Molecular detection of microcystin synthetase genes (mcy genes) and semi- quantitative immunological detection of the production of microcystin toxin in vitro-grown pure cultures of cyanobacteria

Molecular detection of microcystin synthetase genes (mcy genes) and semi- quantitative immunological detection of the production of microcystin toxin in vitro-grown pure cultures of cyanobacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular detection of microcystin synthetase genes (mcy genes) and semi- quantitative immunological detection of the production of microcystin toxin in vitro-grown pure cultures of cyanobacteria Prashant Chaturvedi, Divya Singh, Renu Pathak, Purnima Beohar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4371317/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Laboratory mass cultures were established for cyanobacterial strains M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongates. The growth of these cultures was assessed by monitoring turbidity, chlorophyll concentration, and protein content. After an 18-day inoculation period, the maximum growth of pure cultures was observed. Well-developed cultures were concentrated using centrifugation and subsequently lyophilized to preserve them in powdered form. DNA extraction was performed on the lyophilized cultures, resulting in clear DNA bands just below the wells. The quality of the extracted DNA, as determined by the A260/280 ratio, ranged from 1.6 to 1.8. The genes mcyABDE were successfully amplified in M. aeruginosa and O. laetevirens var. minimus, while A. fertilissima and P. uncinatum showed amplification of mcyABD and mcyABE genes, respectively. No amplification was observed in S. elongatus. Using a semi-quantitative ELISA technique, a significant concentration of Microcystin was detected only in Microcystis aeruginosa, at a level of 0.5 ppb, whereas the other cultures produced trace amounts below 0.5 ppb. Cyanobacterial strains Microcystin Semi-quantitative ELISA mcyABDE genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Growth assessment and maximum growth of cyanobacterial strains: Turbidity, chlorophyll concentration, and protein content were monitored to assess the growth of cyanobacterial strains M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongatus . The cultures exhibited maximum growth after an 18-day inoculation period. Concentration and preservation of well-developed cultures: Well-developed cultures were concentrated using centrifugation and subsequently lyophilized to preserve them in powdered form. This ensured the long-term stability and storage of the cultures. DNA extraction and gene amplification: DNA extraction from the lyophilized cultures resulted in clear DNA bands. The quality of the extracted DNA, assessed by the A260/280 ratio, indicated good quality DNA. The genes mcyABDE, mcyABD, and mcyABE were successfully amplified in M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, and P. uncinatum, respectively. However, no amplification was observed in S. elongatus. Additionally, using a semi-quantitative ELISA technique, Microcystin was detected only in Microcystis aeruginosa at a concentration of 0.5 ppb, while the other cultures produced trace amounts below 0.5 ppb. Introduction Cyanobacteria serve as photosynthetic agents in aquatic ecosystems, but their excessive proliferation can lead to irritations. These proliferations, known as blooms, form colorful mats on the water's surface. Blooms cause low oxygen levels in water bodies, resulting in hypoxia, which can lead to increased mortality rates among invertebrates, shellfish, fish, and plants. Additionally, blooms can obstruct light from reaching the benthic region of the ecosystem, reducing productivity. Some blooms produce harmful toxins that pose risks to aquatic creatures, livestock, and humans, and are referred to as Harmful Algal Blooms (Zhang et al., 2009; Berry et al., 2011; Paerl and Otten, 2013). The most commonly reported cyanobacterial toxin is microcystin, which belongs to a family of cyclic pentapeptides with over 90 structural variants. Microcystin's general structure is cyclo (D-Ala-X-D-MeAsp-Z-Adda-D-Glu-Mdha), where X and Z represent variable L amino acids. Microcystin-producing genera in freshwater cyanobacteria include Microcystis, Planktothrix, and Anabaena (Sivonen and Jones, 1999 ). Terrestrial strains like Nostoc and Hapalosiphon (Prinsep et al., 1992; Oksanen et al., 2004) and benthic Phormidium (Izaguirre et al., 2007) have also been identified as microcystin producers. Microcystin forms an irreversible covalent bond with the cysteine of eukaryotic protein phosphatase type 1 and 2A (Runnegar et al., 1995 a). Specifically, the Mdha moiety in microcystin binds covalently to cysteine 273 of the protein phosphatase active site (Welker and Von Dohren, 2006; Wharton et al., 2019). Microcystin acts as a tumor promoter, while nodularin functions as a carcinogen (Nishiwaki-Matsushima et al., 1992; Ohta et al., 1994). It can cause damage to cell tissue and organs, particularly affecting the mitochondrion, leading to the loss of cytochrome-C, calcium ions, and inter-membrane proteins (Lankiewicz et al., 2000; Nodberg and Anner, 2001). The release of cytochrome-C triggers caspase cascades, including caspase-3 via caspase-9. Microcystin also plays a role in mitosis regulation (Snaith et al., 1996) and induces oxidative stress in vitro conditions in rat hepatocytic cells and fish tissues (Li et al., 2003). In plants, microcystin inhibits the activity of Protein Phosphatase 1 and Protein Phosphatase 2A, affecting antioxidant enzyme peroxidases and superoxide dismutase, leading to oxidative stress, apoptosis, and hepatotoxicity (Chen et al., 2004 ). The diversity of chemotypes observed in various cyanobacterial taxa is due to microcystin-producing cyanobacteria. On average, individual genera have been found to produce four distinct microcystins, but up to 27 have been identified so far. Furthermore, certain cyanobacterial strains do not produce microcystins, and studies have shown that microcystin-producing and non-producing strains coexist in natural populations. While research on microcystins has predominantly focused on terrestrial mammals, there is growing recognition of their effects on aquatic organisms. Fish, being at the top of the aquatic food chain, are particularly susceptible to cyanobacterial toxins, which can be ingested or absorbed through their gills. This poses risks not only to fish but also to humans through the food chain (Malbrouck and Kestemont 2006). Harmful cyanobacterial blooms have adverse ecological and environmental impacts, as well as posing risks to animals and humans. Future climate change scenarios predict an increase in bloom-forming cyanobacteria due to changes in hydrological cycling, rising water temperatures, and nutrient loading. As a result, harmful blooms are expected to become more frequent and last longer. This study investigates diverse cyanobacteria that produce toxins and form blooms, with a focus on their cultivation. In-depth studies have been conducted using both in vivo and in vitro approaches to analyse and compare the concentration of the toxins produced. Furthermore, a comprehensive molecular analysis has been carried out to better understand the characteristics of these toxins. Materials and Methods Culture conditions: - The axenic pure cultures Microcystis aeruginosa (Ghosh et al , 2008a), Oscillatoria laetevirens var. minimus (Ray and Bagchi, 2001) and Anabaena fertilissima Banerjee et al , 2013), Phormidium uncinatum ( Bagchi and Verma, 1997), Synechococcus elo ngates (Saggu et al, 2010), were provided by Cyanobacterial Research Lab, Dept. of P.G. Studies and Research in Biological Science, R. D. University, Jabalpur, Madhya Pradesh, India. The cyanobacterial strain was fully grown and maintained all strains in BG-11 medium and its composition is given below – Composition