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Characterization of Struvite Produced by an Algal Associated Agarolytic Bacterium Exiguobacterium Aestuarii St. SR 101

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Characterization of Struvite Produced by an Algal Associated Agarolytic Bacterium Exiguobacterium Aestuarii St. SR 101 Leela et al. J Pure Appl Microbiol, 13(2), 1227-1234 | June 2019 Article 5588 | https://dx.doi.org/10.22207/JPAM.13.2.64 Print ISSN: 0973-7510; E-ISSN: 2581-690X RESEarcH ARTICLE OPEN ACCESS Characterization of Struvite Produced by an Algal Associated Agarolytic Bacterium Exiguobacterium aestuarii St. SR 101 Sheeja Leela1, Jayappriyan Kothilmozhian Ranishree2*, Ramesh Kannan Perumal2 and Rengasamy Ramasamy2 1Shrimathi Devkunvar Nanalal Bhatt Vaishnav College for Women, Chromepet, Chennai - 600 044, India. 2Centre for Advanced Studies in Botany, University of Madras, Gundy Campus, Chennai - 600 025, India. Abstract Biomineralization phenomenon of bacteria proved to have various biotechnological and environmental applications. Production of magnesium ammonium phosphate (struvite) crystals by the agarolytic bacterium Exiguobacterium aestuarii St. SR 101 isolated from red seaweed, Gracilaria corticata was reported for the first time in the present study. Struvite crystallization occurred in the agar culture medium in the presence of the bacterium. Crystal nucleation and growth occurred apparently as a consequence of the localized ion supersaturation, produced by the microbial metabolites and also by the microbial supply of heterogeneous nuclei resulted in crystallization. The crystals were visible between 10 to 15 days after inoculation. The crystal structure of the struvite characterized by optical microscopy, IR spectroscopy, thermogravimetry, powder X-ray diffractometry, and single crystal X-ray diffractometry. The orthorhombic crystal is with the space group Pmn21 and unit-cell parameters a = 6.9447 Å, b =6.1329 Å, c = 11.2026Å. Exiguobacterium aestuarii St. SR 101 showed to have the capacity of producing struvite based fertilizer by bioremediation of industrial phosphate wastes. Keywords: Exiguobacterium aestuarii; biomineralization; struvite; agarolytic; X-ray diffractometry. *Correspondence: [email protected] (Received: 02 April 2019; accepted: 20 May 2019) Citation:Sheeja Leela, Jayappriyan Kothilmozhian Ranishree, Ramesh Kannan Perumal and Rengasamy Ramasamy, Characterization of Struvite Produced By an Algal Associated Agarolytic Bacterium Exiguobacterium aestuarii St. SR 101, J Pure Appl Microbiol., 2019; 13(2): 1227-1234. doi: 10.22207/JPAM.13.2.64 © The Author(s) 2019. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Journal of Pure and Applied Microbiology 1227 www.microbiologyjournal.org Leela et al. J Pure Appl Microbiol, 13(2), 1227-1234 | June 2019 | DOI 10.22207/JPAM.13.2.64 INTRODUCTION of interest, it was thoroughly investigated Biomineralization refers to a process by mineralogists, chemists, physicians, and which involves the formation of minerals. environmentalists, who studied it from different Bacterial activity related to the precipitation of points of view and described many physical a wide range of minerals including carbonates, and chemical properties. The crystal structure oxides, phosphates, sulfides, and silicates1. The of struvite by Whitaker and Jeffery belongs to extracellular precipitation processes of mineral the orthorhombic space group Pmn21 with two compounds by bacteria are due to metabolic molecules in a unit cell9. There are many structural activity that favors the physicochemical conditions similarities between struvite type materials, which resulting in the formation of biominerals. In have been of great interest because of their natural biomineralization, bacterial metabolic broad and important biological, agricultural and enzymes can generate supersaturated conditions. industrial implications10,11,12. For example, Mg2+ Moreover, it is an established and experimentally cations in these compounds are all coordinated proven fact that bacterial structures participate in octahedrally by six H2O molecules whose H atoms 3- the extra or intracellular accumulation of inorganic are strongly bonded to oxygen atoms in (PO4) 2,3 material . The significant role of bacterial bio- groups. No H2O molecules are shared between mineralization is to deliver structures that serve Mg (H2O)6 octahedra. Another common feature as substrates for heterogeneous crystal nucleation among the struvite type phosphate compounds (with lower energy barriers than homogeneous is the face-sharing between PO4 tetrahedra and nucleation) and stereochemical arrangements of Mg (H2O)6 octahedra through hydrogen bonding, the mineral components. Therefore, providing the although the number of shared faces may vary necessary elements and conditions and the mere from structure to structure. The present study existence of bacteria would themselves suffice reported a phosphate containing, biologically for biomineralization processes4,5. Interaction formed crystal, struvite, which has the chemical between soil bacteria and minerals reported composition MgNH4PO4.6H2O, from the agarolytic having many bio-technological and environmental bacterium E. aestuarii St. SR 101 isolated from the applications6,7. red seaweed, G. corticata. Among phosphate containing bio- minerals, struvite, MgNH4PO4. 