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 cylindrica. 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 nalkanes 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
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 species, 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 Spirulina platensis indicates the presence of hydrocarbons heptadecane 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-alkanes (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.
Introduction Cyanobacteria are Gram-negative photoautotrophic prokaryotes 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:
[email protected] S. Singh School of Studies in Microbiology, Jiwaji University, Gwalior 474011, India
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 culture room at 25 ± 1C and illuminated with day-light fluorescent tubes having the photon fluence rate of
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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 (Dembitsky 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 comparison 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 hydrocarbons 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 major n-alkanes were found to be n-octadecane (C18), n-tetracosane (C24), n-pentacosane (C25), nhexacosane (C26), ntricontane (C30) and n-hentriacontane (C31) of total n-alkanes. 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
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Fig. 1 GC-MS analysis of hydrocarbons showing total ion chromatogram (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 nheptadecane (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 hydrocarbons such as C9–C17 and long chain alkanes in NaCl adapted cyanobacterium A. cylindrica. MS profile of predominant long chain n-alkanes revealed ion m/z at 253, 337, 351, 366, 421 and 434 corresponding 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 corresponding 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
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).
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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.
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