Microcystis flos-aquae as major contributor of Microcystin-RR content in fresh water reservoirs of Varanasi , North India

The toxic Microcystis flos-aquae strain was isolated from the local freshwater blooms and batch culture characteristics were observed in the modified J-medium. The isolation of toxic species was carried out by serial dilution and solid-agar plate adopting plate transfer method. Among the four culture media evaluated, modified Parker’s J-media was found most suitable for batch culturing of Microcystis flos-aquae strains. The use of Na2S and Na2SO3 in solid and liquid medium was found effective in reducing the contamination in culture and for providing better growth conditions as required for Microcystis flos-aquae specifically. The culture of isolated Microcystis flos-aquae showed the typical batch culture characteristics and the specific growth rate was in range of 0.02-0.40 d -1 . The 14 days old batch culture of isolate was used to estimate the toxic components, and besides several other variants, the microcystin RR (MC-RR) was dominantly present (0.16 % of its dry weight biomass) as confirmed by LC-PDA/MS. Thus it was observed in this study that the strain M. flos-aquae was responsible for the MC-RR contents in the local ponds of northern India. © 2016 Elixir All rights reserved. Elixir Environ. & Forestry 95 (2016) 40700-40705 Environment and Forestry Available online at www.elixirpublishers. com (Elixir International Journal) Satya Prakash et al./ Elixir Environ. & Forestry 95 (2016) 40700-40705 40701 (0.05 mg l -1 ), Co (NO3)2.6H2O (0.15 mg l -1 ), Al2K2 (SO4)4.24H2O (0.45 mg l -1 ) and Na2S (1.2 mM solution) were autoclaved in separate and added to the medium aseptically after cooling to the room temperature. Four different concentrations of Na2SO3 were used to optimize its supportive concentration for Microcystis spp. growth. For the selective inhibition of contaminating bacteria, sodium sulphide (Na2S) was added to the J-agar medium by applying it to the surface in petriplate, immediately after inoculation. For removal of fungal contaminants, cyclohexamide 25μg ml -1 was used in liquid and solid form of medium. Isolation and purification of Microcystis species Microcystis spp. blooms were collected from different freshwater ponds of Varanasi city (25.5o N 82.9o E, elevation 279 ft / 85 m) and were identified as per systematic keys given by Desikachary (1959). Adhering mud and other planktons were removed carefully, and the specimen was washed with sterile distilled water (Milli Q). In order to reduce contaminants, two-step centrifugation of inoculums (150×g for 30 min, at room temperature, followed by higher-speed centrifugation; 1,000 to 4,000×g for 5 min, at room temperature) and repetitive sub-culturing of single colony in solid and liquid culture media alternately, were made as suggested by Shirai et al. (1991). Microcystis colonies were disaggregated by vortex mixing in deionized water, as suggested by Parker (1982). After the two-step centrifugation of inoculums, the surface layer was withdrawn with micropipette and inoculated on modified J-agar medium. After incubation of 10 to 12 days in light (22 μE m -2 S -1 ), a contaminant free individual colony was selected with the help of fine bore glass capillary, under binocular microscope and transferred to culture tubes containing 10 ml modified Jmedium. Incubation and maintenance of cultures The culture was maintained by regular sub-culturing in Parker’s J-medium. Cultures were incubated in culture rooms (25±1oC) illuminated with full spectrum fluorescent lights. Fluorescent lamps (Sylvania 40-W cool white) were used in culture rack to provide the light energy necessary for photosynthesis. The average light intensity at the surface of the flasks was measured using a lux-meter (TES1332 digital lux-meter) and kept at 22.5 μE m -2 s -1 . In addition, for the microcystin determination the batch cultures of Microcystis sp. were maintained separately in 500 ml flask with 200 ml Jmedium. The cultures were shaken manually 3 to 4 times