6H2O, has attracted MATERIALS AND METHODS considerable attention because of its common Isolation and identification of the bacterium occurrence in widely diverse environments, such The agarolytic bacterium E. aestuarii St. as bat droppings, decomposed foods, a part of SR 101, isolated from red seaweed G. corticata biofilms that develop inside the pipelines in water collected from Kovalam coast, near Chennai, treatment facilities. Crystallization of nitrogen India. The homogenized algal sample inoculated and phosphorous in the form of struvite has been in medium containing 1.5% agar, 0.05% yeast successfully used for nutrient removal and for extract, 0.5% peptone, 3% NaCl, 0.06% NaH2PO4, obtaining a valuable fertilizer. The mineral struvite and 0.01% CaCl2 at room temperature (24 - 27°C) was named already in 1845 after the Russian and under static condition. Within 3 days, the agar Mineralogist, Heinrich Von Struve. According medium became liquefied due to the production to Robinson’s proposals, struvite production of agarolytic enzyme agarase. After 10 to 15 days could be the consequence of a combination of crystals were present in the medium. The crystals ammonium ions formed by the metabolism of were harvested by washing thoroughly using nitrogenous compounds with phosphate and double distilled water, air dried and stored in glass magnesium present in the environment8. Several vials. The isolated bacterial stain characterized as soil-inhabiting genera like Azotobacter, Bacillus, described previously6,13. Myxococcus, and Pseudomonas have been studied Effect of four different nutrients on the production for their potential to form struvite2. Medical of struvite reports reveal that struvite is well known from the Four different ingredients of the basal bladder and urinary concretions. medium viz., Peptone, yeast extract, NaH2PO4 Due to its importance in many fields and MgSO4 were amended at seven different Journal of Pure and Applied Microbiology 1228 www.microbiologyjournal.org Leela et al. J Pure Appl Microbiol, 13(2), 1227-1234 | June 2019 | DOI 10.22207/JPAM.13.2.64 concentrations (Peptone- 0.4, 0.5, 0.6, 0.7, 0.8, RESULTS AND DISCUSSION 0.9, 1%; Yeast extract - 0.04, 0.05, 0.06, 0.07, 0.08, Culture conditions ofE. aestuarii 0.09, 0.1%; NaH2PO4 -0.05, 0.06, 0.07, 0.08, 0.09, The bacterium E. aestuarii St. SR 101 0.1% and MgSO4 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and grown in different cultural conditions concerning 1%), and the bacterium was inoculated separately the yield of struvite revealed the following under respective concentrations in the medium observations. The amount of struvite produced without CaCl2, NaH2PO4 and MgSO4. The flasks by the bacterium was directly proportional to the were periodically observed for the presence concentration of the ingredients tested. Increasing of crystals, under room temperature at static concentrations of the combination of peptone, condition. The crystals were collected after 15 yeast extract, NaH2PO4, and MgSO4 increased the days and transferred to distilled water and washed yield of struvite under the static condition at room free of impurities. They were air dried, and the temperature. The organism grown in the medium dry weight was recorded. Uninoculated culture contained 0.1% yeast extract, 1.0% peptone, 3.0% medium was considered as negative control. NaCl, 0.2% NaH2PO4, and 1.0% MgSO4 showed Characterization of crystals a maximum of 145.0 mg/100 mL, which was 5 The crystals obtained from the culture fold more than positive control (30 mg/100 mL) medium were ground into a fine powder and (Table 1). In the present study, no precipitation analyzed under powder X-ray diffraction using an occurred in the negative control. These results are X’pert PRO (PAN analytical) diffractometer with similar to those observed on moderately halophilic nickel-filtered CuK radiation in the 2θ range 5-80° bacteria14,15. with scan step size 0.02°/s. Fourier transform The present bacterium did not produce infrared of the crystals was recorded in the range struvite when the medium devoid of any one of -1 of 400-4000 cm using Perkin – Elmer RX1 FT-IR the ingredients viz., CaCl2, NaH2PO4 and MgSO4. spectrophotometer
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