|Magnetotactic bacteria (MTB) are ubiquitous in the aquatic environment and have been identified from almost every continent. Magnetospirillumsp., the most studied MTB was commonly found in estuarine and fresh water habitat. The characterstics of bacteria can be determined by computer-assisted fatty acid profiling. Three strains of Magnetospirillumsp. was grown for 72 hours under defined cultural conditions using Magnetospirillum growth media (MSGM). The cellular lipids were saponified, methylated, and futher it was extracted and purified. The resulting fatty acid methyl esters were then separated, identified and quantified by computer controlled gas chromatography using a software library of known fatty acid methyl esters. Later, all the results obtained of the test strains was compared to that of the commercially available software library of profiles of the different bacteria. Computer-assisted fatty acid profiling offers a cheap, rapid and accurate method for determination of bacteria.
A fatty acid is a carboxylic acid with a saturated or unsaturated aliphatic tail. These are usually derived from phospholipids and triglycerides. Fatty acids are generally named according to the number of carbon atom, location of the double bonds and the type of functional groups present in them. Since many years analysis of short chain fatty acids, that is, volatile fatty acids has become a relevant method for the identification of different bacteria. Specifically, a length of 9 to 20 carbons is chosen for the characterisation of genera and species of Magnetotactic bacteria in different laboratories. Microbial fatty acid analysis is carried out by extraction of lipids from the microbes following the process of Hydrolysis and methylation and finally leading to analysis and quantification of the sample via Gas chromatography. Thus this chromatographic technique has proved to be an essential prerequisite for the analysis of lipid mixtures of the biological origin and helps in the isolation of constituent methyl esters.
Magnetotactic bacteria are a polyphyletic group of bacteria that orient themselves along the Earth’s magnetic field. This is due to the presence of organelles called magnetosomes present in them.The magnetosomes are iron oxide crystals surrounded by a lipidbi- layer membrane, and these membrane bound nano crystals of magnetic iron oxide or iron sulphide crystals are synthesised by the magnetotactic bacteria (MTB). Characteristics of BMs are species specific in composition, size and morphology. The BMs of MTB are composed of either Fe3O4 or Fe3S4 iron mineral arranged in chains. Magnetosomes from MSR1 are cubic in shape and uniform in shape. Magnetosomes membrane is a bilayer lipid membrane originated from cell membrane.
Microbial Identification Inc. (MIDI) introduced in 1985, is one of the automated system available for the identification of bacteria based on the fatty acid profile analysis. The Sherlock Microbial Identification system (MIS) compares the relative percentages of individual fatty acids with its databases present in their libraries. The fatty acid signatures can be used successfully for the identification of the taxa of novel bacterial species isolated from different origins of environmental sources (Welch, 1991; Bertone et al., 1996; Neyts et al., 2000; Hinton et al., 2004; Hoffmann et al., 2010). Library entries are therefore created utilizing MIS as a rapid screening method or for complementary identification. Thus the main aim of this research was to identify and analyse the fatty acid methyl esters with the help of fused silica capillary columns (which allows recovery of hydroxy acids and resolution of many isomers), to identify the wide range of organisms.
MATERIALS AND METHODS
Three novel strains of Magnetospirillum sp. have been used for the screening. Magnetospirillum VITRJS1 (KJ530985), Magnetospirillum VITRJS2 (KJ570852), Magnetospirillum VITRJS5 (KM289194) was obtained from Marine biotechnology and biomedicine lab, VIT University. The strains were sub-cultured in Magnetospirillum growth medium (MSGM) (8) and stored for further experiments.
2.1. Chemicals and media
All the chemicals used in this work are obtained from Himedia laboratories, India until and unless specified. All four strains of MTB were cultured in Magnetospirillum growth medium (MSGM) (Blakemore et al., 1979). Hungate anaerobic technique (Hungate 1950) was followed for the culturing of the bacteria.
CULTURING OF BACTERIA: Media was prepared in 100ml serum bottles and nitrogen gas was sparged to the media to create anoxic condition. The bottles were closed with butyl rubber stoppers and sealed with aluminium caps. The media was further sterilized by autoclaving and cultures were inoculated using hungate anaerobic technique (10). The culture bottles were incubated till the bacterial growth reaches its stationary phase.
HARVESTING OF BACTERIA:-Bacterial culture was transferred to centrifuge tubes and were kept for centrifugation for 20 minutes at 5000 rpm. The supernatant was discarded and pellet was kept in tubes for further fatty acid extraction.
SAPONIFICATION: 1.0ml of Reagent 1was added to each tube containing cells. The tubes were securely sealed with caps, vortexed briefly and heated in a boiling water bath for 5 minutes, again the tubes were vigorously vortexed for 5-10 seconds and returned to the water bath to complete the 30 minute heating.
(REAGENT 1-45g sodium hydroxide ,150ml methanol,150ml distilled water)
METHYLATION: The cooled tubes were uncapped, 2ml of Reagent 2 was added. The tubes were again capped and briefly vortexed. After vortexing, the tubes were heated for 10 ± 1 minutes at 80° ± 1°C. This drops the pH of the solution below 1.5 and causes methylation to occur.The fatty acid methy ester is poorly soluble in the aqueous phase at this point)
(REAGENT- 2 -325 ml centrifuged 6.0N HCL and 275 ml CH3OH )
EXTRACTION: 1.25ml of Reagent 3 was added to the cooled tubes and gentle tumbling on a clinical rotator for about 10 minutes was done. The tubes were uncappedand the aqueous (lower) phase was pipetted out and wasdiscarded. This reagent will help in extraction of fatty acid methyl esters into the organic phase for the use with the gas chromatography
(REAGENT 3- 200ml hexane,200ml methyl-tert-butyl ether)
BASE WASH : Finally, to the organic phase remaining in the tubes, 3ml of reagent 4 was added, the tubes were recapped, and tumbled for 5 minutes. Again uncapping was done, and about 2/3 of the organic phase was pipetted into a GC vial which was capped and ready for analysis.
(REAGENT 4-10.8 g sodium hydroxide in 900ml of distilled water.)
RESULTS AND DISCUSSION:
Results still awaited.