of macronutrient - Independent stock solution each of g/L – Potassium phosphate (4.0), Magnesium sulphate (7.5), Calcium chloride (3.6), Citric acid (0.6), Ferric ammonium citrate (0.6), EDTA (0.1), Sodium carbonate (0.2), Sodium nitrate (85.0). Composition of micronutrient - Composition of combined stock solution in gm/L – Boric acid (2.86), Manganese chloride (1.81), Zinc sulfate (0.22), Sodium molybdate (0.39) Copper sulfate (0.079), Cobalt nitrate (0.0494). For the preparation of one liter of medium 10 ml, each macronutrient and 1 ml of combined micronutrient stock solution was taken, the pH of the medium was adjusted to 7.5 and this was autoclaved at 15 lbs pressure for 20 min at 121˚C. Growing condition:- The culture was kept for growth in an air-conditioned growth chamber at a temperature 28 ± 2˚C under the cool white fluorescent light of a continuous light intensity of approximately 1500 lux. The culture was manually shaken twice a day for aeration. Estimation of growth An increase in the density in the pure culture was taken as a growth parameter and increasing total chlorophyll A and protein content was considered for a more precise estimation of growth. For growth studies the aliquots of 3 ml of culture were taken aseptically at regular intervals and optical density (O.D.) at 750 nm was measured for growth, using a spectrophotometer (digital spectrophotometer, model ME 802). Absorbance of the density of the culture was taken against the BG-11 medium blank. Thereafter another growth parameter was chlorophyll concentration, this was done according to the method of MacKinney., (1941). The final growth parameter was protein concentration within all pure cultures which were done according to the method of Lowry et al ., (1951). Standard curve of Bovine serum albumin: - A standard curve was prepared by using the increasing concentration of bovine serum albumin in the different ranges 100 µg − 1000µg per ml according to Lowry et al., (1951). Absorbance was taken of each concentration against the blank at 750 nm by using digital spectrophotometer (digital spectrophotometer, model ME 802). Preservation of cyanobacterial cultures Pure cultures were centrifuged for 10 min at 6000 × g at 25 ºC to collect the supernatant and the pellets sediment at the bottom surface of the centrifuge tubes. Culture materials were transferred to lyophilization tubes and kept in a pre-freezer and maintained at − 20ºC for 1 hour before lyophilization. Lyophilization tubes (Borosil, India) were fitted at the appropriate position of freeze-drier (NSW, India) and the vacuum was set at − 20ºC. The process was carried out till the material completely dried in the form of a powder which was transferred into airtight storage vials and kept in the refrigerator for further experimental use. Isolation of genomic DNA from cyanobacteria cultures The culture material was lyophilized at − 20ºC until it turned into powder and became brittle. It was stored in cryo-vials at 4ºC. Samples collected at different times or locations were dried and stored. Two methods were standardized for the isolation of genomic DNA from dry culture material. In the first method described by Jungblunt and Neilan, (2006), 25 mg of lyophilized cyanobacteria culture material was heated at 65ºC for 2 h in 3.0 ml of DNA extraction buffer containing 800 mM ammonium acetate, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0), 1% SDS and 1% lysozyme (fresh). Thereafter, 50 µl of RNase from a stock of 10 mg ml -1 was added and further incubation was done at 37 ºC for 30 min. To stop the reaction, the mixture was chilled in an ice bath for 10 min and centrifuged at 12000 × g for 10 min at 4ºC. To one volume of cell, extract was added one volume of ice-cold isopropanol and 0.1 volume of 4 M ammonium acetate and centrifuged at 12000 × g for 10 min at 4ºC to precipitate the DNA. DNA quantification and purity assessment Approximately 10 µl of DNA samples obtained from different protocols was added to 990 µl of sterile double distilled water. Their purity was checked by taking the ratio of their absorbance at A 260 /A 280 nm. The yield of each sample was also calculated by using the following formula: A 260 × dilution factor × 50 µg ml -1 Agarose gel electrophoresis of cyanobacterial genomic DNA One percent agarose gel was prepared in 0.5X TBE running buffer (45 mM Tris-borate/1 mM EDTA). To 100 ml warm (70ºC – 80ºC) sterilized TBE buffer, agarose was slowly added followed by gentle stirring so that the agarose dissolves completely. Thereafter, 50 µl of ethidium bromide from the stock of 10 mg ml -1 was added. The temperature of the solution was decreased to 50ºC and was poured onto a sealed gel caster. A comb was placed in the solidifying gel. After solidification, agarose gel was submerged in a running buffer (0.5X TBE buffer). DNA extracted as above (15 µl) was mixed with 3 µl of bromophenol blue (0.5 mg/ml) and glycerol (5 µl). This was applied to the wells along with the DNA ladder (HI media, India). Electrophoresis was carried out at 70 V and 25 mA for 1 h in a horizontal slab gel electrophoresis apparatus (Bangalore Genei) until the bromophenol blue reached 3/4th of the bottom of the gel. The tank was filled with 0.5X TBE and the gel was kept submerged throughout. After the samples had run to a desired distance the gel was taken out from the electrophoresis unit and placed over a UV transilluminator (Biotch R & D laboratories Yercaud). The orange fluorescent bands of DNA were detected under a UV cut-off filter. These bands were photographed using a digital camera (Sony, Japan). PCR amplification for desired genes of dry cyanobacteria cultures materials. For PCR amplification of mcy ABDE genes, the preparation of the reaction mixture and amplification cycle was carried out as described by Jungblunt and Neilan, (2006) and Kumar et al, (2011). A reaction mixture of 23 µl was prepared using the PCR amplification kit containing a final concentration: 12.5 µl of autoclave distilled water, 2.5 µl of 10 x taq polymerase buffer, 1 µl of 25 mM MgCl 2 , 4 µl of 200 µM of dNTPs mixture solution, 1 µl of 20 pmol forward and reverse primers, 1 µl of 1U taq DNA polymerase enzyme, 2 µl of template DNA sample 50 ng in each case. Thermal cycling was performed using a gradient-type thermal cycler (Merck Genei) with an initial denaturation step at 94 ºC for 5 min, followed by 40 cycles of 94ºC for 1 min, 60.8ºC for 1 min, and 72 ºC for 1 min and a final extension of 10 min at 72ºC. The amplified product was then analyzed using agarose gel electrophoresis as above. The forward and reverse primer pairs (Bagchi and Ghosh, 2010) as follows were procured from (Imperial Life Sciences, India): mcy A Forward 5’-AAAATTAAAAGCCGTATCAAA-3’ Reverse 5’-AAAAGTGTTTTATTAGCGGCTCAT-3’ mcy B Forward 5’-CTATGTTATTTATACATCAGG-3’ Reverse 5’-CTCAGCTTAACTTGATTATC-3’ mcy D Forward 5’-GATCCGATTGAATTAGAAAG-3’ Reverse 5’-GTATTCCCCAAGATTGCC-3’ mcy E Forward 5’-TTTGGGGTTAACTTTTTTGGGCATAGTC-3’ Reverse 5'-AATTCGCCGGTATTAGACGTT-3’ Preparation of pure cyanobacterial culture medium for ELISA test Cyanobacterial pure cultures cultivated at mass scale upto 2 liters in specific growth mediums in the laboratory. Thereafter cyanobacterial cultures were filtered with different kinds of filtration methods used to possibly remove all the finest particles present in the growth medium, as particles may chock ODS cartridges. First of all cyanobacterial growth mediums were filtered with a muslin cloth to remove coarse particles and, thereafter again filtered with Whatman filter paper no. 42 to remove fine impurities. Later, growth mediums were again filtered with the vacuum filtration method using cellulose nitrate filters with pore size 5 µm (Axiva Sichem Biotech, India). In the last step, the growth medium was filtered with fine cellulose nitrate membrane filters of 0.45 µm (Millipore Corporation, Bedford) under a vacuum. In totality, 1 