk-
anes (Barnes et al. 1996). In the present communication, by
using a diazotrophic cyanobacterium Anabaena cylindrica,
which also show plant like photosynthesis, an attempt was
made to evaluate the effect of NaCl stress on n-alkanes
variability.
Materials and methods
Organism and culture conditions
Anabaena cylindrica was axenically grown in BG-11
medium (Rippka et al. 1979) without the addition of
combined-N. The medium was buffered to pH 7.5 with
10 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulphonic
acid (HEPES/NaOH). The cultures were incubated in cul-
ture room at 25 ± 1C and illuminated with day-light
fluorescent tubes having the photon fluence rate of
123
140 World J Microbiol Biotechnol (2008) 24:139–141

50 lmol m–2 s–1 at surface of the culture vessels. All the

experiments were performed with mid-log phase cultures,
having a cell density of 400 lg protein ml–1. Exponentially
growing cells of A. cylindrica grown in BG-11 medium
containing 200 mM NaCl for 6 days were designated as
NaCl adapted cells (Bhadauriya et al. 2007).

Extraction of hydrocarbons

The NaCl-untreated as well as NaCl adapted cells of A.

cylindrica were harvested by centrifugation (5,000 · g for
10 min) and extracted with pentane/dichloromethane/
methanol (40:30:30, v/v) as described previously (Dem-
bitsky et al. 1999). Non-saponifiable fractions were used
for hydrocarbon analysis.

GC-MS analysis

A Varian 2400 Gas Chromotograph with capillary column

coupled to a mass detector Finnigan mat TSQ (700) was
used. Hydrocarbons were analysed by GC on HP-5 column
(30 m, internal diameter 0.32 mm, film thickness
0.25 mm). The GC oven was programmed as follows:
2 min at 50C; 15C min–1–250C min–1; and hold for
5 min at 250C. The injector temperature was kept at
250C (splitless). The flow rate of carrier gas was
2 ml min–1. The MS detector was operated at 150C, with
electron impact ionization energy at 70 eV. The scan range
was m/z 40–650 and scan rate 0.9 scans s–1. Solvent delay
was set at 11 min. Hydrocarbons were identified by com-
parison with those found in Wiley mass spectral library
(7th edition).
Results and discussion
The n-alkanes produced by A. cylindrica were separated by
serially coupled capillary column to mass detector. Figure 1
a, b shows the complete ion chromatograms of hydrocar-
bons by GC-MS analysis of NaCl untreated and NaCl
adapted cells of A. cylindrica, respectively. It is evident
from the data of GC-MS analysis of hydrocarbons that the
major constituents of hydrocarbons in NaCl-untreated cells
of A. cylindrica were very long chain n-alkanes ranging
from n-tricosaene (C23) ton-hentriacontane (C31). The ma-
jor n-alkanes were found to be n-octadecane (C18), n-tetr-
acosane (C24), n-pentacosane (C25), nhexacosane (C26), n-
tricontane (C30) and n-hentriacontane (C31) of total n-alk-
anes. Variability of hydrocarbons with similar range of long
chain hydrocarbons has also been reported in filamentous
cyanobacterium Scytonema spp (Dembitsky and Srebnik
2002). In contrast, NaCl adapted cells of A. cylindrica
showed the dominance of both light n-alkanes and very long
Fig. 1 GC-MS analysis of hydrocarbons showing total ion chro-
matogram (short and long n-alkanes) of NaCl-untreated (a) and NaCl
(200 mM)-adapted (b) cells of A. cylindrica
chain n-alkanes (Fig. 1b). The dominant components of
light aliphatic hydrocarbons in NaCl-adapted cells of A.
cylindrica were found to be n-nonane (C9), n-undecane
(C11), n-dodecane (C12), n-tridecane (C13), n-tetradecane
(C14), n-pentadecane (C15), n-hexadecane (C16) and n-
heptadecane (C17). The other long chain hydrocarbons of
NaCl adapted cells of A. cylindrica were found to be similar
to its NaCl-untreated counterpart in the range of C23–C31.
This is the first report of the occurrence of light hydrocar-
bons such as C9–C17 and long chain alkanes in NaCl
adapted cyanobacterium A. cylindrica.
MS profile of predominant long chain n-alkanes re-
vealed ion m/z at 253, 337, 351, 366, 421 and 434 corre-
sponding to molecular formula C18H38, C24H50, C25H52,
C26H54, C30H62 and C31H64, respectively. Similarly, short
n-alkanes revealed ion m/z at 127, 155, 169, 183, 197, 211,
255 and 240 corresponding to molecular formula C9H20,
C11H24, C12H26, C13H28, C14H30, C15H32, C16H34 and
C17H36, respectively. The molecular ion peak of straight
chain saturated hydrocarbon was always present, though of
low intensity for long chain compounds. The fragmentation
pattern was characterized by cluster of peak and the cor-
responding peak of each cluster was 14 (CH2) mass units
apart. The most abundant peak has been observed at m/z 57
that corresponds to CH3 (CH2) 3+.
The data in Table 1 show the n-alkanes variability in A.
cylindrica under NaCl stress. It is evident from the data of

