Biotechnol Lett (2012) 34:247–251 DOI 10.1007/s10529-011-0771-9
ORIGINAL RESEARCH PAPER
Oxidative stress response and fatty acid changes associated with bioaccumulation of chromium [Cr(VI)] by a fresh water cyanobacterium Chroococcus sp. Muthukannan Satheesh Kumar • Ramasamy Praveenkumar • Asokraja Ilavarasi Kamaraj Rajeshwari • Nooruddin Thajuddin
•
Received: 16 June 2011 / Accepted: 5 October 2011 / Published online: 16 October 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Cr(VI) at 2.5, 5, 7.5 and 10 mg/l was removed over 1–5 days by a freshwater cyanobacterium, Chroococcus sp. 2.5 mg Cr(VI)/l gave the optimum rate. With 5 mg Cr(VI)/l, activities of superoxide dismutase and catalase were increased. Amounts of palmitic (16:0), stearic (18:0) and oleic acid (18:1) in the cell also increased after exposure to Cr(VI). Keywords Catalase Chroococcus sp. Fatty acid changes Oxidative stress Superoxide dismutase
Introduction Hexavalent chromium in the environment is almost totally derived from anthropogenic activities. The high concentrations of Cr(VI) are toxic, carcinogenic (Singh et al. 1998) and genotoxic (Godet et al. 1996).
M. S. Kumar R. Praveenkumar A. Ilavarasi K. Rajeshwari N. Thajuddin (&) Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India e-mail:
[email protected] M. S. Kumar e-mail:
[email protected]
Hence there is a need to combat its toxicity. Conventional methods of metal ion removal often remain expensive and ineffective with metal ions at 1–100 mg/l (Volesky 1994). Hence biosorption and/or bioaccumulation have emerged as an alternative method for metal removal. Microorganisms can be used as biosorbents for metal removal. Cyanobacteria have gained consideration for their potent use in pollution abatement (Thajuddin and Subramanian 2005) and also for degradation of aromatic hydrocarbons (Kumar et al. 2009), pesticide degradation (Subramanian et al. 1994) and metal biosorption (Kumar et al. 2011; Rajeshwari et al. 2011). Often environmental stress may induce overproduction of reactive oxygen species (ROS) and results oxidative damage (Smirnoff 1993). Cellular systems scavenge these active oxygen species by invoking increased anti-oxidative machinery such as enzymes superoxide dismutase (SOD), catalase (CAT) and peroxidase (Prasad et al. 2005). The unsaturated fatty acids of cell membrane are primary targets of peroxidation which subsequently leads to cell death, whereas organisms employ numerous approaches to limit their damage. Hence, a low degree of fatty acid unsaturation may protect the cell against oxidative damage. There have been no reports dealing with such adaptations in fatty acid content under Cr(VI) induced oxidative stress. We have therefore investigated the possible changes in antioxidative enzymes and adaptive changes in fatty acid content of Chroococcus sp. under Cr(VI) accumulating conditions.
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Materials and methods Test organism and molecular characterization Chroococcus sp. NTMS09 was isolated from a tannery effluent contaminated site. Morphological investigation by Dr. N. Thajuddin affirmed that the organism belongs to Chroococcales. Total genomic DNA was prepared by the method of Neilan et al. (1995) and was used as template for amplification of 16S rRNA gene. The two oligonucleotide primers used for amplification of 16S rRNA gene were: forward primer CYA106F 50 -CGGACGGGTGAGTAACG CGTGA-30 and CYA781R 50 -GACTACTGGGGT ATCTAATCCCATT-30 as reverse primer. The target sites of these oligonucleotide primers are conserved for cyanobacteria (Nubel et al. 1997). The amplified product was sequenced and deposited in GenBank with accession no. HQ219684. Preparation of Cr(VI) solution K2Cr2O7 (AR grade) was dissolved in double distilled water and working standards were obtained by further dilutions.
Biotechnol Lett (2012) 34:247–251
with water and centrifuged to remove upper aqueous phase. The organic phase was rinsed twice with equal volume of methanol/water (1:1, v/v) and the lipid collected from the solvent phase and evaporated under vacuum. Preparation of fatty acid methyl esters (FAME) Lipid was refluxed with methanolic sulphuric acid (2% v/v H2SO4 in methanol) for 4 h. The contents were mixed an equal volume of distilled water and the aqueous layer was extracted twice with ethyl acetate. The collected ethyl acetate extract containing FAME was dried over anhydrous Na2SO4 and concentrated under vacuum. FAMEs were analyzed by GC using a FAMEwax column (Restek, USA). Statistical analysis All the data were statistically analyzed (SPSS package, IBM corporation) to express the results as mean ± standard deviation of three independent trials.
