Effect of Mercury on the Growth and Biochemical Behavior of Nostoc Muscorum and Anabaena Variabilis

Int J Pharma Bio Sci 2019 July; 10(3): (B) 11-21

Original Research Article Microbiology

International Journal of Pharma and Bio Sciences ISSN 0975-6299

EFFECT OF MERCURY ON THE GROWTH AND BIOCHEMICAL BEHAVIOR OF NOSTOC MUSCORUM AND ANABAENA VARIABILIS

YOGESH NEGI1, SUNIL SHARMA2, BARIHUN THYRNIANG3, FRANKIE J. LALOO3 AND SAMRAT ADHIKARI4*

1Senior Research Fellow, Advanced Level Biotech Hub Facility, Department of Biotechnology, St. Edmund’s College Shillong-793003, Meghalaya, India 2Research Associate, Advanced Level Biotech Hub Facility, Department of Biotechnology, St. Edmund’s College Shillong-793003, Meghalaya, India 3UG Student, Department of Biotechnology, St. Edmund’s College, Shillong-793003, Meghalaya, India 4*Associate Professor, Head, Department of Biotechnology, Coordinator, Advanced Level Biotech Hub Facility, St. Edmund’s College, Shillong-793003, Meghalaya, India

ABSTRACT

Cyanobacteria are a highly diversified group of photosynthetic organisms which can be exploited as a potential candidate for bioremediation processes. The present study evaluates the toxic effect of a heavy metal, mercury (Hg2+ (II)), on the growth and biochemical aspects of Nostoc muscorum and Anabaena variabilis. The results depicted negative effects of Hg2+ on cyanobacteria by hampering normal growth and physiological activities. The treated cells showed diminishing growth pattern and decrease in the photosynthetic pigments. Significant decline was also revealed in the biomass and lipid content. The LC50 value of Hg was determined to be 0.6µM for both the cultures. Activity of the enzymes involve in nitrogen metabolism such as Glutamine Synthetase (GS), Nitrate Reductase (NR) and Nitrite Reductase (NIR) were studied and showed reduced activity. This decreased activity also correlates with the reduction in the heterocyst frequency as obtained in the results. In contrast, the enzyme superoxide dismutase (SOD) and proline content concerned with stress management in cyanobacterial cells showed significant increase in activity. The result obtained in the present study exemplifies the cytotoxic environment created by the Hg2+ ions on the microbial cultures by altering their membrane’s physiological functions. Thus, causing leaching of photosynthetic pigments leading to decreased photosynthetic efficiency and consequently restraining growth. The growth inhibiting effect of Hg2+ ions was also evident from the reduced activity of enzymes entailed with assimilation and uptake of nitrogen, resulting in decline in heterocyst frequency. Furthermore, the apparent increase in cell’s SOD and proline content can be ascribed to the production of reactive oxygen species (ROS) due to heavy metal stress created by the Hg2+ ions.

KEYWORDS: Cyanobacteria, Hg2+, Heavy metal stress, Growth, Biochemical assays, antioxidant

SAMRAT ADHIKARI*

Associate Professor, Head, Department of Biotechnology, Coordinator, Advanced Level Biotech Hub Facility, St. Edmund’s College, Shillong-793003, Meghalaya, India

Received on: 29-03-2019 Revised and Accepted on: 16-05-2019 DOI: http://dx.doi.org/10.22376/ijpbs.2019.10.3.b11-21

Creative commons version 4.0

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INTRODUCTION MATERIALS AND METHODS

