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Abstract: Blooms of Cyanobacteria in freshwater lakes are a recurrent phenomenon throughout the world. Permanent blooms of Microcystis aeruginosa are observed in many temple tanks of Tamilnadu and Orissa. Microcystin, a fast death factor, produced by Microcystis aeruginosa is found in alkaline water bodies and forms scums. Microcystin is a low molecular weight peptide and has a tumor promoting property in fishes, birds, mammals and in humans. It is released into water bodies when the cell is lysed and water treatment plants do not normally remove the toxin from water. In the current study Microcystis aeruginosa was isolated from water samples collected from Thenneri Lake and cultured under optimal laboratory conditions and microcystin, a toxin produced by Microcystis aeruginosa was isolated and characterized by high-performance liquid chromatography (HPLC) and Electron spray ionization mass spectroscopic technique.
Keywords: Cyanobacteria, Microcystis aeruginosa, Microcystin, HPLC, ESI-Mass Spectrometry.
Cyanobacterial bloom or surface bloom in lakes and reservoirs causes a significant water quality problem around the world. Increased eutrophication leads to increased occurrence of toxic cyanobacterial blooms in which the certain species of cyanobacteria are capable of producing several types of toxins. The toxins are very diverse in their chemical structure and toxicity (Dow et.al, 2000). These have been detected in a great number of water samples from nearly every region on earth. Among which microcystins are the most common cyanotoxins and can be expected wherever blooms of cyanobacteria occur in surface water. Microcystins are known to be produced by several strains from the genera Anabaena, Microcystis, Plantothrix and two strains of genus Nostoc (Sivonen and Jones, 1999). Microcystins form the largest group with more than 70 variants. These are monocyclic heptapeptides with the general chemical structure containing erythro-B-methylaspartic acid (D-MeAsp), alanine (D-Ala), glutamic acid (D-Glu), N-methyldehy-droalanine and ADDA, a hydrophobic 20 carbon chain (3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6 dienoic acid). Two variable L-amino acids, which differ for the different variants of microcystins. Single letter abbreviations are used in the naming of the toxin in order to indicate the various amino acids that are present in the toxin, i.e. Microcystin-LR contains leucine (L) and arginine (R) (Figure 1).
Figure 1. Structure of Microcystin-LR
MC-LR is amphiphatic molecule. Hydrophilic functional groups, including carboxyl groups on glutamic acid and methylaspartic acid, as well as the amino group on arginine, while the Adda residue is hydrophobic. Microcystin-LR was chosen as a representative microcystin in this study because it is the most toxic and abundant variant. Moreover, the commercial availability of MC-LR standard makes it easy to gain reproducible results.
Microcystins are actively taken up by the liver in fish where they disrupt normal cellular activity by inhibiting protein phosphatases. Inhibition of these enzymes in fish can ultimately result in widespread cellular death and loss of liver structure (Malbrouck et.al, 2006). Microcystins are toxic to fish at concentrations as low as a few micrograms per liter (µg/L) or possibly even fractional µg/L. Fish typically either ingest cyanobacteria or prey that have fed on cyanobacteria (Tencall et.al, 1994). To a lesser extent, they can absorb the toxins directly from the water. Histopathological examination of the liver of H. fossilis after oral exposure to Microcystis sp. showed that the parenchymal architecture of the liver is disturbed and hepatocytes show dissociation after 7 days of exposure, in which the hepatocytes appear swollen and cytoplasm appears granula (Sandeep Mehra et.al, 2009). Most of the microcystin to be in the liver suggested that liver necrosis was probably the main factor in causing death (Chakib Djediat et.al. 2010). In the present study Microcystis aeruginosa was isolated from water samples collected from Thenneri Lake and cultured under optimal laboratory conditions and microcystin, a toxin produced by microcystis aeruginosa was isolated and characterized by high-performance liquid chromatography (HPLC) and (ESI-MS) Electron spray ionization mass spectroscopic technique.
Materials and Methods
All chemicals were of either HPLC or analytical grade. The microcystin - LR standard was purchased from BioVision Research Products (California). Methanol and Formic acid were purchased from Merck.