Results and discussion Metal removal Metal ion removal 100 mg (dry cell wt) cyanobacteria from a stationary phase culture was inoculated in 100 ml fresh BG11 medium at pH 7 supplemented with Cr(VI) at 2.5, 5, 7.5, or 10 mg/l. Cells without Cr(VI) served as control.
Activities of SOD (Marklund and Marklund 1974) and CAT (Sinha 1972) were measured.
Chroococcus sp. NTMS09 effectively adsorbs and accumulates Cr(VI) ions (Fig. 1). Immobilized Oscillatoria sp. and Phormidium sp. are also good sorbents of Cr(VI)/l (Rajeshwari et al. 2011). The test organism showed decreased removal at higher Cr(VI) concentrations. This result corresponds to the work of Kumar et al. (2009) which described the same phenomenon in cyanobacteria and the condition was attributed to cellular degradation or adaptability of cyanobacteria to the stressful environment. The maximum metal removal was at 24 and 48 h and then gradually decreased. This may be due to desorption of metal which is adsorbed on the surface of the organism.
Lipid extraction
Adsorption isotherms
Biomass was collected, freeze dried and the powder extracted with methanol/chloroform (2:1, v/v). The mixture was filtered and organic phase was washed
Freundlich and Langmuir (Crist et al. 1981) isotherms have been investigated in this study. Freundlich adsorption isotherm had a higher correlation coefficient
Determination of Cr(VI) Cr(VI) was assayed at 540 nm using 1,5-diphenyl carbazide in acid solution as a complexing agent (Clesceri et al. 1996). Enzyme activities
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The activity of SOD and CAT was increased in the test organism under Cr(VI) accumulated condition than control. Maximum SOD and CAT activity in Plectonema boryanum was at 10 lg endosulfan/ml (Kumar et al. 2008). The results suggest that SOD and CAT play greater roles in detoxification of ROS and increasing the activities of antioxidant enzymes such as SOD and CAT may be a key defense mechanism against stress.
Cr (VI) removal (%)
100 80 60 40 20 0 24
48
72
96
120
Fatty acid analysis
Time (h) 2.5 mg/l
5 mg/l
7.5 mg/l
10 mg/l
Fig. 1 Dynamics of Cr(VI) uptake by Chroococcus sp. NTMS09 in pH-static experiment. Culture was grown in fresh BG11 medium with initial pH 7 at 26°C, 27 lE m-2 s-1 and 14/10 h light/dark cycle. Initial Cr(VI) was from 2.5 to 10 mg/l. Culture supernatant was collected to measure Cr(VI) ion. 100% relative value = 2.5–10 mg Cr(VI)/l at 0 h. Data represent the mean ± standard deviation (w/v) of three separate trials
than the Langmuir isotherm. The mechanisms involved in Cr(VI) removal by Chroococcus sp. are therefore discussed based on Freundlich isotherm parameters. Maximum Cr(VI) adsorption capacity and adsorption intensity were 9.3 and 1.1, respectively. Antioxidant enzymes
Presence of Cr(VI) altered the fatty acid profiles in the test organism (Table 2). Presence of Cr(VI) increased
Table 2 Effect of 5 mg Cr(VI)/l on fatty acid profiles in Chroococcus sp. NTMS09 after 48 h of treatment Fatty acidsa
Table 1 Effect of 5 mg Cr(VI)/l on SOD and CAT activity in Chroococcus sp. NTMS09 after 48 h of treatment Culture condition
Normala Cr(VI) treated
b
Cr(VI) treatedc % (w/w)
10:0
2 ± 0.03
14:0
1 ± 0.02
2 ± 0.06
16:0
1 ± 0.09
18 ± 0.07
ND
17:0
1 ± 0.05
4 ± 0.01
18:0 20:0
8 ± 0.12 26 ± 0.08
18 ± 0.03 2 ± 0.05
24:0
ND
Othersd
Photosynthetic organisms fight against the toxic effects of metal-induced free radicals by increasing their antioxidative defense mechanisms (Kumar et al. 2008). Thus metal-induced O2- and H2O2 triggered the activity of antioxidant enzymes, such as SOD and CAT, in Chroococcus sp. at 5 mg Cr(VI)/l (Table 1).