“Heavy metal” refers to the group of metals and Study Area metalloids with an atomic density greater than 4g/cm³. According to the Department of Mining and Geology, Some of the heavy metals such as Cu, Zn, Mg, Mn etc., Directorate of Mineral Resources, Government of are required by the living organisms at lower Meghalaya, deposits of coal in Meghalaya is estimated concentrations for proper physiological functioning; to 576.48 million tons (2017). The Jaintia Hills alone however, at an elevated level they can act as potentially have about nine significant coal deposits of which two toxic agents. Activities such as of anthropogenic origin sites namely- Khliehriat (25.35˚N; 92.36˚E) and as well as industrialization have resulted in the Shangpung (25.48˚N; 92.34˚E) were selected for introduction of various heavy metals into the collection of cyanobacterial samples. Coal mining in this environment, consequently contaminating the food area is privately controlled by small-scale ventures and chain1-2. As such, the discharges from mining, smelting being the most rampant and profitable business, and metal treatment industries have become a matter of scientific mining has resulted in deterioration of the public as well as environmental health concern3. environment around the area. Cyanobacteria or "blue-green algae" (Cyanophyceae) represent one of the largest subgroups of Gram Isolation, purification and cultivation of negative bacteria4. They are the one of the earliest cyanobacteria photosynthetic organisms known to have originated in The isolated samples collected from different sites were early Precambrian supereon of the geological time scale grown in BG110 medium or solid medium (with 1.5% (about 2.6-3.5 billion years ago)5. Besides performing agar) in an air conditioned chamber at 24 + 2oC with a photosynthesis and fix atmospheric nitrogen, photon fluence rate of 50µmol m-2 s-1 and control cyanobacteria are also known to improve soil stability, illumination of 12 hours light and 12 hours dark 4 with mitigate the emission of greenhouse gases and most slight modifications. Pure cultures of cyanobacterial importantly help in bioremediation of metal pollutants. isolates were obtained from processed samples by Being primary producers in the food chain, serial dilution technique and direct streak plate method. cyanobacteria additionally play an important role in Axenic cultures of cyanobacteria thus obtained were assessment of ecosystem response to heavy metal then inoculated into sterile selective culture medium and contamination6. Various studies have also reported the used for the present studies. effects of heavy metal toxicity on cyanobacteria7-16. Mercury is known to be one of the most toxic heavy Microscopy analysis metals. Though it occurs in different forms in the As preliminary investigation, the isolated cyanobacterial environment, its function in the biological systems samples were initially examined morphologically using remains unknown. Thus, environment pollution due to Olympus 40X light microscope with a digital camera. mercury contamination has been the focus of research in the past few decades. It is a well established fact that DNA extraction, amplification, sequencing and mercury obstructs the normal photosynthetic process by phylogenetic analysis disrupting the flow of electrons at various sites17. In The cyanobacterial samples were characterized at addition, activities such as mining as well as natural molecular level for which genomic DNA was extracted calamities e.g., forest fires, have augmented the levels using Wizard® Genomic DNA purification Kit (Promega of mercury in the biogeochemical cycle18-21. Microalgae Corporation Inc, USA) following the manufacturer’s and cyanobacteria are also known to release mercury protocol. For molecular annotation, 16S rRNA gene was into the atmosphere by the Hg reduction pathway22. PCR amplified using three sets of universal primers as Biological agents such as algae, bacteria, fungi and peat described elsewhere 28-30 and sequenced at 1st BASE moss are known to have the potential to eliminate toxic DNA Sequencing Services, Malaysia. The contigs heavy metals from the environment23 and thus, obtained using the three sets of primers were checked considered as excellent agents of bioremediation24. With for overlaps and assembled in DNA Baser Sequence regard to bioremediation, cyanobacteria are also Assembler v 3.5.3 (www.dnabaser.com/). The considered as an important candidate owing to their sequences were finally submitted to GenBank and their ability to rapidly adapt to various environmental stresses accessions obtained. In order to determine the including heavy metal contamination25-26. In particular, phylogenetic status of the cyanobacterial species toxicity of mercury and its effect on the various collected, evolutionary tree was constructed using physiological processes have been extensively Bayesian Inference in MrBayes v3.1.2 31. studied2,8,17,27. The Jaintia Hills district of Meghalaya has a diverse bunch of natural mineral resources which in Hg2+ treatment and determination of Lethal the past had been exploited through various unscientific Concentration (LC50) smelting operations. The region has also been the Cells in their exponential growth phase were inoculated epicenter of various cement industries which in a way or in BG-11o medium supplemented with graded other releases various heavy metals into the concentrations of HgCl2, (0.2, 0.4, 0.6, 0.8 and 1.0 µM surrounding, thereby polluting the environment. Hence, for N. muscorum and 0.2, 0.4, 0.6, 0.8 and 1.0µM for A. the present study was undertaken to evaluate the effect variabilis respectively). LC50 values were subsequently of the heavy metal, mercury, on commonly occurring determined in terms of optical density (at 663 nm taken cyanobacterial species-Nostoc muscorum and on every 2nd day till the 16th day), chlorophyll ‘a’ (Chl. a) Anabaena variabilis that were isolated from the habitats content extracted on the 12th day following Mackinney32 in and around the mining sites in Jaintia Hills, and phycobilisome content as described elsewhere33. Meghalaya.