liter of above-filtered lake water was passed through ODS cartridges (Merck LiChrolut ® RP-18). Concentrating the cyanobacterial growth medium for the detection of Microcystin The ODS cartridges were fitted on top with syringes using tubings and manual pressure was applied to the contents of the cartridges. Firstly, the cartridges were pre-washed with 10 ml of 100% methanol in order to equilibrate and subsequently, they were washed with 10 ml of distilled water. Gradually the entire 1 liter of pure culture was passed through the cartridges. The bound material was first washed with 10 ml of 20% methanol which was discarded. Microcystin was eluted using 10 ml of 100% methanol. The eluted 100% methanolic extract was kept for air drying for 1 to 2 days until methanol was evaporated. The dried matter was suspended in 1 ml of water containing 10% methanol and stored under refrigeration until used for ELISA test. Pure cultures for ELISA Two grams of lyophilized pure culture material and pelleted cyanobacterial cultures were used for the detection of the microcystin amount. The first step of extraction was done with 50 ml of 100% methanol. A mixture of dried cyanobacteria powder and methanol was stirred using a magnetic stirrer for 1 h. After decanting off the methanol the residues were again extracted under stirring with an additional 50 ml of methanol for 1 hour. Both extracts were pooled and kept in solvent until dry. The residues were dissolved in 20% methanol and kept at 4 o C until subjected to ELISA test. ELISA test for Microcystin Envirologix inc., (USA) provided the completely available detection ELISA kit. The QualiTube kit (Envirologix Inc., USA) was used for microcystin analysis. In this test, microcystin in a sample competes with enzyme (horseradish peroxidase)-labeled microcystin for a limited number of antibody binding sites on the inside surface of the test tubes. Following list of the chemicals were provided in the kit. Procedure, result and interpretation were done according to the manufacturer’s instructions. Result 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 Discussion In this study, laboratory cultures of M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongatus were grown under mass cultivation conditions. The growth of these cultures was estimated based on parameters such as absorbance, protein concentration, and chlorophyll concentration. Visual observations revealed that fully grown cultures of these cyanobacteria produced various biochemical substances, including proteins and enzymes, for cellular metabolic processes, as well as secondary metabolites and microcystin. Microcystin is a toxin synthesized by a multi-enzyme complex system without mRNA translation. In current study M. aeruginosa and O. laetevirens var. minimus showed amplification of genes mcyABDE, while A. fertilissima exhibited amplification of genes mcyABD, and P. uncinatum carried out amplification of genes mcyABE. In contrast, S. elongatus did not show any amplification of mcy genes. These differences between natural and laboratory growing conditions can lead to variations in the activation and deactivation of microcystin synthase genes, resulting in changes in microcystin toxicity (Pearson et al., 2004; Christiansen et al., 2006). In eutrophicated lakes, harmful cyanobacterial blooms thrive rapidly when there are elevated levels of nitrate and phosphate due to industrial, agricultural, sewage, and human activities. These algal blooms have a detrimental impact on the natural ecology and disrupt the food chain (Dolman et al., 2012; WHO, 2003; Anderson et al., 2002). In a eutrophicated environment, many harmful and toxic cyanobacterial bloom and mat-forming species compete with normal cyanobacteria, bacterial species, microbial flora, coliforms, and water-borne bacteria present in the lake's natural aquatic environment. To maintain an antagonistic relationship with other microbes, these cyanobacteria produce higher levels of microcystin. Natural conditions activate mcy genes to a greater extent, allowing for maximum microcystin synthesis (Londt and Pflugmacher, 2020; Chia et al., 2019; Paerl, 2018; Zhang et al., 2009; Christiansen et al., 2006). Pure cultures of M. aeruginosa cultivated in controlled laboratory conditions, with a defined and limited nutrient supply and no antagonistic relations with other organisms, result in lower amounts of microcystin compared to natural bloom samples collected from various lakes, ponds, and reservoirs in previous surveys (Chaturvedi et al., 2015; Agrawal et al., 2006; Ghosh et al., 2008b). O. laetevirens var. minimus, A. fertilissima, and P. uncinatum produced trace amounts of microcystin under laboratory conditions due to the absence of other microorganisms for an ecological antagonistic relationship. Similarly, earlier studies have reported higher microcystin levels in natural scums and mat samples containing these genera (Chaturvedi et al., 2017; Singh et al., 2017). Conclusion The laboratory-cultivated cyanobacterial pure cultures were subjected to controlled growth conditions in a specific growth medium, allowing for continuous monitoring within a defined time frame. Three key parameters, namely turbidity, chlorophyll concentration, and protein concentration, were measured to assess the growth. The eighteenth day of growth was found to exhibit maximum growth. Subsequently, the well-developed cultures were concentrated and preserved for DNA extraction. High-quality DNA was successfully extracted from these pure cultures and utilized for PCR amplification of mcy gene(s). Interestingly, most cyanobacterial DNA samples displayed amplification of mcy genes, except for S. elongates which did not show any gene amplification. Despite the presence of amplified mcy genes, the majority of cyanobacterial species produced only trace amounts of microcystin, measuring below 0.5 ppb. Notably, M. aeruginosa K1 exhibited relatively higher levels of microcystin production ranging between 0.5–3.0 ppb. Declarations Acknowledgment We express our sincere gratitude to Prof. S. N. Bagchi, Head and Professor of the Department of P.G. Studies and Research in Biological Science at R.D. University, Jabalpur (M.P.), for generously providing us with access to the laboratory facility. Ethical Approval-NA Consent to Publish- Yes Consent to participate-Yes Authors Contributions-All authors contributed to the study conception and design. Prashant Chaturvedi conducted the experiments. Divya Singh conceptualised and prepared the primary draft. Renu Pathak and Purnima Beohar edited contributed in making the final draft. All authors commented on previous versions of the manuscript. 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Miller MA, Kudela RM, Mekebri A, Crane D, Oates SC, Tinker MT, Staedler M, Miller WA, Toy-Choutka S, Dominik C, Hardin D, Jensen B, Kwan E, Catton C, Murray M, Ward K, Mertes L, Murray K, Ward B, Schaefer A, Elrod S, Smith LW (2010) Evidence for a novel marine harmful algal bloom: Cyanotoxin (microcystin) transfer from land to sea otters. PLoS ONE 5(9): e12576. Misdorp R, Kruitwagen G, Jonkers N, Murk AJ, Smidt H, van Osselaer SMJ, de Boer J (2015) Microcystin biodegradation by a natural bacterial community. Water Res. 84: 1–8. Osswald J, Rellán S, Gago A, Vasconcelos V (2007) Anaerobic digestion of cylindrospermopsin- and microcystin-LR-containing cyanobacterial biomass: Fate of cyanotoxins and effect on methane production. Water Res. 41(7): 1379–1387. Pflugmacher S (2002) Promotion of apoptosis by microcystins. Toxicon 40(7): 971–975. Pflugmacher S, Aulhorn M, Grimm B (2007) Influence of environmental factors on the influence of the production of microcystins of cyanobacteria: A laboratory study. Ecotoxicology 16(3): 185–192. Prieto AI, Jos A, Pichardo S, Moreno I, Cameán AM, Moyano R (2008) Effects of oral exposure to microcystin-LR on reproductive performance in male mice. Toxicon 52(1): 93–103. Pu XY, Dai XY, Zhang J, Li RW, Zhang QL, Luo ZH, Zhang HJ (2013) Microcystin-LR causes sexual hormone disturbance and induces histopathological changes in gonads of male zebrafish. J. Environ. Sci. Health B 48(4): 296–304. Pu X, Xie P, Li L, Zhang D, Liu Y, Li M, Li S (2011) Oxidative stress responses and apoptosis induction in Microcystis aeruginosa exposed to microcystins. Chem. Res. Toxicol. 