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 This article can be downloaded from www.ijpbs.net B-11 Int J Pharma Bio Sci 2019 July; 10(3): (B) 11-21 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. This article can be downloaded from www.ijpbs.net B-12 Int J Pharma Bio Sci 2019 July; 10(3): (B) 11-21 Lipid and cell dry weight