123
World J Microbiol Biotechnol (2008) 24:139–141
Table 1 n-Alkane variability in NaCl-untreated and NaCl adapted
cells of A. cylindrica
n-Alkanes Percent variability
NaCl Untreated NaCl-adapted (200 mM)
C9–C17 0.2 43
C17–C23 5.4 34
C23–C31 Up to 94 23
Table 1 that in NaCl adapted cells of A. cylindrica, light
chain n-alkanes in the range of C9–C17 (43%) and heavy
chain saturated alkanes in the range of C17–C23 (34%) and
C23–C31 (23%) were identified as the major components of
total hydrocarbons. In contrast, NaCl-untreated cells of A.
cylindrica had dominance of only long chain n-alkanes in
the range of C23–C31 hydrocarbons comprising about 94%
of its total n-alkanes. The persistence of a high level (43%)
of short chain n-alkanes (C9–C17) in NaCl adapted cells A.
cylindrica as compared to its negligible level (0.2%) in its
NaCl untreated counterpart clearly indicates that NaCl
stress causes the A. cylindrica cells to shift towards the
synthesis of short chain n-alkanes which may be involved
in maintaining the vital cellular functions in A. cylindrica
under NaCl stress. Similar results on UV-B caused changes
in Nicotiana tobacum on wax synthesis was also reported
by Barnes et al. (1996).
Acknowledgements We would like to thank Defense Research
Development Establishment, Gwalior, M.P., India for providing
instrumentation facility and Prof. N. K. Sah, Head, Department of
Biotechnology, MITS, Gwalior, M.P., India for his support.
141
References
Barnes JD, Percy KE, Paul ND et al (1996) The influence of UV-B
radiation on the physicochemical nature of tobacco (Nicotiana
tabacum L.) leaf surfaces. J Exp Bot 47:99–109
Bhadauriya P, Gupta R, Singh S et al (2007) Physiological and
biochemical alterations in a diazotrophic cyanobacterium An-
abaena cylindrica under NaCl stress. Curr Microbiol (in press)
Burja AM, Banaigs B, Abou-Mansour E et al (2001) Marine
Cyanobacteria—a profile source of natural products. Tetrahe-
dron 57:9347–9377
Dembitsky VM, Shkrob I, Dor I (1999) Separation and MSD
identification of hydrocarbons and volatile metabolites of blue
green algae Nostoc sp. by serially columns with consecutive
nonpolar and semipolar stationary phases. J Chromatogr
862:221–229
Dembitsky VM, Srebnik M (2002) Variability of hydrocarbon and
fatty acid component in cultures of the filamentous cyanobac-
terium Scytonema sp. isolated from microbial community black
cover of limestone walls in Jerusalem. Biochem (Moscow)
67:1276–1282
Fay P (1992) Oxygen relations of nitrogen fixation in cyanobacteria.
Microbiol Rev 56:340–373
Ozdemir G, Karabay NU, Dalay MC et al (2004) Antibacterial
activity of volatile component and various extracts of Spirulina
platensis. Phytother Res 18(9):754–757
Rippka R, Deruelles J, Waterbury JB et al (1979) Generic
assignments, strain histories and properties of pure cultures of
cyanobacteria. J Gen Microbiol 111:1–61
Stewart WDP (1980) Some aspects of structure and function in N2-
fixing cyanobacteria. Annu Rev Microbiol 34:497–536
Tandeau de Marsac N, Houmard J (1993) Adaptation of cyanobac-
teria to environmental stimuli: new steps towards molecular
mechanisms. FEMS Microbiol Rev 104:119–190
123