24(5): 747–754. Quesada A, Moreno-Garrido I, Giménez-Casalduero F, Carrasco-Navarro V, Camacho A, Perona E (2016) Effects of microcystin-LR on the early life stage of zebrafish (Danio rerio): Antioxidant response, osmoregulatory disturbance, liver damage, and hepatocyte ultrastructural changes. Chemosphere 147: 180–190. Rapala J, Robertson A, Negri AP, Berg KA, Tuomi P, Lyra C, Erkomaa K, Lahti K, Hoppu K, Lepistö L, Sivonen K (2005) First report of saxitoxin in Finnish lakes and possible associated effects on human health. Environ. Toxicol. 20(3): 331–340. Rinehart KL, Harada KI, Namikoshi M, Chen C, Harvis CA, Munro MH, Blunt JW, Mulligan PE, Beasley VR, Dahlem AM (1988) Nodularin, microcystin, and the configuration of Adda. J. Am. Chem. Soc. 110(3): 855–856. Rinehart KL, Namikoshi M, Choi BW, Stotts RR, Lanekoff I (1994) Isolation, structure determination, and synthesis of a toxic peptide from the cyanobacterium Microcystis aeruginosa. J. Org. Chem. 59(20): 5922–5927. Roegner AF, Bernard C, Martin JL, McGuire MA, Merel S, Lagadic L, Beisel JN (2007) Effects of microcystin-LR on the energy metabolism of common carp: Changes in metabolic pathways and mitochondrial function. Aquat. Toxicol. 82(1): 16–26. Runnegar MT, Falconer IR (1982) Congener-dependent hydrolysis of synthetic cyclic peptide hepatotoxins in vivo in the mouse. Biochem. J. 204(2): 381–384. Runnegar MT, Kong SM, Zhong YZ (1995) Inhibition of reduced glutathione synthesis by cyanobacterial alkaloid, cylindrospermopsin. Biochem. Biophys. Res. Commun. 216(2): 162–167. Sivonen K (1990) Effects of light, temperature, nitrate, orthophosphate, and bacteria on growth of and hepatotoxin production by Oscillatoria agardhii strains. Appl. Environ. Microbiol. 56(9): 2658–2666. Sivonen K, Halinen K, Sihvonen LM, Koskenniemi K, Sinkko H, Rantasärkkä K, Moisander PH (2007) Bacterial diversity and function in the Baltic Sea with an emphasis on cyanobacteria. Ambio 36(2–3): 180–185. Sivonen K, Jones G (1999) Cyanobacterial toxins. In: Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring, and Management. Chorus I, Bartram J, editors. CRC Press: London, UK, pp. 41–111. Sivonen K, Kononen K, Carmichael WW, Dahlem AM, Rinehart KL, Kiviranta J, Niemelä SI (1989) Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the toxin. Appl. Environ. Microbiol. 55(8): 1990–1995. Soares RM, Yuan M, Servaites JC, Delgado AG, Magalhães VF, Hilborn ED, Carmichael WW, Azevedo SM (2006) The role of microcystins in carcinogenesis and tumor promotion. Life Sci. 79(3): 203–207. Stewart I, Seawright AA, Shaw GR (2008) Cyanobacterial poisoning in livestock, wild mammals and birds - an overview. In: Harmful Cyanobacteria. Huisman J, Matthijs HCP, Visser PM, editors. Springer: Dordrecht, The Netherlands, pp. 613–637. Stewart I, Seawright AA, Shaw GR (2009) Cyanobacterial poisoning in livestock, wild mammals and birds - an overview. Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs. Hudnell HK, editor. Springer: New York, NY, USA, pp. 613–637. Teneva I, Blishchik V, Vassilev V, Berndtsson R (2003) Photodegradation of microcystin-LR in laboratory water and in water from a shallow hypertrophic reservoir. Water Res. 37(19): 4599–4610. Vasconcelos VM, Saker ML, Neilan BA, Lawrence JF (1995) Multiple toxins from a single cyanobacterial source: Saxitoxins and cylindrospermopsins from Cylindrospermopsis raciborskii from Lake Awe, Scotland. Toxicon 33(11): 1565–1575. Yuan M, Carmichael WW, Hilborn ED, Lyra TM, Soares RM, Ahmad A, Azevedo SM (2006) Co-occurrence of microcystins and taste-and-odor compounds in a drinking water source in Brazil. Water Res. 40(3): 565–574. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4371317","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299205298,"identity":"1c810c1c-bb8d-4bb7-a7d6-976173c3aaa0","order_by":0,"name":"Prashant Chaturvedi","email":"","orcid":"","institution":"Rani Durgavati University","correspondingAuthor":false,"prefix":"","firstName":"Prashant","middleName":"","lastName":"Chaturvedi","suffix":""},{"id":299205299,"identity":"7576dc12-a035-40df-bbc8-9f0636230e20","order_by":1,"name":"Divya Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDCCwyDCwEbO/njzASBLQoZILQVpxgxnjiWAtPAQ1gIymuHD4cSGGzkGICZhLXzHecwkfhikJTbOyPn86kaNBQ8D++GjG/BpkTzMYybZY2Bj3Mzzdpt1zjGgw3jS0m7g02JwmC1NgscgTbaNPXebcQ4bUIsEjxlBLZJ/DA4z9jDkPDPO+UeUFuZj0jwGhxVncOQwP85tI0KL5GHmw9YyBmnGBjzHzJhz+yR42Aj5he/8wcabb/7YyBmwNz/+nPOtTo6f/fAxvFqAgEUCymADM9gIKAcB5g/ojFEwCkbBKBgFKAAAtmRJMK35l70AAAAASUVORK5CYII=","orcid":"","institution":"Rani Durgavati University","correspondingAuthor":true,"prefix":"","firstName":"Divya","middleName":"","lastName":"Singh","suffix":""},{"id":299205300,"identity":"3a1e4329-d6a2-4260-8b3c-84abd0a1b76d","order_by":2,"name":"Renu Pathak","email":"","orcid":"","institution":"Rani Durgavati 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from BSA protein against blank by spectrophotometeric analysis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4371317/v1/912410c58795feb81ab8173a.png"},{"id":55979543,"identity":"3899e55a-4c7f-4734-a0e3-ff289995df98","added_by":"auto","created_at":"2024-05-07 06:25:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth curve of cynobacterial pure cultures on the basis of turbidity development.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4371317/v1/e90e822d76e328f992437ec4.png"},{"id":55979545,"identity":"f27a52ce-ddb9-4f4b-9cf9-bc55e178bb60","added_by":"auto","created_at":"2024-05-07 06:25:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth curve of cynobacterial pure cultures on the basis of chlorophyll concentration.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4371317/v1/24575fef82cabc176b371ae7.png"},{"id":55979258,"identity":"70611939-af4d-41dd-ae52-627ccff16abc","added_by":"auto","created_at":"2024-05-07 06:17:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth curve of cynobacterial pure cultures on the basis of protein concentration.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4371317/v1/3ba2ed0ad3270fa88a491730.png"},{"id":55979253,"identity":"5275b6d8-5402-4761-868b-eec94567ae25","added_by":"auto","created_at":"2024-05-07 06:17:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":83715,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4371317/v1/db8d3f6ff8899d0335455498.png"},{"id":55979265,"identity":"434cd408-66cd-478f-81a3-47dbe9210ac0","added_by":"auto","created_at":"2024-05-07 06:17:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":223809,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4371317/v1/3e981dd185937af27a681c63.png"},{"id":55979549,"identity":"edeb0fee-a08f-49ad-9a27-9e7d233797a1","added_by":"auto","created_at":"2024-05-07 06:25:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1170244,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4371317/v1/cbc9fc1e-c73f-4553-b33b-6e28f69a774c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular detection of microcystin synthetase genes (mcy genes) and semi- quantitative immunological detection of the production of microcystin toxin in vitro-grown pure cultures of cyanobacteria","fulltext":[{"header":"Highlights","content":"\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003eGrowth assessment and maximum growth of cyanobacterial strains: Turbidity, chlorophyll concentration, and protein content were monitored to assess the growth of cyanobacterial strains \u003cem\u003eM. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongatus\u003c/em\u003e. The cultures exhibited maximum growth after an 18-day inoculation period.