Normalb % (w/w)
Total saturated fatty acids
3 ± 0.35 42 ± 0.05
11 ± 0.08 ND 55 ± 0.05
15:1(c)
1 ± 0.04
17:1(c)
4 ± 0.06
ND
18:1 (n - 9t)
1 ± 0.02
ND
18:1 (n - 9c)
28 ± 0.12
18:2 (n - 6t)
2 ± 0.09
18:2 (n - 6c)
8 ± 0.04
18:3 (n - 6c)
11 ± 0.02
2 ± 0.01
36 ± 0.02 ND 2 ± 0.02 ND
20:2c
2 ± 0.05
Enzyme activity (mg protein-1)
Othersd
1 ± 0.03
SOD
CAT
Total unsaturated fatty acids
8.9 ± 0.7
4.3 ± 0.2
ND Not detected. Data represent the mean ± standard deviation of three separate trials
11.7 ± 0.7
5.6 ± 0.3
a
SOD Superoxide dismutase, CAT catalase. They were incubated at 25 ± 2°C, 14/10 h light/dark cycle and 27 lE m-2 s-1. Data represent the mean ± standard deviation of three separate trials a
Culture was grown on BG 11 medium
b
Culture was grown on BG 11 medium with 5 mg Cr(VI)/l
58 ± 0.05
5 ± 0.07 ND 45 ± 0.03
FAME identification was by comparison with authentic standards (Supelco)
b
Culture incubated in BG 11 medium
c
Culture incubated in BG 11 medium with 5 mg Cr(VI)/l
d Trace amounts (\1%, w/w) of 6:0, 8:0, 11:0, 12:0, 13:0, 15:0, 16:1, 20:1 were detected under normal condition where as not detected under Cr(VI) treated condition
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the level of saturated fatty acids (42 to 55%) and decreased unsaturated fatty acid content (58 to 45%). These changes might result from suppression of activities of fatty acid desaturase enzymes. Palmitic (16:0) and stearic acids (18:0) were the major affected saturated fatty acids when compared to the control. Unusually, lignoceric acid (24:0) was accumulated in treated organism but absent in control. This would arise by the action of a C20 elongase which transforms arachidic acid to lignoceric acid. The changes in fatty acid profile of Aureobasidium pullulans with addition of cadmium has been reported (Certik et al. 2005). Since the membrane is the site of primary contact of the cell with its environment, perturbation in the membrane structure induced by metals may result in significant disruption of its physiological function (Certik et al. 2005) and certain alteration of fatty acid profiles. As inferred from earlier reports, as in this case, the disruption of the physiological functions of the cell was mainly due to formation of ROS, thereby induced oxidative stress. The primary targets of thus formed ROS are cell membrane polyunsaturated fatty acid, resulting in lipid peroxidation, which in turn lead to damage in cell structure and function (Floyd 1990). Hence as a way out to overcome the adversity, the organism may undergo adaptive change to decrease the level of unsaturated fatty acids. The result clearly reveals that under Cr(VI) exposed conditions the test organism evidenced with lowering of fatty acid unsaturation. This suggests that the organism undergo an adaptive mechanism thereby lowering the expression of fatty acid desaturases which involves the formation of unsaturated fatty acids. The changes in cyanobacterial fatty acids, as an adaptive mechanism towards metal stress have never been investigated before. Thus in this context the present work stands first of its kind.
Conclusion Chroococcus sp. NTMS09 was effective for Cr(VI) removal. A strong inference was made on increase in the activity of antioxidative enzymes SOD and CAT and lowering the level of unsaturated fatty acids under Cr(VI) accumulated conditions. These significant changes are the defense mechanisms against stress. Thus, this organism holds great promise in helping to mitigate environmental pollution.
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Biotechnol Lett (2012) 34:247–251 Acknowledgments M.S. acknowledges the award of University Grants Commission–Rajiv Gandhi National Fellowship (UGC-RGNF). The financial support from the Department of Biotechnology (DBT), Government of India is gratefully acknowledged (Project reference: BT/PR/11316/PBD/26/164/ 2008).
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