Physicological Characterization of M.VITRJS5

Physicological Characterization of M.VITRJS5Monica Samal, Teena Patra, Suthindhiran KAbstractMagnetospirillium sp. is a thousand negative -proteobacterium which has the ability to move towards the geo magnetized field lines of Earths magnetic field. The convocation of bacterium is collectively c completelyed Magnetotactic bacteria (MTB). MTB produce intracellular organelles c eached magnetosomes which swear out them in its search for optimal living marks in complex env urgement. MTB is a gradient requiring microorganism that atomic number 18 difficult to replicate in ontogenesis medium in the laboratory. In this work the physiological characterization of MTB attain Magnetospirillium sp. VITRJS5 was done as wholesome as a modified festering medium for the emergence of Magnetospirillium sp. VITRJS5 was developed. The media components be studied base on the physiological diagnostics of the bacteria. The role of negatron presenters, electron acceptors, nitrogen charac ters, carbon arisings, urge on containing compounds, phosphates, cut down agents, on the out harvesting of the bacteria in the MS1 media was studied. The produce of the bacteria in the novel modified MS media was analysed and compared with commercial MTB media such as MSGM (Magnetospirillum harvest-tide media) and Schulers oxygen mho gradient media. An increase in growth is discover in the modified MS media compared to the commercially available media.1. asylumMagnetotactic bacteria are fastidious microorganisms that re acquaint a morphologically and physiologically various group of bacteria. The front man of intracellular, membrane bound nano-sized magnetic particles called magnetosomes assists the bacteria in sustaining an optimal position in redox gradient habitats. Magnetosomes are crystals of iron mineral which consist of either iron oxide (Fe3O4) or iron sulphide (Fe3S4). A combination of aerotaxis, magnetotaxis and chemotaxis helps MTB to move away from oxygen in s urface water, thus directing them in primary(prenominal)taining an optimal position in and around the oxic-anoxic interface (OAI). Magnetospirillum sp. is the most(prenominal) studied and well understood group of bacteria among MTB. Magnetospirillum sp. was mostly isolated from fresh water habitats and many handsome civilizations were available in many laboratories around the world.Magnetotactic Bacteria (MTB) species have characteristic organelles called magnetosomes that contain magnetic crystals. These magnetosomes give them the ability to move in resolution to the earths magnetic field (magnetotaxis). MTB were first observed by Salvatore Bellini in the year 1963 and collected from antithetic freshwater environments near Pavia, Italy 1,2. The most studied species of Magnetospirillum genus are species of Ms. gryphiswaldense 5, Ms. Magneticum7, Ms. magnetotacticum 8. The MTB strains isolated from marine or brackish environments hold the coccoid strains MO-19 , Magnetococcus marinus strain MC-110 and Magnetofaba australis (IT-1)11 the vibrioid strain Magnetovibrio blakemorei strain MV-112 and the spirilla Magnetospira thiophila (MMS-1)13 and Magnetospira sp. strain QH-214 all of which belong to the Alphaproteobacteria. Most magnetotactic bacteria (MTB) are known to be phylogenetically similar to the Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria classes of the Proteobacteria and the Nitrospirae phylum with response to magnetosome formation6.Magnetotactic bacteria are known to thrive in sediments or chemically stratified water columns, where they chance mostly at the oxic-anoxic interface (OAI), the anoxic regions of the habitat, or some(prenominal) 3. Although the detection of these in samples is comparatively simple 4, magnetotactic bacteria are a fastidious group of prokaryotes, and supererogatory culture conditions are necessary for their isolation and cultivation. in that respect is clear march that the availability and div ersity of MTB in distinguishable environments is majorly influenced by salinity and also, iron availability, temperature and soaking ups of sulfur and nitrogen compounds are also seen to be clearly chief(prenominal) in the distribution of MTB. The respiratory forms of metabolism of genus Magnetospirillum are either chemoorganoheterotropic, apply organic acids and carbon source or electron bestowers. Mostly Magnetospirillum sp. fitting of autotrophic and mixotrophic growth and utilize treat as alternative storage oxygen acceptor. Magnetospirillum are obligate microaerophile that requires oxygen even when nitrate is present in the medium.The present study,examined the phylogenetic affiliations and physiological characteristics of Magnetospirillium sp. VITRJS5. To characterize physiological parameters, microaerophillic mickle experiments were performed to determine the following (1) growth pH and temperature ranges, (2) Optimum electronic giver/carbon source and electron acce ptor. (3) optimal nitrogen and phosphate source (4) optimal Iron source (5) optimum reducing agent/ sulphur source. Once the preliminary screening of the compounds over diametrical meannesss of the nutritional requirements is firm to develop a modified growth media.2. Methods and materials2.1. Media and microorganismMagnetospirillum VITRJS5 (Accession number KM289194) is a novel MTB isolated from the fresh water sediment. They are spiral in