Effect of Reducing Agents on the Growth of Magnetospirilla

Effect of Reducing Agents on the Growth of Magnetospirilla Shubhashri Chakraborthy, Pauline Shekina, Upasana Dey and Suthindhiran Krish Abstract: Magnetotactic Bacteria are a fascinating group of microorganisms. Magnetospirilla is a microaerophilic bacterium. The effect of reducing agents varies with the cell depending on the properties of the reagent. They also produce different redox potentials which force a substrate to follow specific metabolic pathway in order to change the product or rate of the reaction. So it is essential to study the effect of reducing agents on the growth of Magnetospirilla. Two strains of this bacterium, MSR-1 and RJS-1 were selected for the study. Reducing agents like Ascorbic Acid, Cysteine and Glutathione were prepared in various concentrations and the bacteria were cultured. The ideal concentration of the reducing agents required for the growth of these two strains was identified. Glutathione at the concentration 0.250mg/ml is found to be ideal for the growth of MSR-1 and Cysteine at the concentration of 0.250mg/ml is found to be ideal for the growth of RJS-1 strain of Magnetospirilla Keywords: Magnetotactic bacteria; Ascorbic Acid; Cysteine; Glutathione; Reducing agents. Introduction: Magnetotactic bacteria (orMTB) are a  polyphyletic  group of  bacteria that orient along themagnetic field lines of Earth’s magnetic field with the help of organelles called magnetosomes. Magnetosomes are iron crystals of magnetite or greigite. The movement of microorganisms in response to the magnetic characteristics of the environment is known as  magnetotaxis. Many efforts have been made to optimize the growth conditions of MTB.[4] Magnetospirilla are facultative anaerobic magnetic bacterium. Reducing agents can have very different effects on the microbial cells depending on the properties of the reagent and of the properties of the cell itself. Reducing agents can produce different redox potentials which might be force a substrate to follow a specific metabolic pathway in order to change the rate or product of the reaction.[6] Previous studies showed that changing the redox environment had a strong impact in the applications of Magnetospirilla. Therefore we studied the effect of different reducing agents at different concentrations on the growth of Magnetospirilla. Materials and Methods: Culturing the bacteria Two strains of Magnetospirilla, MSR-1 (DSMZ, Germany) and RJS-1(Marine Biotechnology Lab, VIT University) were grown in liquid Magnetic Spirillum Growth Medium (MSGM).[1] A stock of 500ml of the MSGM media was prepared without reducing agents and Resazurin. 7ml of the media were transferred in small vials and it was purged with nitrogen gas for 3 minutes to provide microaerophilic condition. The vials were sealed with butyl rubber septa and aluminium centre holed caps. These vials were autoclaved at 121 °C for 15 minutes. Stock solution of the reducing agents Ascorbic Acid, Cysteine, Glutathione and Sodium Sulfide (10mg/10ml) were prepared and filter sterilized. Heat sensitive materials such as vitamins were also filter sterilized. 70ÃŽ ¼l of filtered vitamin solution and 14ÃŽ ¼l of filtered ferric quinate solution were taken in sterile eppendorf tubes. Different concentrations of reducing agents (0.125mg/ml, 0.250mg/ml, 0.375mg/ml and 0.500mg/ml) were also taken in the eppendorf t ubes containing the vitamin and ferric quinate solutions. Using sterile needles and syringes these solutions were transferred into the vials. These vials were incubated at 37 °C in a shaker. Checking the growth of the bacteria The growth of the bacteria were observed using spectrophotometer. Optical Density (OD) was taken at 595nm in 24, 26, 29, 38, 44 and 48 hours of incubation. The values are plotted in graph to find the maximum growth rate. Results and Discussion: A graph was plotted against time in hours and the OD values taken for different concentrations of reducing agents. Figure 1. Graph showing the growth of Magnetospirilla (MSR-1) at various concentrations of Ascorbic Acid. The growth of MSR-1 was maximum at the concentration 0.500mg/ml of Ascorbic acid. Figure 2. Graph showing the growth of Magnetospirilla (MSR-1) at various concentrations of Cysteine. The growth of MSR-1 was maximum at the concentrations 0.250mg/ml of Cysteine. Figure 3. Graph showing the growth of Magnetospirilla (MSR-1) at various concentrations of Glutathione. The growth of MSR-1 was maximum at the concentration 0.250mg/ml of Glutathione. Figure 4. Graph showing the growth of Magnetospirilla (MSR-1) at various concentrations of Sodium Sulfide. The growth of MSR-1 was maximum at the concentration 0.500mg/ml of Sodium Sulfide. Comparing all the OD values the growth of MSR-1 was maximum at the concentration 0.250mg/ml of Glutathione. Figure 5. Graph showing the growth of Magnetospirilla (RJS-1) at various concentrations of Ascorbic Acid. The growth of RJS-1 was maximum at the concentration 0.375mg/ml of Ascorbic Acid. Figure 6. Graph showing the growth of Magnetospirilla (RJS-1) at various concentrations of Cysteine. The growth of RJS-1 was maximum at the concentration 0.250mg/ml of Cysteine. Figure 7. Graph showing the growth of Magnetospirilla (RJS-1) at various concentrations of Glutathione. The growth of RJS-1 was maximum at the concentration 0.375mg/ml of Glutathione. Figure 8. Graph showing the growth of Magnetospirilla (RJS-1) at various concentrations of Sodium Sulfide. The growth of RJS-1 was maximum at the concentration 0.500mg/ml of Sodium Sulfide. Comparing all the OD values the growth of RJS-1 was maximum at the concentration 0.250mg/ml of Cysteine. Our studies showed that optimal concentration of reducing agents showed increased growth. When the concentration of reducing agents is increased further it inhibits the growth because of toxicity. Optimal concentration of reducing agents reduces the lag time and enhances the cell density. Conclusion: There are many other reducing agents which are used for growing various species of Magnetotactic bacteria. From the results we got it is suggested that Glutathione at the concentration 0.250mg/ml is ideal for the growth of MSR-1 and Cysteine at the concentration of 0.250mg/ml is ideal for the growth of RJS-1 strain of Magnetospirilla. References: [1] Blakemore R P, Maratea D and Wolfe R S, â€Å"Isolation and Pure Culture of a Freshwater Magnetic Spirillum in Chemically Defined Medium,† Journal of Bacteriology, 140(2), 720, 1979. [2] Cody Nash, â€Å"Mechanisms and Evolution of Magnetotactic Bacteria,† 2008. [3] Damien Faivre and Dirk Schuler, â€Å"Magnetotactic Bacteria and Magnetosomes,† Chem.Rev, 108, 4875–4898, 2008. [4] John F. Stolz, â€Å" Magnetosomes,† Journal of General Microbiology, 139, 1663-1670,1993. [5] Tadashi Matsunaga and Toshifumi Sakaguchi, â€Å" Molecular Mechanism of Magnet Formation in Bacteria,† Journal of Bioscience and Bioengineering, 90, 1-13, 2000. [6] Xiangqian Li, Qilei Wang and Yemin Xue, â€Å" On the Change in Bacterial Growth and Magnetosome Formation for Magnetosprillum Sp. Strain AMB-1 Under Different Concentrations of Reducing Agents,† Jorunal of Nanoscience and Nanotechnology,13, 1392-1398, 2013. [7] Yoshihito Uchino and Suzuki Ken-Ichiro, â€Å"A Simple Preparation of Liquid Media for the Cultivation of Strict Anaerobes,† Journal of Petroleum and Environmental Biotechnology, S3-001, 2157-2160, 2011.