Key Words

FAME, GC column, Fatty acids.

Introduction

The Ocean, which is called the ‘mother of origin of life’, is also the source of structurally unique natural products that are mainly accumulated in living organisms. Marine biotechnology is the science in which marine organisms are used in full or partially to make or modify products, to improve plants or animals or to develop microorganisms for specific uses. From the past few years, interest in marine bacteria has grown considerably. The importance of marine bacteria and as sources of valuable bioactive metabolites is very well established for more than 20 years. However, the identification of fresh marine isolates, a preliminary step in microbiological research, is a continuing challenge for marine microbiologists. The identification of marine bacteria is commonly based on a wide range of biochemical and physiological tests. The major difficulties found with this traditional approach are the need for an easily cultivable strain and the time required for the preparation of cultures. The availability of miniaturized multi test systems that allow the simultaneous determination of numerous phenotypic characters has overcome the previous difficulties. However, the identification of environmental bacteria remains problematic because there is often a lack of agreement between the biochemical tests reported in the literature for the type strain and the results obtained in the laboratory with fresh isolates (1), (2), (3).The reason stems from the similar physiological and biochemical characters shared by aerobic marine bacteria (1) and from the differences in the biochemical features of fresh and stored isolates. In fact, not only may plasmid DNA be lost during storage, but also fresh isolates may have greater enzymatic activity than their counterparts which have been stored on laboratory media (4) More sensitive and reliable techniques such as genomic fingerprinting hybridization analysis based on specific cleavage of DNA by restriction endonucleases and all PCR-based variants are generally difficult to perform, require many expensive reagents, and do not reduce the problem of long culture preparation times; therefore, they are not of practical use in routine analyses. Recent advances in the biochemistry of microorganisms revealed that analysis of cell components, such as proteins and fatty acids, can be effectively applied to bacterial identification, providing the basis for chemotaxonomy (5),(6). The microbial identification system produced by MIDI (Newark, DE, USA) is widely used for identification of microorganisms by fatty acid analysis (7),(8).The use of fatty acid analysis by gas chromatography for the identification of bacteria since its initial introduction of (9) has given results in agreement with DNA-DNA hybridization data(9)(10)(11)is currently widely used for the identification of both clinical and environmental isolates(12) ,(13),(14),(15),(16), (17). This technique has practical advantages, such as the simplicity of the analytical method, 38 | JRAAS: 26,37-42, 2011 the speed of analysis, and the low cost of materials. Moreover, the whole-cell fatty acid content is a direct and stable expression of the cellular genome. In fact, the cellular fatty acid pattern is a phenotypic character that is not affected by mutations or acquisition or loss of plasmids.A method for the rapid and reliable identification of marine bacteria has yet to be developed. In the present study, we describe a method for identification at the genus level of marine bacteria belonging to the genera Vibrio by using gas-chromatographic profiles of fatty acid methyl esters (FAME). To elaborate the FAME data the fatty acid pattern based on an artificial neural network was chosen. Neural computing is the study of networks of adaptable nodes which, through a process of learning from task examples, store experimental knowledge and make it available for use (18) seek to reproduce the style of computing of the brain and can be regarded as computer models in which the nodes correspond to neurons and the connections between nodes correspond to synapses. Neural networks have the ability to identify unknown patterns after a training phase during which known patterns are shown to the net together with the expected identification.

Figure1: a) The individual colonies isolated from sea water b) Plate shows the separated from the colony as streaking

Materials and Methods

Sample collection

The samples were collected from Nellore costal region, Krishna Patanam beach in Andhra Pradesh, India, during the month of October 2010. 100µl of water sample was spreaded over the surface of the marine agar (Zobell Marine agar) with composition of Peptone 5.0 g, Yeast Extract. 1.0 g, Ferric Citrate 0.1 g, Sodium Chloride 19.45 g, Magnesium Chloride 8.8 g, Sodium Sulfate 3.24 g, Calcium Chloride1.8g, Potassium Chloride 0.55 g, Sodium Bicarbonate 0.16g, Potassium Bromide.0.08 g, Strontium Chloride 34.0 mg, Boric Acid 2.0 mg, Sodium Silicate 4.0 mg, Sodium Fluoride 2.4 mg, Ammonium Nitrate 1.6 mg, Disodium Phosphate 8.0 mg, Agar 15.0. The individual colonies (Fig.1) were taken and maintained as stock cultures at 30ºC in marine agar test tubes slants. The selected strain was streaked on the petri plate and continued for the further process.

Fatty Acid Analysis

Reagents Reparation

Four reagents are required to cleave the fatty acids from lipids: prepared according to the MIDI Sherlock protocol (19).

Reagent 1: Saponification—45g sodium hydroxide, 150ml methanol, and 150ml distilled water. Dispensing with an autopippete assures reproducibility and allows to perform large numbers of assays in a day.

Reagent 2: Methylation—325ml 6.0N hydrochloric acid and 275ml methyl alcohol to drop the pH of the solution below 1.5 and causes methylation (for the increased volatility in a partially polar column) of the fatty acid. The 39 | JRAAS: 26,37-42, 2011 fatty acid methyl ester is poorly soluble in the aqueous phase at this point.

