Standard curve of protein
The protein standard curve was generated using Bovine serum albumin (BSA) with known concentrations, following the method described by Lowry et al. (1951). Absorbance measurements were taken at 750 nm using a Digital spectrophotometer, comparing each concentration against the blank. Microsoft Excel was used to create a graph with a regression equation, yielding an R2 value of 0.986. This indicates a confident limit exceeding 98% and confirms the acceptability of the standard curve (Fig-01).
Growth curve
The growth curve was determined through spectrophotometric analysis, which involved monitoring the increasing density of pure cultures of M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongates. The density of the cultures was assessed by measuring the absorbance of light at 750 nm against the blank of the growth medium. These cyanobacterial pure cultures are naturally occurring photosynthetic microorganisms.
The growth pattern of the cyanobacterial culture resembled that of other bacteria, exhibiting distinct phases such as the lag phase, log phase, exponential growth phase, stationary phase, and decline phase. In order to support normal growth, cyanobacteria synthesize proteins, chlorophyll, and various enzymes. Estimating the concentration of protein and chlorophyll is an important parameter for measuring growth.
The growth, chlorophyll, and protein concentrations were estimated over a period of 15 to 18 days from the day of inoculation(Fig-02).
Concentration of chlorophyll
Photosynthetic microorganisms contain the green chlorophyll pigment in their cellular structure, which contributes to the intensity of the green color observed in cyanobacterial cultures. The concentration of chlorophyll pigment was estimated using the spectrophotometric method at 665 nm, with absorbance readings taken against the methanol blank. The maximum chlorophyll concentration was observed on the eighteenth day of growth in pure cultures of M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongates, approximately measuring 27.21, 26.13, 25.12, 27.97, and 24.21 µg/ml, respectively (Fig-03).
Concentration of protein
The protein concentration was estimated in fully grown cyanobacterial cultures following the method described by Lowry et al (1951). A digital spectrophotometer was used at 750 nm, and absorbance readings were taken against the blank. Cyanobacterial cultures not only synthesized proteins but also various enzymes to utilize the different nutrients present in the growth medium. The highest protein concentration was estimated on the eighteenth day of growth in cyanobacterial cultures of M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongates, approximately measuring 62.88, 64.34, 56.60, 61.42, and 59.11 µg/ml, respectively(Fig-04)
Microcystin-producing genotypes in isolated pure cultures of cyanobacteria
Pure cultures of cyanobacteria were examined for the presence of microcystin-producing genotypes. Five pure cultures, namely Microcystis aeruginosa, Oscillatoria laetevirens var. minimus, Anabaena fertilissima, Phormidium uncinatum, and Synechococcus elongatus, were obtained from the laboratory-grown culture collection. These cultures were isolated from different water bodies and identified through molecular analysis (e.g., 16S rDNA analysis). They were maintained in specific culture media for over three years (Table-1)
Isolation of total DNA and its purity analysis in pure culture of cyanobacterium
DNA extraction was performed from freeze-dried cells of the pure cultures, and the purity of the extracted DNA was assessed by determining the A260, A280, and the A260/A280 ratio. The A260/A280 ratio ranged between 1.6 and 1.8, indicating good DNA purity. The DNA concentration in the cyanobacterial pure cultures ranged from 75 to 150 µg/ml. Agarose gel electrophoresis confirmed the presence of intact genomic DNA, showing a single sharp band just below the starting point without any smearing or lower molecular weight DNA bands (Fig-05)
Occurrence of mcy genes in pure cultures
Amplification of microcystin synthetase genes was observed in M. aeruginosa and O. laetevirens var. minimus, with the presence of mcyA, mcyB, mcyD, and mcyE genes. In A. fertilissima, three genes (mcyA, mcyB, and mcyD) were amplified, while in P. uncinatum, the mcyA, mcyB, and mcyE genes were amplified. No mcy genes were detected in the DNA sample of S. elongatus. The amplified amplicons of mcyABDE exhibited sizes of 291-297 bp, 800 bp, 818 bp, and 472 bp, respectively (Fig-06)
Table 1. Level of cell bound microcystin in pure culture
|
S.No.
|
Pure Culture in Laboratory
|
Microcystin level (ppb)
|
*
|
0.5 ppb calibrator
|
0.5 ppb
|
*
|
3.0 ppb calibrator
|
3.0 ppb
|
01.
|
M. aeruginosa
|
≥0.5 ppb ; ≤3.0 ppb
|
02.
|
O.laetevirens var. minimus
|
≤ 0.5 ppb
|
03.
|
A. fertilissima
|
≤ 0.5 ppb
|
04.
|
P. uncinatum
|
≤ 0.5 ppb
|
05.
|
S. elongatus
|
≤ 0.5 ppb
|