Collection and Growth of Microcystis aeruginosa:
Microcystis bloom scum was collected from the Thenneri lake, Kancheepuram district, Tamilnadu, India, using a phytoplankton net (25-mm diameter mesh) The micro algal cultures were microscopically examined using Olympus (HB) microscope and Microcystis algal species was identified as described by Lee et al. (1997). Microcystis aeruginosa was isolated by serial dilution and cultivated in Fogg's medium at Krishnamurthy Institute of Algology, Anna nagar, Chennai. The culture were grown at 24+10C in a thermo-statically controlled room and illuminated with cool white inflorescence lamps (Philips 40W, Cool daylight 6500K) at an intensity of 2000 lux in a 12 hours light dark regime.
Extraction and pre-purification of Microcystin:
Microcystis aeruginosa cells were grown for 30 days, harvested by centrifugation at 5000 rpm for 15 min, lyophilized and stored at -20 oC. The lyophilized cells (150 mg) were added to 70% methanol solution at 50 µL per mg of dried mass (Phelan R. R. et.al. 2007). The mixture was sonicated three times for 5 min. The cell debris was removed by centrifugation at 2000 rpm for 10 min and the pellet was further extracted twice. The supernatant was evaporated to dryness and resuspended with 3 mL of 15% methanol. The crude extract was concentrated on Bond Elut C18/Vac 3 cc, 500 mg solid phase extraction (SPE) cartridge (Varian, North America) using an extractor and vacuum pump at a flow rate of 5 mL/min. 3 mL of crude extract in 15% methanol was applied to preconditioned SPE cartridge. The preconditioning step included washing with 10 mL of methanol followed by 10 mL of deionised water. The cartridge was then washed with 10 mL of 15% methanol and 10 mL of deionised water to remove the undesired impurities. Finally, the sample was eluted using 3 mL of 95% of methanol and then evaporated to dryness (Kim et.al, 2009).
The detection of microcystins was performed using Agilent 1100 HPLC system equipped with quaternary pump, automatic injector, thermostatized column compartment and photodiode array detector. The chromatographic separation was performed on C-18 column (Zorbax C-18, 4.6 X 250 mm i.d., 5 μm particle size, Agilent technologies) using a mobile phase of methanol and water at a flow rate of 0.5 ml/min under isocratic condition (Waya Sengpracha et.al. 2006). The volume of sample injected was 20 µL and photodiode array detector was set at 238 nm. Microcystins were quantified with calibration curve obtained from authentic Microcystin-LR. A mass spectrum was recorded using a Thermo Finnigan LCQ Advantage MAX 6000 (ESI-MS) Electron Spray Ionization mass spectrometer.
Results and Discussion
From the HPLC-PDA results, the retention time of standard Microcystin-LR was about 5.127 min. as shown in Figure 2 (a). A typical chromatogram of the microcystins containing cyanobacterial extract is shown in Figure 2 (b), in which the peak present at retention time 4.970 minute was closely related to the retention time of standard Microcystin-LR. These results show the presence of Microcystin-LR in purified cyanobacterial extract.
Figure 2. (a) HPLC chromatogram of standard toxins (Microcystin-LR) and (b) HPLC Chromatogram of purified toxins from Microcystis aeruginosa Species
Figure 3. Mass spectrum of isolated Microcystin-LR
It was further confirmed by ESI-Mass spectrometry. The mass spectrum of the isolated compound showed a molecular ion peak at m/z 995.93 (M+H)+ as shown in Figure 3. The direct quantification of Microcystin-LR showed that isolated Microcystis aeruginosa produced the concentration of 0.15 g of sample contained 3.68 x 10-5 g of Microcystin-LR.
In conclusion, the hepatotoxin Microcystin-LR in Microcystis aeruginosa collected from Thenneri Lake in Kancheepuram District, Tamilnadu, India has been determined using HPLC with methanol-water as mobile phase and further confirmed by (ESI-MS) Electron spray ionization Mass spectrometry.
The authors would like to thank Mr. K.Divakar, Research student of Dr. P. Gautham for HPLC analysis. The authors also would like to thank Research team of Dr. K. Palanivelu for providing SPE analysis. The authors gratefully acknowledge Dr.M.Palanichamy for providing constant support throughout the course of the investigation. The authors are grateful to University Grants Commission, New Delhi for financial support.