Reagent 3: Extraction—200 ml hexane and 200 ml methyl tert-butyl ether. This will extract the fatty acid methyl esters into the organic phase for use with the gas chromatograph.

Reagent 4: Sample Cleanup—10.8 g sodium hydroxide dissolved in 900ml distilled water. This procedure reduces contamination of the injection port liner, the column, and the detector. More than 10,000 analyses can be performed on a column prior to needing any maintenance.

Sample Processing

Steps to prepare GC ready extracts are illustrated in Figure 3.

Fig3 : The flow chart explains the extraction process of fatty acids

Harvesting—a 4mm loop is used to harvest about 40mg of bacterial cells from the third quadrant (second or first quadrant if slow growing) of the quadrant streaked plate. The cells are placed in a clean 13×100 culture tubeSaponification—1.0ml of Reagent 1 is added to each tube containing cells. The tubes are securely sealed with teflon lined caps, vortexed briefly and heated in a boiling water bath for ca. 5 minutes, at which time the tubes are vigorously vortexed for 5-10 seconds and returned to the water bath to complete the 30 minute heating.

Methylation—the cooled tubes are uncapped, 2ml of Reagent 2 is added. The tubes are capped and briefly vortexed. After vortexing, the tubes are heated for 10 ± 1 minute at 80° ± 1°C. (This step is critical in time and temperature.)

Extraction—Addition of 1.25ml of Reagent 3 to the cooled tubes is followed by recapping and gentle tumbling on a clinical rotator for about 10 minutes. The tubes are uncapped and the aqueous (lower) phase is pipetted out and discarded.

Base Wash—About 3ml of Reagent 4 is added to the organic phase remaining in the tubes, the tubes are recapped, and tumbled for 5 minutes. Following uncapping, about 2/3 of the organic phase is pipetted into a GC vial which is capped and ready for analysis.

ESTIMATION OF FATTY ACID METHYLE ESTERS (FAME)

The FAME were analyzed by using Gas chromatography (GC)-mass spectrophotometry with an HP 6890 gas chromatograph equipped with an HP 5972A mass selective detector (Hewlett Packard Co., Porlo Alto California) and an HP Ultra 2 cross-linked 5% phenyl-methyl silicon capillary column (25m by 0.2mm 0.33m µ m) .The oven temperature was programmed with injection and 1-min hold at 80 C, followed by an increase to 160˚ C at 60 C min-1 , a hold at C for 28 min., and an increase at 5˚C min-1 to 230 C. Individual FAME was identified by comparing their mass spectra with standard kits (Ana-labs North Heven, Conn.) the fatty acid content of the cells was calculated as the average of the three independent cultivations. The method presented here is RTSB50 (Table 1)

Result

The marine bacteria analyzed in the present study was to identify the bacteria based up on the fatty acid composition , the list of the fatty acids composition like 9:0 Pelargonic acid,10:0 capric acid,11:0 undecanoic acid,12:0 lauric acid,13:0 Tridecanoic acid,14:0 myristic acid,15:1 Pentadecenoic acid,16:1Palamitoic acid,17:1 iso mageraleic acid,18:1 Arachidonic acid, 20:4eicosenicacid,15:0 Pentadecanoic acid,16:0 Palmitic acid, 19:0Nanadoconic ,18:0steric acid was given clearly according to the GC report

The chromatogram obtained in this experimental analysis fig 2 is more descriptive and elaborative. It confirms and correlates the presence of saturated and unsaturated forms of fatty acids present in the bacterium with the predetermined fatty acids unknown fatty acids. Our experimental data matches and establishes the similar results mentioned in the report of MIDI Sherlock software databases and given the similarity were matched with Vibrio gazogenes (RTSBA 66.00). The mostly concerned genus Vibrio. (20) Reported that 16:1, 16:0, and 18:1 were the major fatty acids in the Vibrio species and tested, with 16:1 being predominant.

Table 1

Discussion

Natural lipids consist of complex mixtures of molecular species, which are found in association with cell membranes, lipo proteins and other subcellular structures. The composition differs among different cell and tissue types. The GC of fatty acids by James and Martin 1956 provided the first success in dealing with the complexity of the hydrolysis products of fats and oils.

Fatty acid profiling is a popular method for characterizing microbial communities of natural systems. Direct extraction of microbial fatty acids in situ would be useful compared with methods that extracted lipids first and subsequently release fatty acids from lipids. The occurrence and abundance of microbial fatty acids have been used by many investigators for the identification of microorganisms in microbial communities (21). The Microbial Identification System (MIS, MIDI, Newark, DE) for fatty acid methyl ester (FAME) analysis is a standard method for identification of microorganisms. Whole cell fatty acids are converted to methyl

Fig: 3 Chromatogram of fatty acids

esters and analyzed by gas chromatography. The fatty acid composition of the unknown is compared to a library of known organisms in order to find the closest match.