\u003c/li\u003e\n \u003cli\u003eConcentration and preservation of well-developed cultures: Well-developed cultures were concentrated using centrifugation and subsequently lyophilized to preserve them in powdered form. This ensured the long-term stability and storage of the cultures.\u003c/li\u003e\n \u003cli\u003eDNA extraction and gene amplification: DNA extraction from the lyophilized cultures resulted in clear DNA bands. The quality of the extracted DNA, assessed by the A260/280 ratio, indicated good quality DNA. The genes mcyABDE, mcyABD, and mcyABE were successfully amplified in M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, and P. uncinatum, respectively. However, no amplification was observed in S. elongatus. Additionally, using a semi-quantitative ELISA technique, Microcystin was detected only in Microcystis aeruginosa at a concentration of 0.5 ppb, while the other cultures produced trace amounts below 0.5 ppb.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eCyanobacteria serve as photosynthetic agents in aquatic ecosystems, but their excessive proliferation can lead to irritations. These proliferations, known as blooms, form colorful mats on the water's surface. Blooms cause low oxygen levels in water bodies, resulting in hypoxia, which can lead to increased mortality rates among invertebrates, shellfish, fish, and plants. Additionally, blooms can obstruct light from reaching the benthic region of the ecosystem, reducing productivity. Some blooms produce harmful toxins that pose risks to aquatic creatures, livestock, and humans, and are referred to as Harmful Algal Blooms (Zhang et al., 2009; Berry et al., 2011; Paerl and Otten, 2013).\u003c/p\u003e \u003cp\u003eThe most commonly reported cyanobacterial toxin is microcystin, which belongs to a family of cyclic pentapeptides with over 90 structural variants. Microcystin's general structure is cyclo (D-Ala-X-D-MeAsp-Z-Adda-D-Glu-Mdha), where X and Z represent variable L amino acids. Microcystin-producing genera in freshwater cyanobacteria include Microcystis, Planktothrix, and Anabaena (Sivonen and Jones, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Terrestrial strains like Nostoc and Hapalosiphon (Prinsep et al., 1992; Oksanen et al., 2004) and benthic Phormidium (Izaguirre et al., 2007) have also been identified as microcystin producers.\u003c/p\u003e \u003cp\u003eMicrocystin forms an irreversible covalent bond with the cysteine of eukaryotic protein phosphatase type 1 and 2A (Runnegar et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1995\u003c/span\u003ea). Specifically, the Mdha moiety in microcystin binds covalently to cysteine 273 of the protein phosphatase active site (Welker and Von Dohren, 2006; Wharton et al., 2019). Microcystin acts as a tumor promoter, while nodularin functions as a carcinogen (Nishiwaki-Matsushima et al., 1992; Ohta et al., 1994). It can cause damage to cell tissue and organs, particularly affecting the mitochondrion, leading to the loss of cytochrome-C, calcium ions, and inter-membrane proteins (Lankiewicz et al., 2000; Nodberg and Anner, 2001). The release of cytochrome-C triggers caspase cascades, including caspase-3 via caspase-9. Microcystin also plays a role in mitosis regulation (Snaith et al., 1996) and induces oxidative stress in vitro conditions in rat hepatocytic cells and fish tissues (Li et al., 2003). In plants, microcystin inhibits the activity of Protein Phosphatase 1 and Protein Phosphatase 2A, affecting antioxidant enzyme peroxidases and superoxide dismutase, leading to oxidative stress, apoptosis, and hepatotoxicity (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe diversity of chemotypes observed in various cyanobacterial taxa is due to microcystin-producing cyanobacteria. On average, individual genera have been found to produce four distinct microcystins, but up to 27 have been identified so far. Furthermore, certain cyanobacterial strains do not produce microcystins, and studies have shown that microcystin-producing and non-producing strains coexist in natural populations.\u003c/p\u003e \u003cp\u003eWhile research on microcystins has predominantly focused on terrestrial mammals, there is growing recognition of their effects on aquatic organisms. Fish, being at the top of the aquatic food chain, are particularly susceptible to cyanobacterial toxins, which can be ingested or absorbed through their gills. This poses risks not only to fish but also to humans through the food chain (Malbrouck and Kestemont 2006).\u003c/p\u003e \u003cp\u003eHarmful cyanobacterial blooms have adverse ecological and environmental impacts, as well as posing risks to animals and humans. Future climate change scenarios predict an increase in bloom-forming cyanobacteria due to changes in hydrological cycling, rising water temperatures, and nutrient loading. As a result, harmful blooms are expected to become more frequent and last longer.\u003c/p\u003e \u003cp\u003eThis study investigates diverse cyanobacteria that produce toxins and form blooms, with a focus on their cultivation. In-depth studies have been conducted using both in vivo and in vitro approaches to analyse and compare the concentration of the toxins produced. Furthermore, a comprehensive molecular analysis has been carried out to better understand the characteristics of these toxins.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCulture conditions: -\u003c/h2\u003e\n \u003cp\u003eThe axenic pure cultures \u003cem\u003eMicrocystis aeruginosa\u003c/em\u003e (Ghosh \u003cem\u003eet al\u003c/em\u003e, 2008a), \u003cem\u003eOscillatoria laetevirens var. minimus\u003c/em\u003e (Ray and Bagchi, 2001) and \u003cem\u003eAnabaena fertilissima\u003c/em\u003e Banerjee \u003cem\u003eet al\u003c/em\u003e, 2013), \u003cem\u003ePhormidium uncinatum\u003c/em\u003e ( Bagchi and Verma, 1997), \u003cem\u003eSynechococcus elo\u003c/em\u003engates (Saggu et al, 2010), were provided by Cyanobacterial Research Lab, Dept. of P.G. Studies and Research in Biological Science, R. D. University, Jabalpur, Madhya Pradesh, India. The cyanobacterial strain was fully grown and maintained all strains in BG-11 medium and its composition is given below \u0026ndash;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eComposition of macronutrient\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e- Independent stock solution each of g/L \u0026ndash; Potassium phosphate (4.0), Magnesium sulphate (7.5), Calcium chloride (3.6), Citric acid (0.6), Ferric ammonium citrate (0.6), EDTA (0.1), Sodium carbonate (0.2), Sodium nitrate (85.0).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eComposition of micronutrient\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e- Composition of combined stock solution in gm/L \u0026ndash; Boric acid (2.86), Manganese chloride (1.81), Zinc sulfate (0.22), Sodium molybdate (0.39) Copper sulfate (0.079), Cobalt nitrate (0.0494).\u003c/p\u003e\n \u003cp\u003eFor the preparation of one liter of medium 10 ml, each macronutrient and 1 ml of combined micronutrient stock solution was taken, the pH of the medium was adjusted to 7.5 and this was autoclaved at 15 lbs pressure for 20 min at 121˚C.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGrowing condition:-\u003c/strong\u003e The culture was kept for growth in an air-conditioned growth chamber at a temperature 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2˚C under the cool white fluorescent light of a continuous light intensity of approximately 1500 lux. The culture was manually shaken twice a day for aeration.