each day. Growth measurement A batch culture experiment with isolated Microcystis spp. was conducted at room temperature in 100ml Erlenmeyer flasks (14×3 flasks), with working volume of 35 ml. Each flask was inoculated at 5×10 4 cells ml -1 of the prepared inoculums. Typical growth experiments were carried out in triplicates with the flasks being agitated manually 3 or 4 times in a day. To assure the same light conditions throughout the experiment, the flasks were placed at exactly the same distance from the light source and at the same height. Growth was monitored after 1 day interval by both cell counting in hemocytometer and by estimating Chl-a. One set of culture (three flasks) were used for estimating cell number and Chl-a, while other set was used to estimate the microcystin content. For Chl-a estimation, the cultured biomass were extracted in 5.0 ml of 80 % acetone and kept overnight at 4 oC in refrigerator. Thereafter, it was homozenized and centrifuged at 4000 rpm for 10 min. The final volume of the supernatant was made to 25 ml with 80 % acetone and the absorbance of extract was read at 663 nm using Spectronic-20 colorimeter (Bausch and Lomb). Chlorophyll a, content was calculated according to the formula proposed by Talling and Driver (1963) with the modification in extinction coefficient (12.7) of Chl-a at 663 nm as given by Mackinney (1941). μg Chl-a per sample = 12.7(D663) V/L, Where, D663 = absorbance (OD) reading at 663 nm, V = volume of acetone extract in ml, and L = length of spectrophotometer cell i.e. 1 cm. Specific growth rate (μ) of the batch culture was determined using following equation, according to its definition (APHA, 1998) μ. d -1 = ln (X2-X1)/ t2-t1 Where X2 = biomass concentration i.e. (cells no. ml -1 ) or Chl-a (μg ml -1 ) at the end of selected time interval; X1 = biomass concentration at the beginning of selected time interval; t2 t1= elapsed time between selected interval, d. Characterization and quantification of microcystin The batch culture of the Microcystis sp. in exponential phase was used for microcystin extraction. After centrifugation (5000 rpm; 10 min) of the culture the pellet was extracted with 80% methanol (5 ml). For proper extraction three times extractions were performed and supernatant were pooled before vacuum evaporation. The samples with methanol were sonicated for 5 min and subsequently shaken for 45 min during each extraction step. After centrifugation (at 5000 rpm, 15 min.) the supernatants were pooled and concentrated at 42 oC by vacuum centrifugation. The extracts were re-dissolved in 80 % HPLC grade methanol and before HPLC analysis the samples were filtered through 0.22 μm cellulose filter. Identification and quantification of microcystins by LCPDA/MS The extracted samples were analyzed by HPLC with photodiode array (PDA) and mass (MS) detection. A Waters Alliance 2695 solvent delivery system equipped with a 2996 PDA and ZQ 2000 MS detector (in series) was used for all analysis (Waters, Elstree, UK). Separation was carried out on a Sunfire C18 column (2.1 mm id × 150 mm long; 5 μm particle size) which was maintained at 40 oC. Mobile phase was Milli-Q water (A) and acetonitrile (B) both containing 0.05 % trifluoroacetic acid (TFA). Test samples (20 μl) were separated using a gradient increasing from 15 % to 60 % B over 25 min at a flow rate of 0.3 ml min -1 followed by ramp up to 100 % B and re-equilibration over the next 10 min. Eluent was monitored from 200-300 nm with a resolution 1.2 nm and peptides were quantified by external standard at 238 nm. MS analyses were all performed in positive ion electrospray mode, scanning from m/z 100 to 1600 with a scan time of 2s and inter-scan delay of 0.1s. Ion source parameters, sprayer voltage, 3.07 kV; cone voltage, 70 eV; desolvation temperature, 300 oC; and source temperature, 100 oC. Instrument control, data acquisition and processing were achieved using Masslynx v4.0. Cone-induced dissociation (CID) mass data were obtained using a cone voltage of 100 eV to obtain sufficient fragmentation for identification.