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Lipid and cell dry weight quantification Nitrite Reductase assay (NIR) (E C 1.7.1.4) Lipid extraction was carried out on the 16th day of NIR activity was measured as per methods described by inoculation from the control as well as treated groups as Arizmendi and Serra40. The reaction mixture in addition per the method given34. The extracted total lipid yield to cell free extract in a total volume of 1 ml contained 0.5 weight was determined using weighing balance. Cell dry M Tris-HCl (pH 7.0), 1.6 mM NaNO3, and 4µmol Methyl weight was also determined by filtering out 5 mL of the viologen. The reaction was then allowed to proceed for culture using Whatman GC/F filters, dried overnight at 15 mins at room temperature with the addition of 250 60 °C and measured gravimetrically. mM of sodium dithionite and bicarbonate buffer. Finally the reaction was terminated by shaking vigorously in a Preparation of cell free extracts vortex shaker until blue color developed. The amount of th - - On the 16 day of inoculation, the cyanobacterial cells nitrite formed (expressed as nmol NO2 consumed min treated with different concentration of Hg2+ along with 1mg-1 protein) was obtained from the standard curve for the control group were harvested by centrifugation at which the absorbance was taken at 540 nm following 10,000g for 10 minutes at 4˚C and washed twice in the method of Snell and Snell 41. Phosphate Buffered Saline (1X PBS, pH 7.4). The pellet was resuspended in 5mL 1X PBS followed by sonication Heterocyst frequency using Riviera, Germany at 120 W for 5mins in ice bath. The heterocyst frequency was calculated per 100 The whole cell lysate was subjected to centrifugation at vegetative cells on the 9th day of inoculation using 15, 000g for 10 min. The supernatant thus obtained was Olympus 40X light microscope with a digital camera. used for estimation of protein and for further enzymatic assays. Superoxide dismutases (SOD) assay (E C 1.15.1.1) SOD activity was determined as per the established Protein estimation protocol of Beauchamp & Fridovich42. One unit of SOD Total protein content of the cell free extract was activity is defined as the amount of enzyme that caused estimated as suggested by Lowry et al.35 using Bovine 50% inhibition of the rate of reduction of Nitro Blue Serum Albumin (BSA) as standard. Tetrazolium (NBT) under the assay conditions and expressed in units/mg protein. Determination of electron transport system activities Proline quantification For measurement of photochemical activity estimated in Two mL of cell free extract was taken in a test tube and terms of O2 exchange expressed as nmoles O2 evolved 2 mL each of glacial acetic acid and acidic ninhydrin or consumed µg-1 Chl. a hr-1, the cell free extract was were added. The mixture was heated in a water bath at suspended in reaction buffer (25 mM HEPES-NaOH, pH 100°C for 1 hour and the reaction was stopped by 7.5, 20 mM NaCl) and used for measuring oxygen placing the test tubes to an ice-bath. 4 mL toluene was evolution and oxygen consumption as described by then added to the reaction mixture and stirred for about Robinson et al.36. 30 seconds; the toluene layer was separated and warmed to room temperature. Absorbance was Glutamine Synthetase (transferase) (GS) assay (EC measured at 520 nm and the amount of proline content 6.3.1.2) was quantified from the standard curve obtained using The GS enzyme was assayed using the method of proline as reference43. Sampaio et al.37. The reaction mixture containing the enzyme extract in a total volume of 1 ml contained 40 STATISTICAL ANALYSIS mM Tris-HCl (pH 7.0), 3 mM MnCl2, 20 mM K-arsenate, 0.4 mM Na-ADP, 60 mM Hydroxylamine neutralize with The data generated from the experiments were 2N NaOH, 30 mM Glutamine. The reaction was allowed ○ analysed using one-way analysis of variance (ANOVA) to proceed for 10 mins at 37 C in dark and finally with Tukey-Kramer procedure. Readings were terminated by adding 2.0 ml of stop mixture (10 % considered significant when p- value was <0.05. FeCl3, 24 % TCA, 6 N HCl, double distilled water). The ɤ-Glutamyl Hydroxamate thus formed was estimated by RESULTS measuring the absorbance at 540 nm and the amount of