shape and move towards the earths magnetic field. They have arctic flagellum at each end of the cell. The strain was obtained from Marine biotech and biomedicine lab, VIT University. The bacteria were sub-cultured in Magnetospirillum growth media (MSGM) under microaerophillic conditionsMagnetospirillum VITRJS5 was initially grown in previously standardized minimal media MS1 to analyse the growth conditions in the presence of electron conferrers and acceptors. MS1 media per litre containing K2HPO4 850mg, Na2HPO4 850mg, NH4H2PO4 500mg, Mg SO4 100mg, FeSO4 7H2O 5mg, Na2MoO4 0.2mg, EDTA 10mg and Mineral solution 10ml electron giver and acceptor has to be added fit inly. From the earlier studies, the optimum temperature and pH for the growth of Magnetospirillum is 28C and 7.5 pH.2.2 aim of electron acceptors and ingress of selected acceptorFor this, 50 ml of MS-1 media was added in five blood serum bottles and 50 mg of electron donor which in this case is atomic number 11 acetate (CH3COONa) was added to each bottle. Then 50 mg of five disparate acceptors were added in different bottles. These five acceptors are nitrite, selenite, nitrate, thiosulphate and sulphate. Nitrogen was sparged to the media to make it anaerobic, unsympathetic with butyl rubber stopper and sealed with aluminium caps. All the anaerobic culturing was performed according to Hungate 195015. The Magnetospirillum VITRJS5 was inoculated to MS1 media. These bottles were kept for overnight pensiveness in a shaker incubator. directly the diff erent niggardlinesss of selected acceptor is determined .It is done for concentration of 5mM, 10 mM , 15mM, 20 mM and 30 mM . Steps for anaerobic culture was repeated. The absorbance is checkered in UV-VIS spectrophotometer at wavelength of 595 nm.2.3. Determination of electron donors and concentration of selected donorFor this, 50 ml of MS-1 media was added in five serum bottles and 50 mg of electron acceptor which in this case is nitrate was added to each bottle. Then different donors were added in different bottles. These donors are methanol (250 l), ethanol( 50l), glycerol (50l), pyruvate (82.5 mg/ 15mM) , citrate (221 mg/ 15mM),succinate (202.6 mg/15mM), lactate(110l/15mM), glucose (135.12 mg/15mM), sucrose (256.9 mg/15mM), ferric chloride (121.65 mg/15mM)and thiosulphate (186.1 mg/15mM).Nitrogen gas was bubbled into each bottle for anaerobic condition and sample VITRJS5 strain was inoculated in each bottle. These bottles were kept for overnight incubation in a shaker incub ator. Narrowing down the number of donors based on the growth of sample. Different concentrations of selected donor are determined. Selected donors are added in different concentration of 5mM, 10mM, 15mM, 20mM and 30 mM. The steps for anaerobic culture are repeated. The absorbance is checked in UV-VIS spectrophotometer at 595 nm.2.4. Determination of nitrogen source for optimum growthMS-1 Media was prepared without nitrogen source (MS-1 media without NH4H2PO4 ) and with the selected concentration of electron donor and acceptor. For this, activity of NH4H2PO4 was checked in different concentrations (5mM, 10mM, 15mM, 20mM and 30 mM) against different concentrations of NH4Cl(5mM, 10mM, 15mM, 20mM and 30 mM).The above described procedure of anaerobic growth was repeated. The absorbance was checked at a wavelength of 595 nm.2. 5. Determination of reducing for optimum growthMS-1 media with selected concentration of donor and acceptor was prepared. For this, activity of thiol glycolate wa s checked in different concentrations of 5mM, 10mM, 15mM, 20mM and 30 mM against cysteine-HCl 4% at concentrations of 50 l , 100 l, 150 l, 200 l and 500 l. The procedure for anaerobic culture growth was repeated. The absorbance was checked at 595 nm.2.6. Determination of iron source for optimum growthMS-1 media with selected concentration of donor and acceptor and without FeSO4 . 7H2O was prepared. For this, activity of ferrous sulphate was checked in different concentrations of 5mM, 10mM and 15mM against ferric citrate at concentrations of 0.5mM, 1.0mM and 1.5mM and against ferric quinate at concentrations of 0.5mM, 1.0mM and 1.5mM. The procedure for anaerobic culture growth was repeated. The absorbance was checked at 595 nm.3. Result3.1 Electron acceptor and its concentration for optimum growthOn incubating the strain M.VITRJS5 in the MS1 media with the conglomerate electron acceptors, we obtained various results as seen in elude1.From the table, we find that Nitrite, Nitrate an d Selenite good electron acceptors for the strain. however we negate the role of Nitrite and Selenite as in nitrite, growth is observed after 2 weeks and selenite is considered toxic. Thus, Nitrate is considered as the most probable electron donor.Next, different concentrations of Nitrate are added to MS1 media to see which concentration of nitrate supports maximum growth (as seen in Table 2 and fig.1)Table 1. Tabular commission of growth with various electron acceptor.Table 2. Tabular standard of absorbance of different concentration of nitrateFig.1 Graphical commission of the growth with different concentration of nitrate From the above table and fig.1, it is seen that 5mM of nitrate shows maximum growth on all three days. Thus, there is optimum growth seen in 5mM serum bottle.3.2. Electron donor and its concentration for optimum growthOn incubating the strain M.VITRJS5 in the MS1 media