FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES , 2003
COMPLETE SPECIFICATION
(See Section 10; rule 13)
PROCESS FOR BIODIESEL PRODUCTION FROM
CRYPTOCOCCUS CUR VA TVS
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at
Dhirubhai Ambani Life Sciences Centre,
R-282, TTC Area of M1DC,
Thane Belapur Road, Rabale,
Navi Mumbai - 400 701
Maharashtra India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

TECHNICAL FIELD
The present disclosure relates to the production of biodiesel from oleaginous yeast such as Cryptococcus curvatus. The present invention also describes a process for the production of biodiesel from oil extracted from oleaginous yeast such as Cryptococcus curvatus.
BACKGROUND
Biodiesel is an environmentally benign solution for global warming, the energy crisis and depleted fossil fuel supplies. Today, biodiesel (e.g., fatty acid methyl esters) is the name given to clean burning alternative fuels, produced from biological, renewable resources that are biodegradable and non-toxic. Biodiesel can be used directly or can be blended at any level with petroleum products, such as petroleum diesel.
Biofuels have clear benefits in addressing environmental concerns related to greenhouse gases, and offer new income to farmers. However, traditional oil-rich crops are limited by land availability, and environmental and social issues regarding the use of feed and food crops for fuel. An alternative way to produce biodiesel in a green and sustainable manner without competing with food crops is to use microbes. There are a few microorganisms in nature that have the inherent ability to accumulate or store oil/lipid up to 60% of their dry weight, when grown under nitrogen-limited conditions. These lipids usually consist of 80%-90% triacylglycerols with a fatty acid composition similar to many plant seed oils (Ratledge 1984; Ykema et al., 1988). These organisms are called oleaginous microorganisms.
Microbial oils, also called single cell oils, are produced by some oleaginous microorganisms, such as yeast, fungi, bacteria, and microalgae (Ma et al., 2006). It has been demonstrated that such microbial oils can be used as feedstock for biodiesel production. In comparison to other vegetable oils and animal fats, the production of microbial oil has many advantages: microbes have a short life cycle as compared to plants so the time to harvest is shorter, there is, less labor required, microbial oil production is less affected by venue, season and climate, and scale-up is easier (Li and Wang., 1997). Therefore, microbial oil has a tremendous potential to become one of the major oil feedstock for biodiesel production in the future. Although not a new concept, work in this area has been very limited (Li et al., 2008).

Microbial cells studied to date for use in biodiesel production include bacteria, yeast and fungi. Preferred fungus genera include Mortierella, Phycormyces, Entomophthora, Pythium, Thraustochytrium, Blakeslea, Rhizomucor and Aspergillus. Exemplary bacteria that have been studied include those of the genus Propionibacterium. Studied algae include dinoflagellate and/or belong to the genus Crypthecodinium, Porphyridium or Nitschia, for example Crypthecodinium cohnii.
Yeasts such as Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces have been studied for their microbial oil properties and among these oleaginous yeasts, Cryptococcus curvatus has attracted attention because it can accumulate large amounts of oil, up to 60% of the cell's dry weight (Ratledge 1991), utilizing cheap carbon sources like whey permeate (Ykema 1989) and other carbohydrate-rich agricultural or food processing wastes. The yeast oil produced by C. curvatus resembles plant seed oils like palm oil (Davies 1988).
Currently, the production of yeast oil is more expensive than the production of vegetable oils. Therefore, single-cell oil fermentations will be economically feasible when a particular oil can be produced with high added value. Accordingly, previous approaches have used process engineering to yield a higher lipid production rate, a higher cellular lipid content, and higher biomass production, all geared to make the process more economically feasible. Different cultivation modes, including fed-batch and continuous fermentations, have been reported to increase the cell density of oleaginous microbes in culture. However, none of the prior art focuses on the efficient extraction of oil from fermentation media.
SUMMARY
Disclosed herein are certain methods of fermenting oleaginous yeast such as Cryptococcus curvatus, methods of fermenting oleaginous yeast such as Cryptococcus curvatus, methods of extracting the oil produced by oleaginous yeast such as Cryptococcus curvatus, and methods of producing biodiesel from oleaginous yeasts such as Cryptococcus curvatus. In some embodiments, the methods use inexpensive and economical components and processes.