ɤ-Glutamyl Hydroxymate formed (expressed as nmol of -1 -1 Morphological examination followed by molecular ɤ-Glutamyl Hydroxymate formed min mg protein) was identification using phylogenetic analysis of the 16S determined from the standard curve. rRNA sequence data, identified the two cyanobacterial

species as Nostoc muscorum and Anabaena variabilis Nitrate Reductase assay (NR) (E C 1.7.1.1) of the family Nostocaceae (Figs. 1 and 2). Their 16 NR activity was assayed following the method of 38 rRNA sequences were submitted to GenBank, NCBI Manzano et al. with slight modification as described by 39 and their respective accession numbers. (KR709143 Bagchi and Singh . The enzyme activity was measured and KR709140) were obtained. Both the species were spectrophotometrically following the appearance of successfully cultivated under the aforementioned culture nitrite using methyl viologen, chemically reduced by conditions and used for further analysis. The growth dithionite as electron donor. The amount of nitrite curve showed an overall increase in growth behaviour formed was determined from the standard curve and th - -1 -1 among the control group (i.e., 0.03 on 0 day to 0.9 on was expressed as nmol NO formed min mg protein. 2 16th day) however, the cultures treated with graded

concentrations of mercuric (Hg2+) ions (0.2-1.0µM),

depicted certain decline in the growth status despite

taking similar initial inoculums for both the species (Figs. This article can be downloaded from www.ijpbs.net B-13

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3, a and b). The cultures of N. muscorum and A. Hg2+ (II) stress on various enzymes entailed in nitrogen variabilis appeared to have the ability to survive the metabolism, the present study emphasized on the heavy metal treatment up to the concentration of 0.8µM; activities of GS, NR, NIR and SOD enzymes. At LC50, but at the concentration of 1.0µM, the growth seemed the GS activity was reduced by 45.1% and 44.6% in N. severely inhibited, leading to cell lysis and death. The muscorum and A. variabilis respectively. Similarly, the ‘Chl. a’ and phycobilisomes content estimation also NR activity also showed a decrease of 44.0% for N. suggested similar diminishing pattern among the treated muscurom and 58.0% for A. variabilis. The NIR activity group in comparison to the control groups (Figs. 4, 5a illustrated a greater percentage decrease of 94.2% and and b). Evidently, from the growth curve, ‘Chl. a’ and 93.5% for N. muscorum and A. variabilis, respectively as phycobilisomes content, the LC50 value was found to be compared to the control group (Table 2). While GS, NR 0.6µM for the treated cultures and thus used for further and NIR showed overall decrease in their respective analyses and enzymatic assays. At LC50, the average activities, the SOD activity on the contrary showed lipid content (mg/L) measured at the stationary phase drastic increase in both N. muscorum and A. variabilis. (16th day), was found to be 134±15 (control) and For instance, in N. muscorum the SOD activity was 38.75±1.5 (treated) for N. muscorum and 148±18 found to be 0.12 U/mg on the 8th day which increased to (control) and 56.77±5.0 (treated) for A. variabilis. In 1.6 U/mg on the 16th day of inoculation. In case of A. correspondence with the results of lipid content, the variabilis, 0.5 U/mg SOD activity was observed on the biomass quantification (mg L-1day-1) as well showed 8th day which elevated to 2.0 U/mg on the 16th day (Fig. decrease productivity of 6.25±1.5 (N. muscorum) and 6). A similar increase was also noticed in the proline 10.29±0.5 (A. variabilis) in comparison to their content of treated cultures. A marked increase of 11.8 respective controls with biomass content of 25±1.5 (N. and 15.4 µmol proline/g fresh weight was observed for muscorum) and 35±1.5 (A. variabilis) (Table 1). The N. muscorum and 16.0 to 24.0 µmol proline/g fresh th th electron transport activity estimated in terms of O2 weight for A. variabilis on the 8 and 16 day of exchange showed a significant drop at 81% and 85.5% inoculation, respectively as compared to the untreated for N. muscurom and A. variabilis, respectively as group (Fig. 7). compared to the control (Table 2). To study the effect of

Figure 1 Micro Pictograph of the Cyanobacteria species used in the study; (a). N. muscorum and (b). A.variabilis.

