IJCRR - 4(8), April, 2012
Pages: 175-184
Date of Publication: 25-Apr-2012
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PRODUCTION AND OPTIMIZATION OF SINGLE CELL OIL BY OLEAGINOUS BACTERIA ISOLATED FROM OIL CONTAMINATED ENVIRONMENTS
Author: T. Murugan, D. Saravanan, R.Balagurunathan
Category: General Sciences
Abstract:Natural sources of oils and fatty acids are derived from plants, animals and microorganism. The oils or lipids, thus produced from microorganisms are known as ?Single Cell Oil?. This present study aimed to isolate lipid accumulating bacteria for the production and optimization of single cell oil. In this preliminary investigation three soil samples were collected in around Kanchipuram District, 37 different bacterial strains were isolated and screened by Sudan black stain of that 3 strains (AOM13, AOM17 & MEW3) showed maximum lipid accumulation. They were used for lipid production of by shake flask method. The lipid was extracted by Folch method using cell dry matters. Among 3 strains AOM17 was identified as the efficient producers which produce about 23% of lipid content. It was confirmed as lipid by solubility, saponification test. Further the lipid was subjected to antimicrobial activity against human pathogens by well diffusion method, the strain MEW3 showed good activity against Bacillus sp., Staphylococcus aureus and Proteus sp. In optimization, all three strains showed maximum lipid accumulation at 15.5 g/L of glucose, 0.4 g/L of ammonium sulphate, pH 8 and 96 hours incubation time. The three potential strains AOM13, AOM17 and MEW3 were identified as Cellulomanas sp., Arthrobacter sp., Acromicrobium sp., respectively.
Keywords: Single cell oil, Oil production, oleaginous microbes
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INTRODUCTION
Oils, fats and lipids from the natural compounds are serves as sources of energy and are considered an important component of our food3 .The demand for oils and fats is largely met form plant and animal sources 19. Natural sources of oils and fatty acids are derived from plants, animals and microorganism9, 19. The demand for oils and fats in general is largely met from plants and animals sources3 . The main drawback is, these sources alone cannot be able to meet the total requirement of lipids. The drawback behind from these sources, a complex mixture of fatty acids with varying lengths (Docosahexaenoic acid) and degree of unsaturation were obtained and it needs expensive lipid purification. Furthermore, the fish or animals oil gets contaminated by environmental factors, which leads to typical smell and unpleasant taste 19 . Microorganisms will be the suitable alternatives, as they have the ability to convert a number of waste materials into a series of valuable-added products 3 . The oils or lipids, thus produced from microorganisms are known as ?Single cell oil? and the microbes are called ?Oleaginous microbes‘, since they accumulate more than 20% of their biomass as lipids 9 . The lipid which accumulates in oleaginous microorganisms is mainly triacylglycerols16. Oleaginous microorganism could provide an economically feasible source of poly unsaturated fatty acids 15 . Some essential fatty acids (EPA) produced by microorganism are a direct precursor for a number of biologically active compounds 19 such as prostaglandins, leukotrienes, thromboxanes and other related metabolites. It exhibits regulatory effects on lipoprotein metabolism, blood rheology, vascular tone, leukocyte function, plate activation and cell growth 21 . The exploitation of oleaginous microorganism for the production of single cell oil is value added products that has relevance and importance to our nation‘s economy. Most of the work has so far been, produced single cell oil mainly from yeasts. Prokaryotic microorganism should now also consider as a lipids with potential application in the oil industry. In this view, the present study was aimed for isolation and screening for SCO producing bacteria from different soil samples and to produce single cell oils in maximum quantity using different carbon sources based on the optimization of various factors. MATERIALS AND METHODS Collection of Samples For this study, soil samples were collected from three different oil contaminated places in around Kanchipuram District and aseptically transferred to laboratories for processing. Isolation and Purification of Bacterial Strains The samples were serially diluted using sterile distilled water blanks. Spread plated was made on Mineral salt agar medium9 and incubated for 24-48 hours at room temperature. Morphologically different bacterial isolates were selected and purified on Nutrient agar slants and subsequently analysed for micro morphological characteristics. Screening Methods for Single Cell Oil production Sudan black B staining: The isolated strains were stained with Sudan Black B staining 15; 2 . the smear was fixed in the slide and was flooded with Sudan Black B stain for 15 minutes and rinsed twice in xylene, followed by counter stained with diluted safranin for 15 seconds. Then it was washed with distilled water. The air dried slide was observed under a phase contrast microscope on oil immersion for presence of blue or grayish coloured fat globules within the cell. Production of single cell oil All the Sudan black positive isolates were studied for the production of lipids. About 2ml of 24 hours bacterial culture were inoculated in 100 ml of sterilized Supplemented Nutrient Broth (SNB) medium and placed in a shaker at 200 rpm for 3 to 5 days at 30º C 20 . Cell dry matter After three days of incubation, the broth was centrifuged at 5000 rpm for 15 minutes for separation of pellet. The pellet was washed three times with sterile distilled water and dried overnight at 80ºC 8, 9. Then the biomass was weighed for the determination of cell dry matter 15 Total lipid extraction Total lipid content of cell dry weight was extracted by Folch methods 9 . The cell dry matter was extracted with chloroform and methanol mixture solution (2:1), twice at room temperature by mixing it for 15-20 minutes in a shaker and centrifuged at 2000 rpm for 10 minutes. 0.9% Nacl solution was added to the pellet and vortexed for few seconds, again it was centrifuged at 2000 rpm for 10 minute.
