Physicological Characterization of M.VITRJS5

Physicological Characterization of M.VITRJS5Monica Samal, Teena Patra, Suthindhiran KAbstractMagnetospirillium sp. is a Gram negative -proteobacterium which has the ability to move towards the geomagnetic field lines of undercoats magnetic field. The group of bacterium is collectively called Magnetotactic bacteria (MTB). MTB produce intracellular organelles called magnetosomes which help them in its search for optimal living conditions in complex environment. MTB is a gradient requiring microorganism that ar awkward to replicate in outdevelopment medium in the laboratory. In this work the physiological characterization of MTB pass Magnetospirillium sp. VITRJS5 was done as well as a modified maturation medium for the reaping of Magnetospirillium sp. VITRJS5 was developed. The media components ar studied based on the physiological characteristics of the bacteria. The role of negatron donors, electron acceptors, nitrogen computer addresss, ascorbic acid sources, iron cont aining compounds, orthophosphates, reducing agents, on the developing of the bacteria in the MS1 media was studied. The baffleth of the bacteria in the myth modified MS media was analysed and comp atomic number 18d with commercial MTB media such as MSGM (Magnetospirillum growth media) and Schulers oxygen treat gradient media. An increase in growth is observed in the modified MS media comp atomic number 18d to the commercially available media.1. doorMagnetotactic bacteria ar fastidious microorganisms that represent a morphologically and physiologically diverse group of bacteria. The presence 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 surface water, thus directing them in maintaining an optimal position in and near the oxic-anoxic port wine (OAI). Magnetospirillum sp. is the approximately studied and well understood group of bacteria among MTB. Magnetospirillum sp. was mostly isolated from fresh water habitats and many pure cultures 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 response to the earths magnetic field (magnetotaxis). MTB were starting observed by Salvatore Bellini in the year 1963 and collected from antithetical 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 unpalatable environments include the coccoid strains MO-19 , Magnetococcus marinus strain MC-110 and Magnetofaba au stralis (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 occur mostly at the oxic-anoxic interface (OAI), the anoxic regions of the habitat, or both 3. Although the detection of these in samples is relatively simple 4, magnetotactic bacteria are a fastidious group of prokaryotes, and special culture conditions are necessary for their isolation and cultivation. There is clear evidence that the availability and diversity of MTB in different environments is majorly influenced by salinity an d also, iron availability, temperature and compactnesss of sulfur and nitrogen compounds are also seen to be clearly important in the distribution of MTB. The respiratory forms of metabolism of genus Magnetospirillum are either chemoorganoheterotropic, apply organic acids and carbon source or electron donors. Mostly Magnetospirillum sp. capable of autotrophic and mixotrophic growth and utilize nitrate as alternative terminal oxygen acceptor. Magnetospirillum are obligate microaerophile that requires oxygen notwithstanding 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 batch experiments were performed to determine the following (1) growth pH and temperature ranges, (2) Optimum electronic donor/carbon source and electron acceptor. (3) optimum nitrogen and phosphate source (4) optimum Iron source (5) optimum re ducing agent/ sulphur source. Once the preliminary screening of the compounds over different concentrations of the nutritional requirements is determined 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 polar flagellum at separately end of the cell. The strain was obtained from Marine biotechnology 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 marginal media MS1 to analyse the growth conditions in the presence of electron donors and acceptors. MS1 media per litre containing K2HPO4 850mg, Na2HPO4 850mg, NH4H2PO4 500mg, MgSO4 100mg, FeSO4 7H2O 5mg, Na2MoO4 0.2mg, EDTA 10mg and Mineral soluti on 10ml Electron donor and acceptor has to be added accordingly. From the earlier studies, the optimum temperature and pH for the growth of Magnetospirillum is 28C and 7.5 pH.2.2 Determination of electron acceptors and concentration of selected acceptorFor this, 50 ml of MS-1 media was added in five serum bottles and 50 mg of electron donor which in this racing shell is sodium acetate (CH3COONa) was added to each bottle. Then 50 mg of five different acceptors were added in different bottles. These five acceptors are nitrite, selenite, nitrate, thio sulphate and sulphate. Nitrogen was sparged to the media to stain it anaerobic, closed 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 incubation in a shaker incubator. straightway the different concentrations of selected acceptor is determined .It is done for c oncentration of 5mM, 10 mM , 15mM, 20 mM and 30 mM . Steps for anaerobic culture was repeated. The absorbance is checked 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), Ferrous 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 incubator. Narrowing down the number of donors based on the growth of sample. Diffe rent 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, action mechanism 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 preceding(prenominal) described cognitive operation 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 was 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 ferrous 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. egress3.1 Electron acceptor and its concentration for optimum growthOn incubating the strain M.VITRJS5 in the MS1 media with the various electron acceptors, we obtained various results as seen in table1.From the table, we find that Nitrite, Nitrate and Selenite wide electron acceptors for the st rain. But 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 digit.1)Table 1. Tabular representation of growth with various electron acceptor.Table 2. Tabular representation of absorbance of different concentration of nitrateFig.1 Graphical representation 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 table 3, it is seen that growth of M.VI TRJS5 is more pronounced with electron donors Ethanol, Glycerol, Pyruvate, Succinate, Lactate, Glucose and Sucrose along with Acetate which was already being used as an electron donor for other 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. Representation of growth with various donorsTable 4. Tabular Representation of absorbance of growth with different concentrations of pyruvate Fig 2. Graphical representation 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 and pyruvate.3. Nitrogen source and its concentration for optim um growthThe next step is incubation of M.VITRJS5 strain in MS-1 media(without NH4H2PO4) with ammonium chloride and Ammonium hydrogen 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 thioglycolate and cysteine HCl 4% to determine the better sulphur source.Fig 6. Grap hical 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 Sodium 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 minimum 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 o f brand- mod 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, cause 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, etc.From 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 Ammonium hydrogen phosphate (nitrogen source and phosphorus source).Also, the growth of the stra in was seen to be minimalized in case of Sodium thioglycolate and Cysteine HCl as these are strong 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 motivated a new field of seek involving scientific and biomedical applications of MTB and diverse commercial which could be improved using magnetic nanocrystals. But the main drawba cks 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 control for magnetosome synthesis.