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eEstimation of growth\u003c/h2\u003e\n \u003cp\u003eAn increase in the density in the pure culture was taken as a growth parameter and increasing total chlorophyll A and protein content was considered for a more precise estimation of growth. For growth studies the aliquots of 3 ml of culture were taken aseptically at regular intervals and optical density (O.D.) at 750 nm was measured for growth, using a spectrophotometer (digital spectrophotometer, model ME 802). Absorbance of the density of the culture was taken against the BG-11 medium blank. Thereafter another growth parameter was chlorophyll concentration, this was done according to the method of MacKinney., (1941). The final growth parameter was protein concentration within all pure cultures which were done according to the method of Lowry \u003cem\u003eet al\u003c/em\u003e., (1951).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eStandard curve of Bovine serum albumin: -\u003c/strong\u003e A standard curve was prepared by using the increasing concentration of bovine serum albumin in the different ranges 100 \u0026micro;g \u0026minus;\u0026thinsp;1000\u0026micro;g per ml according to Lowry et al., (1951). Absorbance was taken of each concentration against the blank at 750 nm by using digital spectrophotometer (digital spectrophotometer, model ME 802).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003ePreservation of cyanobacterial cultures\u003c/h2\u003e\n \u003cp\u003ePure cultures were centrifuged for 10 min at 6000 \u0026times; g at 25 \u0026ordm;C to collect the supernatant and the pellets sediment at the bottom surface of the centrifuge tubes. Culture materials were transferred to lyophilization tubes and kept in a pre-freezer and maintained at \u0026minus;\u0026thinsp;20\u0026ordm;C for 1 hour before lyophilization. Lyophilization tubes (Borosil, India) were fitted at the appropriate position of freeze-drier (NSW, India) and the vacuum was set at \u0026minus;\u0026thinsp;20\u0026ordm;C. The process was carried out till the material completely dried in the form of a powder which was transferred into airtight storage vials and kept in the refrigerator for further experimental use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003eIsolation of genomic DNA from cyanobacteria cultures\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eThe culture material was lyophilized at \u0026minus;\u0026thinsp;20\u0026ordm;C until it turned into powder and became brittle. It was stored in cryo-vials at 4\u0026ordm;C. Samples collected at different times or locations were dried and stored. Two methods were standardized for the isolation of genomic DNA from dry culture material. In the first method described by Jungblunt and Neilan, (2006), 25 mg of lyophilized cyanobacteria culture material was heated at 65\u0026ordm;C for 2 h in 3.0 ml of DNA extraction buffer containing 800 mM ammonium acetate, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0), 1% SDS and 1% lysozyme (fresh). Thereafter, 50 \u0026micro;l of RNase from a stock of 10 mg ml\u003csup\u003e-1\u003c/sup\u003e was added and further incubation was done at 37 \u0026ordm;C for 30 min. To stop the reaction, the mixture was chilled in an ice bath for 10 min and centrifuged at 12000 \u0026times; g for 10 min at 4\u0026ordm;C. To one volume of cell, extract was added one volume of ice-cold isopropanol and 0.1 volume of 4 M ammonium acetate and centrifuged at 12000 \u0026times; g for 10 min at 4\u0026ordm;C to precipitate the DNA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eDNA quantification and purity assessment\u003c/h2\u003e\n \u003cp\u003eApproximately 10 \u0026micro;l of DNA samples obtained from different protocols was added to 990 \u0026micro;l of sterile double distilled water. Their purity was checked by taking the ratio of their absorbance at A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e280\u003c/sub\u003e nm. The yield of each sample was also calculated by using the following formula:\u003c/p\u003e\n \u003cp\u003eA\u003csub\u003e260\u003c/sub\u003e \u0026times; dilution factor \u0026times; 50 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eAgarose gel electrophoresis of cyanobacterial genomic DNA\u003c/h2\u003e\n \u003cp\u003eOne percent agarose gel was prepared in 0.5X TBE running buffer (45 mM Tris-borate/1 mM EDTA). To 100 ml warm (70\u0026ordm;C \u0026ndash; 80\u0026ordm;C) sterilized TBE buffer, agarose was slowly added followed by gentle stirring so that the agarose dissolves completely. Thereafter, 50 \u0026micro;l of ethidium bromide from the stock of 10 mg ml\u003csup\u003e-1\u003c/sup\u003e was added. The temperature of the solution was decreased to 50\u0026ordm;C and was poured onto a sealed gel caster. A comb was placed in the solidifying gel. After solidification, agarose gel was submerged in a running buffer (0.5X TBE buffer). DNA extracted as above (15 \u0026micro;l) was mixed with 3 \u0026micro;l of bromophenol blue (0.5 mg/ml) and glycerol (5 \u0026micro;l). This was applied to the wells along with the DNA ladder (HI media, India). Electrophoresis was carried out at 70 V and 25 mA for 1 h in a horizontal slab gel electrophoresis apparatus (Bangalore Genei) until the bromophenol blue reached 3/4th of the bottom of the gel. The tank was filled with 0.5X TBE and the gel was kept submerged throughout.\u003c/p\u003e\n \u003cp\u003eAfter the samples had run to a desired distance the gel was taken out from the electrophoresis unit and placed over a UV transilluminator (Biotch R \u0026amp; D laboratories Yercaud). The orange fluorescent bands of DNA were detected under a UV cut-off filter. These bands were photographed using a digital camera (Sony, Japan).\u003c/p\u003e\n \u003cp\u003ePCR amplification for desired genes of dry cyanobacteria cultures materials.\u003c/p\u003e\n \u003cp\u003eFor PCR amplification of \u003cem\u003emcy\u003c/em\u003eABDE genes, the preparation of the reaction mixture and amplification cycle was carried out as described by Jungblunt and Neilan, (2006) and Kumar et al, (2011). A reaction mixture of 23 \u0026micro;l was prepared using the PCR amplification kit containing a final concentration: 12.5 \u0026micro;l of autoclave distilled water, 2.5 \u0026micro;l of 10 x taq polymerase buffer, 1 \u0026micro;l of 25 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 4 \u0026micro;l of 200 \u0026micro;M of dNTPs mixture solution, 1 \u0026micro;l of 20 pmol forward and reverse primers, 1 \u0026micro;l of 1U taq DNA polymerase enzyme, 2 \u0026micro;l of template DNA sample 50 ng in each case. Thermal cycling was performed using a gradient-type thermal cycler (Merck Genei) with an initial denaturation step at 94 \u0026ordm;C for 5 min, followed by 40 cycles of 94\u0026ordm;C for 1 min, 60.8\u0026ordm;C for 1 min, and 72 \u0026ordm;C for 1 min and a final extension of 10 min at 72\u0026ordm;C. The amplified product was then analyzed using agarose gel electrophoresis as above. The forward and reverse primer pairs (Bagchi and Ghosh, 2010) as follows were procured from (Imperial Life Sciences, India):\u003c/p\u003e\n \u003cp\u003e\u003cem\u003emcy\u003c/em\u003eA\u003c/p\u003e\n \u003cp\u003eForward 5\u0026rsquo;-AAAATTAAAAGCCGTATCAAA-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eReverse 5\u0026rsquo;-AAAAGTGTTTTATTAGCGGCTCAT-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003emcy\u003c/em\u003eB\u003c/p\u003e\n \u003cp\u003eForward 5\u0026rsquo;-CTATGTTATTTATACATCAGG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eReverse 5\u0026rsquo;-CTCAGCTTAACTTGATTATC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003emcy\u003c/em\u003eD\u003c/p\u003e\n \u003cp\u003eForward 5\u0026rsquo;-GATCCGATTGAATTAGAAAG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eReverse 5\u0026rsquo;-GTATTCCCCAAGATTGCC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003emcy\u003c/em\u003eE\u003c/p\u003e\n \u003cp\u003eForward 5\u0026rsquo;-TTTGGGGTTAACTTTTTTGGGCATAGTC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eReverse 5\u0026apos;-AATTCGCCGGTATTAGACGTT-3\u0026rsquo;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of pure cyanobacterial culture medium for ELISA test\u003c/h2\u003e\n \u003cp\u003eCyanobacterial pure cultures cultivated at mass scale upto 2 liters in specific growth mediums in the laboratory. Thereafter cyanobacterial cultures were filtered with different kinds of filtration methods used to possibly remove all the finest particles present in the growth medium, as particles may chock ODS cartridges. First of all cyanobacterial growth mediums were filtered with a muslin cloth to remove coarse particles and, thereafter again filtered with Whatman filter paper no. 42 to remove fine impurities. Later, growth mediums were again filtered with the vacuum filtration method using cellulose nitrate filters with pore size 5 \u0026micro;m (Axiva Sichem Biotech, India). In the last step, the growth medium was filtered with fine cellulose nitrate membrane filters of 0.45 \u0026micro;m (Millipore Corporation, Bedford) under a vacuum. In totality, 1 liter of above-filtered lake water was passed through ODS cartridges (Merck LiChrolut \u0026reg; RP-18).