The lower chloroform layer containing lipid was obtained and allowed for solvents evaporation to determine the lipid weight 9 . Based on the percentage of the lipid accumulation from the total cell dry matter 15, different potential isolates were selected as mentioned in the (Table 2), and the extracted lipids were stored for further studies. Confirmation analysis of lipids Solubility test: A small amount of extracted lipid from all bacterial isolated were taken in three test tubes and each of three tubes was added with water, alcohol, chloroform and vortexed well to detect the solubility nature. Saponification test: about 2 ml of 2% NaoH solution was added to all the small amount of lipid extracted from potential isolated and mixed well to emulsify the lipid. Positive result observed by the formation of soapy solution.
Antibacterial activity of lipids
The single cell oil (lipid) extracted from the potential isolates were tested for antimicrobial activity against human bacterial pathogens by well diffusion method. The 18 hours culture of Bacillus sp., Staphylococcus sp., Escherichia coli and Proteus sp., were swabbed on MHA plates. In each well, the crude lipid (50µl) was added and incubated at 30º C for 24 hours 7 . Selection of potential strains Based on the dry weight percentage of total lipid extracted from the cell dry matter of isolates, the potential isolates were selected. The isolated strain was further used for maximum production of single cell oil and optimization studies. Optimization of single cell oil production To enhance production of single cell oil, the potential strains AOM13, AOM17 and MEW3 were subjected to optimization studies. Production of SCO was optimized by studying various nutritional factors (such as carbon and nitrogen) and environmental (such as pH and incubation time) was incorporated in Minimal unbalanced agar media 12, 13, 18 . To identify the maximum lipid accumulation in potential strains, the incubated plates were flooded with Sudan black staining solution (0.02 % of Sudan black + 96 % Ethanol) for 30 – 60 minutes and washed with 96 % ethanol and observed for the maximum stain absorption 18 . Biosurfactant activity Hemolysis tests: To observe the hemolytic activity, the potential isolates were inoculated on blood agar plates and incubated at 30º C for 72 hours 20 . Emulsification tests: The pure cultures of potential strains were suspended in test tubes containing 2ml of Mineral salt solution. After 48 of incubation, 2 ml of kerosene (hydrocarbon) was added to each tube and the mixtures were vortexed at high speed for 1 minute and were allowed to stand for 24 hours to observe emulsification activity 20 . Characterization of potential isolates Micro morphological test such as Gram staining, Motility, Spore staining, Metachromatic granule staining and biochemical test such as starch hydrolysis, Gelatin liquifization, Nitrate reduction and sugar fermentation tests, etc., were performed to identify the potential bacterial strains.
RESULTS
Isolation and purification of bacterial strains
A total of 37 morphologically different strains were isolated from three samples. Among 37 isolates, 17 isolates from soil sample-I, 8 isolates from soil sample-II and 12 isolates from soil sample-III were isolated and subcultured. Among the 37 isolates 35 strains were showed Gram positive and only 2 strains were showed Gram negative (Table 1).
Screening methods for Single Cell Oil production Sudan black B staining: Out of 37 isolates, 6 strains were showed positive. 3 strains from soil sample-I isolate (AOM2, AOM13 and AOM17), 2 strains from soil sample-II isolate (MEW3 and MEW8) and 1 strain from soil sample-III isolates (VOM12).
Production of single cell oil
The six positive strains namely, AOM2, AOM13, AOM17, MEW3, MEW8 and VOM12 were used for the production single cell oil. The percentage of total lipid content was calculated from total cell dry matter and they showed 10%, 18%, 23%, 21%, 16% and 8% respectively (Table 2).
Confirmation for lipids analysis
Solubility test:
The extracted lipids were insoluble in water but soluble in alcohol and chloroform.