with the various electrondonors, we obtained various results as seen in table1.From the tab le 3, it is seen that growth of M.VITRJS5 is more pronounced with electron donors Ethanol, Glycerol, Pyruvate, Succinate, Lactate, Glucose and saccharose along with Acetate which was already being used as an electron donor for some early(a) MTBs. Among these, Pyruvate, Sucrose and Acetate showed almost double the growth as seen in other electron donors. But we negate Sucrose as an electron donor as it is not economical.Later, different concentrations of Nitrate are added to MS1 media to see which concentration of nitrate supports maximum growth (as seen in Table 2 and fig.1)Table3. internal theatrical performance of growth with various donorsTable 4. Tabular Representation of absorbance of growth with different concentrations of pyruvate Fig 2. Graphical standard of absorbance of growth with different concentrations of pyruvate As seen in the above two graphs and tables, 30mM concentration of pyruvate shows highest growth in comparison with the other concentrations of acetate a nd pyruvate.3. Nitrogen source and its concentration for optimum growthThe adjacent step is incubation of M.VITRJS5 strain in MS-1 media(without NH4H2PO4) with ammonium ion chloride and Ammonium total heat phosphate to determine the better nitrogen source.Fig 4. Graphical representation of absorbance of growth with different concentrations of NH4H2PO4As seen in figure 4, 5 and tables 6,7, it is observed that the best growth is seen in bottles of Ammonium hydrogen phosphate(NH4H2PO4) rather than that of Ammonium chloride(NH4Cl). The most consistence growth is seen in 20 mM concentration of NH4H2PO4 as compared with other concentrations.3.4. Carbon sourceSince it has already been confirmed that pyruvate acts as the best electron donor, the same can act as a carbon source.3.5. Phosphorus sourceAmmonium hydrogen phosphate acts both as a nitrogen source as well as phosphorys source.3.6. Sulphur sourceNext the M.VITRJS5 strain was incubated in prepared MS-1 media with sodium thioglycol ate and cysteine HCl 4% to determine the better sulphur source.Fig 6. Graphical representation of absorbance of growth with different concentrations of sodium thioglycolateAs seen in fig 6,7 and table 8,9, 50l concentration of Cys HCl shows highest growth as compared to the other concentrations. There was no growth in other concentrations due to the reducing properties of Cysteine HCl and atomic number 11 thioglycolate. MTBs are microaerophilic and hence, would not grow in anaerobic condition. Sulphur is a reducing agent and hence, would remove oxygen species from the media. Therefore, there is less or minimal growth of the bacteria.3.7 Iron source Table 10. Tabular representation of absorbance of growth with different concentrations of Ferric citrateFig 8. Graphical representation of absorbance of growth with different concentrations of Ferric citrateTable 11. Tabular representation of absorbance of growth with different concentrations of quinateFig 9. Graphical representation of absorbance of growth with different concentrations of Ferric quinate3.8 Comparison of saucy media with existing media4. DiscussionAs already mentioned above, Magnetotactic bacteria (MTBs) are microaerophilic bacteria which have the ability to biomineralise membrane-encased, single-magnetic-domain mineral crystals (magnetosomes) and hence, pillowcase the cell to orientate along the Earths geomagnetic field. These mostly occur in aquatic bodies ranging from saline to freshwater environment.Our basic objective in this study was to test the growth of the microaerophilic strain using different concentrations of electron donor, electron acceptor, nitrogen source, carbon source, phosphorus source, sulphur source, iron source, and so forthFrom the study, the following observations have been noted downThe strain M.VITRJS5 was seen to grow well in 5mM concentration of Nitrate (electron acceptor), 30mM concentration of Pyruvate (electron donor and carbon source) and 20mM concentration of Amm onium hydrogen phosphate (nitrogen source and phosphorus source).Also, the growth of the strain was seen to be minimalized in case of Sodium thioglycolate and Cysteine HCl as these are pixilated reducing agents and thus, would reduce the amount of oxygen species in the media. As mentioned above, these bacteria are microaerophilic and hence, would not grow in sulphur sources (except in 15mM Sodium Thioglycolate and 50l Cysteine HCl).The comparison of the present media for Magnetospirillum growth media (MSGM) Schler Magnetospirillum isolation media and the new prepared media was done. The new media contained sodium nitrate as acceptor, sodium pyruvate as electron donor and carbon source, ammonium hydrogen phosphate as nitrogen and phosphorus source, ferric citrate as iron source and cysteine HCl as sulphur source.Magnetotactic bacteria has the ability to produce magnetosomes which has stimulated and incite a new field of research involving scientific and biomedical applications of M TB and diverse commercial which could be improved using magnetic nanocrystals. But the main drawbacks to the application of magnetosomes involves the fastidious nature of MTB related to the growth. Thus,it is difficult to culture them on a large scale, and it is important to understand genetic/environmental see to it for magnetosome synthesis.