Lipids from R.glutinis using Agricultural Waste
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Lipids are the molecules which contain hydrocarbons and building blocks of the structure and function of cell. Examples of lipids include vitamins, certain, waxes , and mainly of the non-protein membrane of cells. These are molecules which can be extracted from animals and plants using nonpolar solvents such as acetone, chloroform and aether. Fats belong to this group as do other steroids, phospholipids forming cell membrane components etc. During the past decade,interest in the ability of some heterophic microorganisms to produce substantial quantities of lipid has increased .,Microbial lipids (in particular polyunsaturated fatty acids (PUFA),but also monounsaturated (MUFA) and saturated (SFA) fatty acid ) have potential commercial application as nutraceuticals, pharmaceuticals, and feed ingredients for aquaculture. Considerable effort has gone into isolation of,and optimisation of lipuid production bt,oleaginous strains (Ratledge, 1993; Leman,1997). Biodiesel is an alternative, biodegradable, nontoxic, and clean renewable fuel with properties similar to conventional diesel. The raw materials for biodiesel production now mainly include biological sources such as vegetable and fruit waste,palm seed oil,soyabean oil and some recovered animal fats and oils (Tonboriboon et al., 2010).However the cost of biodiesel is high due to the high cost of the raw materials (about 70-75% of the total cost). A cheaper raw material for biodiesel production would help to reduce the total cost.Microlipids,namely single cell oils,produced by oleaginous microorganism including bacteria.yeast,moulds and algae,are nowof interest as promising potential feedstocks for biodiesel production due to their similar fatty acid composition to that of vegetables oil (Li et al., 2007; Saenge et al., 2011a,b; Yen and yang, 2012). In the course of the ongoing endeavour
Oleaginous fungi, and particularly yeast, are very efficient in the accumulation of intracellular TAG and it is expected that they will be exploited by the bio fuel industry in the future.Nevertheless, the costs of microbial lipids are still too high necessarily to be used as carbon sources for the cultivation of these microorganisms and the performance of the bioprocess has to be further improved in terms of both the yield and the productivity.
In the present study of production of lipids from R.glutinis using agricultural wastes, various vegetables and fruit peels were taken i.e.orange,banana,pineaaple ,pea and cabbage .Their peel extracts were prepared via different pre-treatment and various analytical tests were performed to estimate total sugar,reducing sugar and nitrogen content in the peel extracts .The extracts prepared were further used to grow R.glutinis for the production of lipids .The amount of lipids accumulation by the organism in the different culture condition were then estimated as well as the fatty acid composition of the accumulated lipids were also determined .The results suggests that the composition of lipids is such that it is suitable for conversion to biodiesel.Therefore,the results in the present study suggest that agricultural waste such as vegetables and fruits peels can be used as feedstock for production of lipids form R.glutinis ,which can further be converted to biodiesel.However, we further suggest research efforts on the following aspects: screening of other agriculture wastes as a potential feedstock for microbial oil production; novel pre-treatment methods to obtain sugar rich hydrolysates from lignocelluloses biomass to enhance the production of lipids and designing a commercially feasible method for large scale production of biodiesel from oleaginous microorganisms.
Peels of fruits and vegetables were collected from local mandis, domestic waste, food industry waste and various other places and their extracts of different concentration (25%w/v, 50%w/v, 75%w/v) were prepared using steaming under pressure method. Their sugar content was analyzed using DNS method for reducing sugar estimation and phenol-sulfuric acid method for total sugar estimation. Kjeldahl method was performed to assay the amount of nitrogen present in the extracts. The culture was grown on the extracts and the growth curve was studied at an interval of every 24hrs. Lipid extraction was carried out using various methods when the growth reaches to exponential phase
The culture of the Rhodotorula glutinis was obtained from CVM, MSU, USA and was maintained on minimal media containing: glucose 5 g/l ; Na2HPO4 6 g/l ; NaCl 5 g/l ; KH2PO4 3 g/l ; NH4Cl 2 g/l ; MgSO4 0.1 g/l ; yeast extract 2 g/l with pH 5.5 and was preserved at 4° C.
The culture of R.glutinis was sub-cultured in the minimal media containing agar for every 7 days, plates were streaked and preserved at 20°C.The glycerol stocks were also prepared and the culture was preserved in cryo-vials at -80°C.
- Different amounts (25g, 50g, 75g) of peels of orange, banana, pea, pineapple and cauliflower were taken in different beakers separately. 25%w/v extract of all mix, fruit mix and vegetable mix was also prepared separately by mixing orange, banana, pea, pineapple and cauliflower for all mix; orange, banana and pineapple for fruit mix; pea and cauliflower for vegetable mix simultaneously.
- 100 ml of distilled water was added to each beaker.
- The samples were then subjected to autoclaving for 15 minutes at 10 psi.
Add 40g of sodium-potassium tartarate in about 60ml of distilled water and mix well until a clear solution is seen. Do the volume make-up upto 100ml by distilled water
- 6 test tubes containing sugar at varying concentration i.e 0 mg/ml, 0.2 mg/ml, 0.4 mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml were taken to prepare a standard curve. 15 test tubes containing 1ml sample of the extract of peels of each of orange, pea, pineapple, banana, cauliflower were also taken in triplicates.
- The volume make-up to 1ml was done by adding distilled water to the 6 test tubes by adding 1ml, 0.8ml, 0.6ml, 0.4ml, 0.2ml, 0ml distilled water consecutively to each of the test tube.
- 3 ml of DNS reagent was added to each test tube.
- Test tubes were kept for an incubation period of 20 minutes at 60°C to develop the red- brown colour.
- After cooling to room temperature, the absorbance was recorded with a spectrophotometer at 540 nm.
- The O.D. of the samples were plotted on the standard curve to determine their sugar concentration.
Nitrogen is one of the five major elements found in organic materials such as protein. This fact was recognized by a Danish chemist, Johan Kjeldahl, who used it as a method of determining the amount of protein in samples taken from wide verity of organisms.
The central basis used in this procedure is the oxidation of the organic compound using strong sulphuric acid. As the organic material is oxidized the carbon it contain is converted to carbon dioxide and the hydrogen is converted in to water .The nitrogen, from the amine groups found in the peptide bonds of the polypeptide chains, is converted to ammonium ion, which dissolves in the oxidizing solution, and later be converted to ammonia gas.
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