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eConcentrating the cyanobacterial growth medium for the detection of Microcystin\u003c/h2\u003e\n \u003cp\u003eThe ODS cartridges were fitted on top with syringes using tubings and manual pressure was applied to the contents of the cartridges. Firstly, the cartridges were pre-washed with 10 ml of 100% methanol in order to equilibrate and subsequently, they were washed with 10 ml of distilled water. Gradually the entire 1 liter of pure culture was passed through the cartridges. The bound material was first washed with 10 ml of 20% methanol which was discarded. Microcystin was eluted using 10 ml of 100% methanol. The eluted 100% methanolic extract was kept for air drying for 1 to 2 days until methanol was evaporated. The dried matter was suspended in 1 ml of water containing 10% methanol and stored under refrigeration until used for ELISA test.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003ePure cultures for ELISA\u003c/h2\u003e\n \u003cp\u003eTwo grams of lyophilized pure culture material and pelleted cyanobacterial cultures were used for the detection of the microcystin amount. The first step of extraction was done with 50 ml of 100% methanol. A mixture of dried cyanobacteria powder and methanol was stirred using a magnetic stirrer for 1 h. After decanting off the methanol the residues were again extracted under stirring with an additional 50 ml of methanol for 1 hour. Both extracts were pooled and kept in solvent until dry. The residues were dissolved in 20% methanol and kept at 4\u003csup\u003eo\u003c/sup\u003eC until subjected to ELISA test.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eELISA test for Microcystin\u003c/h2\u003e\n \u003cp\u003eEnvirologix inc., (USA) provided the completely available detection ELISA kit. The QualiTube kit (Envirologix Inc., USA) was used for microcystin analysis. In this test, microcystin in a sample competes with enzyme (horseradish peroxidase)-labeled microcystin for a limited number of antibody binding sites on the inside surface of the test tubes. Following list of the chemicals were provided in the kit. Procedure, result and interpretation were done according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eStandard curve of protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth curve\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth curve was determined through spectrophotometric analysis, which involved monitoring the increasing density of pure cultures of \u003cem\u003eM. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;S. elongates.\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003eThe growth, chlorophyll, and protein concentrations were estimated over a period of 15 to 18 days from the day of inoculation(Fig-02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConcentration of chlorophyll\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotosynthetic 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 \u0026micro;g/ml, respectively (Fig-03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConcentration of protein\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 \u0026micro;g/ml, respectively(Fig-04)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrocystin-producing genotypes in isolated pure cultures of cyanobacteria\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePure 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)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of total DNA and its purity analysis in pure culture of cyanobacterium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA 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 \u0026micro;g/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)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOccurrence of mcy genes in pure cultures\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmplification 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)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eTable 1. Level of cell bound microcystin in pure culture\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.040955631399317%\" valign=\"top\"\u003e\n \u003cp\u003eS.No.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.44368600682594%\" valign=\"top\"\u003e\n \u003cp\u003ePure Culture in Laboratory\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.515358361774744%\" valign=\"top\"\u003e\n \u003cp\u003eMicrocystin level (ppb)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.040955631399317%\" valign=\"top\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.44368600682594%\" valign=\"top\"\u003e\n \u003cp\u003e0.5 ppb calibrator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.515358361774744%\" valign=\"top\"\u003e\n \u003cp\u003e0.5 ppb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.040955631399317%\" valign=\"top\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.44368600682594%\" valign=\"top\"\u003e\n \u003cp\u003e3.0 ppb calibrator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.515358361774744%\" valign=\"top\"\u003e\n \u003cp\u003e3.0 ppb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.040955631399317%\" valign=\"top\"\u003e\n \u003cp\u003e01.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.44368600682594%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eM. aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.515358361774744%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ge;0.5 ppb ; \u0026le;3.0 ppb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.040955631399317%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;02.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.44368600682594%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eO.laetevirens var. minimus\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.515358361774744%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le; 0.5 ppb\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.040955631399317%\" valign=\"top\"\u003e\n \u003cp\u003e03.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.44368600682594%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eA. fertilissima\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.515358361774744%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026le; 0.5 ppb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.040955631399317%\" valign=\"top\"\u003e\n \u003cp\u003e04.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.44368600682594%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. uncinatum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.515358361774744%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le; 0.5 ppb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.040955631399317%\" valign=\"top\"\u003e\n \u003cp\u003e05.