Saponification test: The NaOH solution saponifies the lipids presented in the extracted compound by the formation of soapy solution indicating the presence of lipids. Antibacterial activity of lipids The crude lipids extracted from potential strain MEW3 showed antibacterial activity against Bacillus sp., Staphylococcus sp., and Proteus sp. (Table 3).
Optimization of single cell oil production Effect of Nutritional sources Effect of carbon sources: Growth of all strains was observed in 4 different concentration of glucose. All strains showed maximum lipid accumulation at 1.55 g/L concentration. Strain AOM13 and MEW3 showed maximum lipid accumulation at increased concentration of glucose (1.6g/L).
Effect of Nitrogen sources: In 4 different concentration of nitrogen source, maximum lipid accumulation was found at low concentration of nitrogen souce (0.4g/L). The results were given in table 5.
Biosurfactant activity Hemolysis tests: Among 3 potential isolates, 2 strains AOM17 and MEW3 were showed hemolytic activity. Emulsification tests: All the three strains studied for emulsification activity, which doesn‘t show any positive result. Characterization of potential isolates Based on the cultural characteristics, microscopic, and biochemical characteristics (mentioned in Table-8) the three potential organisms- AOM13, AOM17 and MEW3 were identified as Cellulomanas sp., Arthrobacter sp., Acromicrobium sp., respectively.
DISCUSSION
Microbial lipophilic compounds called single cell oils (SCO), has been the object of research and industrial interest for many years, due to their specific characteristics. Such SCO products are potential for using as alternative sources of animal or plant oils. According to Gouda et al., and Patnayak and Sree 2005 single cell oil is mainly produced by Fungi and Yeasts, Bacteria for single cell oil production are not well explored. In this concern, the present study has aimed to isolate and identify single cell oil producing bacteria from different oil contaminated sites and to optimize for maximum production. For the collection of sample, three different oil contaminated sites were taken and analysed for isolation of bacteria from oil reservoirs20. In their present study also totally 37 strains were isolated from three different oil contaminated sites. Of this sample-I contained large amount of microbial load when compared with sample-II and sample-III. The SCO producers were screened by sudan black staining and by determination of total lipid content on cell dry matter15. In their study also all the 37 strains were screened by Sudan black stain of that 6 isolates showed positive result for lipid accumulation. The production of bacterial SCO was done by shake flask method according to Gouda et al., 2002. The lipids were extracted by Foltch method which is an efficient method to extract the total lipid content from cell dry matter1, 9, 15. After extraction, based on the percentage of total lipid content, 3 isolates were selected. Among 3 strains AOM17 was identified as the efficient producers which produce about 23% of lipid content. For the confirmation of extracted compounds as lipids, lipid analyzing tests were done and confirmed the presence of lipids. All the 3 strains were taken up for further optimization studies and application studies. The lipids extracted from cell dry matter were analyzed for their antimicrobial properties. In previous work, Gram positive and yeast only showed higher sensitivity to the lipid compounds11 in the present study, the strain MEW3 showed activity against Gram positive bacteria and Gram negative bacteria. The potential strains were optimized for maximum lipid production and tested qualitatively by absorption ability of Sudan black stain. The nutritional factors such as carbon source and nitrogen source and environmental factors such as pH and incubation time were optimized for maximum production of lipid. This study showed the mild increased concentration of glucose enhances the production of lipid. At 15.5 g/L concentration all three strains AOM13, AOM17 and MEW3 showed maximum lipid accumulation when glucose (carbon source) used. Papanikolaou et al. reported 0.5g/L of ammonium sulphate had produced high lipid. In this study, 0.4 g/L had showed maximum lipid accumulation when ammonium sulphate (nitrogen source) used. Hall and Ratledge, reported that pH had little influence on lipid accumulation. In this study pH had influence the accumulation of lipid. Strain AOM17 and MEW3 showed maximum lipid accumulation at pH 8 and strain AOM 13 showed maximum lipid accumulation at pH 7. The effect of incubation time was previously reported by Papanikolaou et al. In this study, all the potential strains showed maximum lipid accumulation at an increased time of incubation. After 96 hours of incubation, the lipid accumulation remains constant. The three potential Single cell oil producers, AOM13, AOM17 and MEW3 were identified as Cellulomanas sp., Arthrobacter sp., Acromicrobium sp., respectively.
CONCLUSION
The present study can be further analyzed by thin layer chromatography and Gas chromatography. In future, many unknown bacteria may result in the strains that are even better source of SCO producers than the ones we know. Efficient production techniques with detailed knowledge of lipid mechanism should be developed. This study offers an insight into exploring the possibility of producing such valuable added single cell oil at an high amount and to use it as a supplementary to other edible fats or to synthesis lipid based products such as biosurfactant, bioplastics, etc.,
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