Likewise, disclosed herein are compositions including crude glycerol, corn steep liquor, yeast autolysate, and oleaginous yeast, wherein the pH of the composition is about 5.5. In some embodiments, the composition comprise crude glycerol at about 1% (w/v), corn steep liquor at about 2% (w/v), yeast autolysate at about 0.5% (w/v), and the oleaginous yeast Cryptococcus curvatus.
In some embodiments, the composition consists essentially, or alternatively consists of, crude glycerol, e.g., about 1% (w/v), corn steep liquor, e.g., about 2% (w/v), yeast autolysate, e.g., about 0.5% (w/v),, and wherein the pH of the medium is about 5.5. In some embodiments, the composition does not comprise malt extract. In other embodiments, the composition does not comprise one or more of malt extract and peptone. In still further embodiments, the composition does not include peptone, tryptone, beef extract, malt extract, galactose, starch, arabinose, glycerol, mannitol, sucrose or fructose. In some embodiments, the oleaginous yeast is Cryptococcus curvatus. In some embodiments, instead of bakers yeast autolysate for example, the composition comprises one or more of previously de-oiled yeast cells, previously de-oiled yeast autolysate, previously lysed Cryptococcus curvatus yeast cells, previously fermented and de-oiled Cryptococcus curvatus yeast cells, and previously de-oiled Cryptococcus curvatus yeast autolysate. For example, in one embodiment, a composition comprises or consists essentially of crude glycerol, corn steep liquor, previously de-oiled yeast cells (such as previously fermented and de-oiled Cryptococcus curvatus), and oleaginous yeast (such as Cryptococcus curvatus).
In some embodiments, the fermentation medium includes a volume of about 25 liters, 27 liters, 50 liters or greater. In some embodiments, the fermentation medium consists of a volume.of about 25 liters or greater. In some embodiments, the oleaginous yeast is Cryptococcus curvatus. In some embodiments, the oleaginous yeast is Cryptococcus curvatus and the yeast cell biomass after fermentation is at least about 40 g/L on a dry weight basis. In some embodiments, the oleaginous yeast is Cryptococcus curvatus and the yeast cell biomass after fermentation is at least about 50 g/L on a dry weight basis. In some embodiments, the fermentation is fed batch fermentation.
In some embodiments, the extraction step includes treating the fermented oleaginous yeast with

one or more of the compounds or combinations selected from the group consisting of dichloromethane; chloroform; n-hexane;,a combination of methanol and chloroform; a combination of methanol and dichloromethane; and a combination of n-hexane and isopropyl alcohol. In some embodiments, the fermented oleaginous yeast is collected by centrifugation prior to the extraction step. In other embodiments, the fermented oleaginous yeast is homogenized prior to the extraction step. In still other embodiments, the fermented oleaginous yeast is dried prior to the extraction step. In further embodiments, the fermented oleaginous yeast is frozen or is freeze-dried prior to the extraction step. In still other embodiments, the fermented oleaginous yeast is collected by centrifugation prior to the homogenization step. In other embodiments, the fermented oleaginous yeast is homogenized without a prior centrifugation collection step. In some embodiments, the extracted oil yield comprises at least about 40% of the dry weight of the fermented yeast. In other embodiments, the extracted oil comprises about 50% of the dry weight of the fermented yeast. In some embodiments, the oleaginous yeast comprises Cryptococcus curvatus.
Also disclosed herein are methods for producing biodiesel. In some embodiments, the methods include fermenting oleaginous yeast in a fermentation medium including crude glycerol, corn steep liquor and yeast autolyaste, wherein the pH of the fermentation medium is about 5.5; extracting the oil from the fermented oleaginous yeast; and transesterifying the extracted oil. For example, in some embodiments, the fermentation medium includes crude glycerol at about 1% (w/v), corn steep liquor at about 2% (w/v), lysed yeast cells, such as yeast autolyaste at about 0.5% (w/v), and wherein the pH of the fermentation medium is about 5.5. In some embodiments, the fermentation medium consists essentially of, or alternatively, consists of crude glycerol at about 1% (w/v), corn steep liquor at about 2% (w/v), yeast autolyaste at about 0.5% (w/v), , and wherein the pH of the fermentation medium is about 5.5. In some embodiments, the fermentation medium does not include malt extract. In other embodiments, the fermentation medium does not include one or more of malt extract and peptone. Thus, in some embodiments, the method does not include fermenting the yeast in a fermentation medium including malt extract. In other embodiments, the method does not include fermenting the yeast in a fermentation medium including peptone or malt extract. In yet other embodiments, the method does not comprise fermenting the yeast in a fermentation medium comprising peptone, tryptone, beef extract, malt extract, galactose, starch, arabinose, glycerol,

mannitol, sucrose or fructose. In some embodiments, instead of bakers yeast autolysate for example, the composition comprises one or more of previously de-oiled yeast.cells, previously de-oiled yeast autolysate, previously lysed Cryptococcus curvatus yeast cells, previously fermented and de-oiled Cryptococcus curvatus yeast cells, and previously de-oiled Cryptococcus curvatus yeast autolysate.
In some embodiments, extracting the oil includes homogenizing the fermented oleaginous yeast without a prior centrifugation collection step. In some embodiments, the oleaginous yeast comprises Cryptococcus curvatus.
Also disclosed herein are compositions including the oil extracted from oleaginous yeast. In some embodiments, the oil includes at least 0.2% myristic acid; at least 25% palmitic acid; at least 0.5% palmitoleic acid; at least 9% stearic acid; at least 45% oleic acid; and at least 5% linoleic acid. For example, in some embodiments, the oil includes about 0.6% myristic acid; about 29.7% palmitic acid; about 1.0% palmitoleic acid; about 9.6% stearic acid; about 50.6% oleic acid; and about 7.5% linoleic acid.
Also disclosed herein are compositions comprising biodiesel produced from oleaginous yeast. In some embodiments, the biodiesel so produced includes the following parameters: acid value (EN14104) of 0.47; soap (ppm) of 50; moisture content (ppm) of 500; density g/ml (ENIS03675) of 878.6; viscosity cm2/sec (EN ISO3104) of 6.061; cloud point (ASTM-D2500) of +S°C flash point (ASTM D93) of > 120°C; Cu strip corrosion (ASTM D130) of la: carbon residue % (ASTM D524) of 0.01; and sulphated ash % (ISI448P:04) 0.02.
Also disclosed herein are methods for isolating oil from fermented oleaginous yeast. In some embodiments, the methods include fermenting Cryptococcus curvatus in a fermentation medium including crude glycerol, corn steep liquor, and yeast autolysate, wherein the pH of the fermentation medium is about 5.5; extracting the oil from the fermented Cryptococcus curvatus; wherein the method does not comprise fermenting the Cryptococcus curvatus in a fermentation medium comprising malt extract or peptone, and yeast cell biomass after fermenting is at least about 40 g/L on a dry weight basis. In other embodiments, the methods include fermenting Cryptococcus curvatus in a fermentation medium that includes crude glycerol, corn steep liquor, and previously de-oiled yeast cells, such as previously fermented