Figure 2 Phylogenetic tree of N. muscorum and A. variabilis and other related cyanobacterial

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species constructed using MrBayes v3.1.2.

Figure 3a Effect of different concentrations of mercury (Hg2+) on growth behaviour of N. muscorum taken after every two days. The values represents mean ± standard deviation (SD) from two independent experiments with two replicates each.

Figure 3b Effect of different concentrations of mercury (Hg2+) on growth behaviour of A. variabilis taken after every two days. The values represents mean ± standard deviation (SD) from two independent experiments with two replicates each.

Figure 4 Effect of mercury (Hg2+) at different concentrations (0.2-1.0µM) on Chlorophyll “a” concentrations (mg/L) of N. muscorum and A. variabilis. Measurements were carried out in duplicates on the 12th day. The values represents mean ± standard deviation (SD) from two independent experiments with two replicates each.

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Figure 5a Quantification of phycobilisome content (mg/L) of N. muscorum following treatments with graded concentrations (0.2-1.0µM) of mercury (Hg2+). Measurements were carried out in duplicates on the 12th day. The values represents mean ± standard deviation (SD) from two independent experiments with two replicates each.

Figure 5b Quantification of phycobilisome content (mg/L) of A. variabilis following treatments with graded concentrations (0.2-1.0µM) of mercury (Hg2+).Measurements were carried out in duplicates on the 12th day. The values represents mean ± standard deviation (SD) from two independent experiments with two replicates each.

Figure 6 2+ SOD activity following treatment with respect to Hg (LC50=0.6µM) on N. muscorum and A. variabilis taken on the 8th and 16th day. The values represents mean ± standard deviation (SD) from two independent experiments with two replicates each.

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Figure 7 2+ Proline quantification following treatment with respect to Hg (LC50=0.6µM) on N. muscorum and A. variabilis taken on the 8th and 16th day. The values represents mean ± standard deviation (SD) B. from two independent experiments with two replicates each.

Table 1 2+ Effect of Hg ions at LC50=0.6µM on dry weight, biomass productivity and lipid content of N. muscorum and A. variabilis taken on day 16th after inoculation. The values represent mean ± standard deviation (SD) from two independent experiments with two replicates each.

Parameters N. muscorum A. variabilis Concentration of Hg2+ Control 0.6µM Control 0.6µM Incubation days 16 16 16 16 Dry weight (g/L) 0.4±1.0 0.1±0.01 0.45±0.015 0.16±0.01* Biomass productivity (mg/L/Day) 25±1.5 6.25±1.5* 35±1.5 10.29±0.5* Average lipid content (mg/L) 134±15 38.75±1.5* 148±18 56.77±5.0* Values are mean±SD; (n=2), *p Value<0.05 when compared with control

Table 2 2+ Effect of different concentrations of Hg ions at LC50=0.6µM on different enzymes involved in nitrogen metabolism of N muscorum. and A. variabilis taken on day 16th after inoculation. The values represent mean ± standard deviation (SD) from two independent experiments with two replicates each.

N. muscorum A. variabilis Characteristics - - - - NO3 NO3 + Hg NO3 NO3 + Hg O2 evolution 109.4±19 21.1±0.12 115.0±15.0 25.1±0.12 O2 consumption 69.4±16 10.1±2.9 79.5±10.0 15.1±2.9 Heterocyst Frequency (%) 14±0.7 5±0.2* 10±0.65 4±0.3* Glutamate synthetase 323.0±0.2 177.3±8.2* 321.80±13.2 177.3±8.2* Nitrate reductase 30.2±3.2 16.9±4.2 35.2±3.2 14.9±4.2 Nitrite reductase 152.3±22 8.8±1.6* 120.3±29 7.5±2.6* Values are mean±SD; (n=2), *p Value<0.05 when compared with control