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.44368600682594%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. elongatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.515358361774744%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le; 0.5 ppb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, laboratory cultures of M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongatus were grown under mass cultivation conditions. The growth of these cultures was estimated based on parameters such as absorbance, protein concentration, and chlorophyll concentration. Visual observations revealed that fully grown cultures of these cyanobacteria produced various biochemical substances, including proteins and enzymes, for cellular metabolic processes, as well as secondary metabolites and microcystin. Microcystin is a toxin synthesized by a multi-enzyme complex system without mRNA translation.\u003c/p\u003e \u003cp\u003eIn current study M. aeruginosa and O. laetevirens var. minimus showed amplification of genes mcyABDE, while A. fertilissima exhibited amplification of genes mcyABD, and P. uncinatum carried out amplification of genes mcyABE. In contrast, S. elongatus did not show any amplification of mcy genes. These differences between natural and laboratory growing conditions can lead to variations in the activation and deactivation of microcystin synthase genes, resulting in changes in microcystin toxicity (Pearson et al., 2004; Christiansen et al., 2006).\u003c/p\u003e \u003cp\u003eIn eutrophicated lakes, harmful cyanobacterial blooms thrive rapidly when there are elevated levels of nitrate and phosphate due to industrial, agricultural, sewage, and human activities. These algal blooms have a detrimental impact on the natural ecology and disrupt the food chain (Dolman et al., 2012; WHO, 2003; Anderson et al., 2002).\u003c/p\u003e \u003cp\u003eIn a eutrophicated environment, many harmful and toxic cyanobacterial bloom and mat-forming species compete with normal cyanobacteria, bacterial species, microbial flora, coliforms, and water-borne bacteria present in the lake's natural aquatic environment. To maintain an antagonistic relationship with other microbes, these cyanobacteria produce higher levels of microcystin. Natural conditions activate mcy genes to a greater extent, allowing for maximum microcystin synthesis (Londt and Pflugmacher, 2020; Chia et al., 2019; Paerl, 2018; Zhang et al., 2009; Christiansen et al., 2006).\u003c/p\u003e \u003cp\u003ePure cultures of M. aeruginosa cultivated in controlled laboratory conditions, with a defined and limited nutrient supply and no antagonistic relations with other organisms, result in lower amounts of microcystin compared to natural bloom samples collected from various lakes, ponds, and reservoirs in previous surveys (Chaturvedi et al., 2015; Agrawal et al., 2006; Ghosh et al., 2008b). O. laetevirens var. minimus, A. fertilissima, and P. uncinatum produced trace amounts of microcystin under laboratory conditions due to the absence of other microorganisms for an ecological antagonistic relationship. Similarly, earlier studies have reported higher microcystin levels in natural scums and mat samples containing these genera (Chaturvedi et al., 2017; Singh et al., 2017).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe laboratory-cultivated cyanobacterial pure cultures were subjected to controlled growth conditions in a specific growth medium, allowing for continuous monitoring within a defined time frame. Three key parameters, namely turbidity, chlorophyll concentration, and protein concentration, were measured to assess the growth. The eighteenth day of growth was found to exhibit maximum growth. Subsequently, the well-developed cultures were concentrated and preserved for DNA extraction. High-quality DNA was successfully extracted from these pure cultures and utilized for PCR amplification of mcy gene(s). Interestingly, most cyanobacterial DNA samples displayed amplification of mcy genes, except for S. elongates which did not show any gene amplification. Despite the presence of amplified mcy genes, the majority of cyanobacterial species produced only trace amounts of microcystin, measuring below 0.5 ppb. Notably, M. aeruginosa K1 exhibited relatively higher levels of microcystin production ranging between 0.5\u0026ndash;3.0 ppb.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our sincere gratitude to Prof. S. N. Bagchi, Head and Professor of the Department of P.G. Studies and Research in Biological Science at R.D. University, Jabalpur (M.P.), for generously providing us with access to the laboratory facility.\u003c/p\u003e\n\u003cp\u003eEthical Approval-NA\u003c/p\u003e\n\u003cp\u003eConsent to Publish- Yes\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Consent to participate-Yes\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; Authors Contributions-All authors contributed to the study conception and design. Prashant Chaturvedi conducted the experiments. Divya Singh conceptualised and prepared the primary draft. Renu Pathak and Purnima Beohar edited contributed in making the final draft. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript\u003cem\u003e.”\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFunding-No funding was received to assist with the preparation of this manuscript\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eCompeting Interests-The authors have no competing interests to declare that are relevant to the content of this article\u003c/li\u003e\n \u003cli\u003eAvailability of data and materials-NA\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAzevedo SMFO, Carmichael WW, Jochimsen EM, Rinehart KL, Lau S, Shaw GR, Eaglesham GK (2002) Human intoxication by microcystins during renal dialysis treatment in Caruaru\u0026mdash;Brazil. 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Water Res. 40(3): 565\u0026ndash;574.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cyanobacterial strains, Microcystin, Semi-quantitative ELISA, mcyABDE genes","lastPublishedDoi":"10.21203/rs.3.rs-4371317/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4371317/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLaboratory mass cultures were established for cyanobacterial strains M. aeruginosa, O. laetevirens var. minimus, A. fertilissima, P. uncinatum, and S. elongates. The growth of these cultures was assessed by monitoring turbidity, chlorophyll concentration, and protein content. After an 18-day inoculation period, the maximum growth of pure cultures was observed. Well-developed cultures were concentrated using centrifugation and subsequently lyophilized to preserve them in powdered form. DNA extraction was performed on the lyophilized cultures, resulting in clear DNA bands just below the wells. The quality of the extracted DNA, as determined by the A260/280 ratio, ranged from 1.6 to 1.8. The genes mcyABDE were successfully amplified in M. aeruginosa and O. laetevirens var. minimus, while A. fertilissima and P. uncinatum showed amplification of mcyABD and mcyABE genes, respectively. No amplification was observed in S. elongatus. Using a semi-quantitative ELISA technique, a significant concentration of Microcystin was detected only in Microcystis aeruginosa, at a level of 0.5 ppb, whereas the other cultures produced trace amounts below 0.5 ppb.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Molecular detection of microcystin synthetase genes (mcy genes) and semi- quantitative immunological detection of the production of microcystin toxin in vitro-grown pure cultures of cyanobacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-07 06:17:43","doi":"10.21203/rs.3.rs-4371317/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd7a41cb-5fc3-44d5-874e-eb97495d8f53","owner":[],"postedDate":"May 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-07T06:17:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-07 06:17:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4371317","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4371317","identity":"rs-4371317","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}