DISCUSSION demonstrated. The adverse effect of Hg2+ on N. muscorum and A. variabilis was certain from the Cyanobacteria are known to grow abundantly under diminishing growth pattern, decrease in photosynthetic limited growth requirement 1. As scavengers of heavy pigments (i.e., ‘Chl. a’ and phycobilisomes) as well as reduction in biomass productivity. Earlier reports have metals, cyanobacteria have been reported to possess 2+ the ability to absorb, filter and transform various also suggested the toxic effects of Hg by inhibiting the 44-47 photosynthesis, nutrient uptake and nitrogen nutrients and metal contaminants . Mercury is a toxic 17,51 heavy metal and a well known environmental pollutant, metabolism thereby affecting cyanobacterial growth . well recognized for its recalcitrance and biomagnification The decline in growth behaviors can also be attributed properties once entered into the food chain 8, 48-50. As to the decrease in overall lipid content, as observed in such, it is of utmost significance to study the effect of the present study. Probably, the exposure to heavy heavy metals on growth and physiological activity on metals must have resulted in deterioration of membrane cyanobacteria. In the present study, the effect of the lipids which can be explained from the fact that heavy metal, mercury, on the growth behaviour and cyanobacterial cell wall component i.e., peptidoglycan biochemistry of the two freshwater cyanobacterial and membrane lipids as well as proteins are known to species namely N. muscorum and A. variabilis was serve as important metal binding sites and thus causing

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Int J Pharma Bio Sci 2019 July; 10(3): (B) 11-21 increased lipid peroxidation and protein structural quantification in this study showed a remarkable deformation 52-54. The present study also demonstrated increase in response to mercuric ions mediated stress drastic reduction in the electron transport activity which also correlates with the previously established estimated in terms of O2 exchange in the treated cells studies on SOD and proline accumulation against heavy 65, 68-69 (expressed as in terms of nmoles of O2 evolved or metal stress in cyanobacteria . consumed). This reduction can be correlated with the drop off in photosynthetic pigments along with CONCLUSION membrane perturbation, consequently hampering the photosynthetic efficiency. Mercury induced inhibition of The present study demonstrated the adversity of heavy electron transport chain and photosynthetic activity has 8,17,27,55 metals, in particular mercuric ions, contamination on the also been reported in earlier studies . Glutamine phytoplanktonic cyanobacteria and environment on a synthetase is an important enzyme involved in wider scale. The present study would also serve as a assimilation of ammonia as well as utilization of nitrate, 56-61 basis for evaluating as well as maintaining the levels of nitrite and urea as a source of nitrogen for the cells . mercury below the threshold concentration, thus, Biochemical studies on enzymes implicated in nitrogen protecting the environment. metabolism showed significant decrease in three of the vital enzymes i.e., GS, NR and NIR, responsible for the FUNDING/ACKNOWLEDGEMENT metabolism of nitrogen. This decrease can probably be the outcome of the stress mediated by the heavy metal The authors extend their gratitude to the Department of contamination, in particular mercuric ions, which is Biotechnology (DBT), Govt. of India for funding the known to inhibit enzymes, thereby obstructing various 62 project, sanctioned to Dr. Samrat Adhikari (Sanction metabolic processes . This is also in accordance with Order No. BT/22/NE/2011), Advanced Level Biotech the decline in the heterocyst frequency, which are the Hub, St. Edmund’s College, Shillong. specialized nitrogen fixing cells of cyanobacteria. Owing to the inhibition of the essential enzymes regulating uptake and assimilation of nitrogen, the heterocyst AUTHORS CONTRIBUTION STATEMENT differentiation likely ceased in the cyanobacterial filaments. Being photoautotrophic, cyanobacteria are Dr. S. Adhikari, Dr. S. Sharma and Mr. Y. Negi known to inevitably produce reactive oxygen species formulated and designed the experiment. Mr. F.J. Laloo (ROS). Besides, the production of ROS gets further and Miss B. Thyrniang performed the experiments under elevated on exposure to various heavy metals including my supervision along with Dr. S. Sharma. The mercuric ions63,64. However, to counteract the oxidative evaluation, analyses and interpretation of data was done damage, certain defence mechanisms have been again by Dr. S. Adhikari, Dr. S. Sharma and Mr. Y. Negi. evolved in cyanobacteria, which include production of The entire manuscript was drafted framed and edited by cascade of antioxidants65,66. One of the antioxidants, Dr. S. Sharma and Dr. S. Adhikari. SOD, is known to act as a first line of defence in this cascade of events 66,67. Other than this, proline is CONFLICT OF INTEREST another important indicator, known to have a significant role in combating the environmental stress in plants as Conflict of interest declared none. well as micro organism, including cyanobacteria68. The result of the SOD enzyme assay and proline

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