and de-oiled Cryptococcus curvatus yeast cells; extracting the oil from the fermented Cryptococcus curvatus; wherein the method does not include fermenting the Cryptococcus curvatus in a fermentation medium comprising malt extract or peptone or bakers yeast autolysate, and wherein the yeast cell biomass after fermenting is at least about 40 g/L on a dry weight basis. In some embodiments, the yeast cell biomass after fermenting is at least about 50 g/L on a dry weight basis. In yet other embodiments, the method does not comprise fermenting the yeast in a fermentation medium comprising peptone, tryptone, beef extract, malt extract, galactose, starch, arabinose, glycerol, mannitol, sucrose or fructose. In some embodiments, instead of bakers yeast autolysate for example, the composition comprises one or more of previously de-oiled yeast cells, previously de-oiled yeast autolysate, previously lysed Cryptococcus curvatus yeast cells, previously fermented and de-oiled Cryptococcus curvatus yeast cells, and previously de-oiled Cryptococcus curvatus yeast autolysate.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows glycerol levels, dry cell weight and optical density in fed batch fermentation of Cryptococcus curvatus, when crude glycerol is used in the fermentation medium.
Figure 2 shows glycerol levels, dry cell weight and optical density in fed batch fermentation of Cryptococcus curvatus when malt extract is absent from the fermentation medium.
Figure 3 shows glycerol levels, dry cell weight and optical density in fed batch fermentation of Cryptococcus curvatus at a 25 liter scale.
Figure 4 shows glycerol levels, dry cell weight and optical density in fed batch fermentation when Cryptocccus yeast lysate is used in the fermentation medium.
DETAILED DESCRIPTION
I. DEFINITIONS
The term "Cryptococcus curvatus" as used herein is an oleaginous yeast, such as one that is able to accumulate about 50% of its weight as oil under oil production conditions.
The term "biodiesel" means a diesel fuel comprising long-chain alkyl (methyl, propyl or ethyl)

esters. A biodiesel may comprise fatty acid methyl esters ("FAMEs") of oil isolated from yeast, including chemical variants or modifications of the FAMEs.
The term "oil" as used herein, with respect to oil isolated from or produced by yeast, refers to lipids (such as fatty acid methyl esters) produced in yeast, or lipids or total lipid content isolated from yeast.
The term "oil production conditions" as used herein refers to growth or fermentation conditions in which yeast will produce oil. Such conditions may include a medium with an excess of carbon and limited amounts of other nutrients, specifically nitrogen. In some embodiments, "oil production conditions" are realized when the ratio of carbon source to nitrogen source in the growth medium is about 40-50. In some embodiments, "oil production conditions" are realized when the tricarboxylic acid cycle is repressed, the metabolic pathway is altered, protein synthesis ceases and the lipid accumulation process is activated. In some embodiments, "oil production conditions" are realized when NAD-IDH activity is decreased or absent in the oleaginous yeast strain as compared to the same yeast strain under control conditions, and the oleaginous yeast strain can no longer utilize acetic acid as a carbon source, but can utilize glycerol or lactic acid as a carbon source.
As used herein, the term "about" and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the invention. As used herein, "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term, including plus or minus 5%, 1%, 0.1%, 0.01%, or refer generally to a standard deviation seen when using standard procedures for measurement.
As used herein, the term "transesterification" or "transesterifying" refers to the process of exchanging the organic group R" of an ester with the organic group R of an alcohol. A schematic reaction is shown below.


As used herein, the term "culturing" means the process by which cells (e.g., yeast cells) are grown under human controlled conditions. In some embodiments, culturing includes fermenting.
As used herein, the term "culture medium" or "growth medium" or "fermentation medium" means a liquid or gel designed to support the growth of microorganisms or cells, for example, yeast cells.
As used herein, the term "yeast cell biomass" refers to yeast cells that are produced by fermentation or by other culturing means. In some embodiments, the yeast cell biomass has been collected by centrifugation and separated to some degree from the culture or fermentation medium. "Fermented yeast cell biomass" refers to yeast cells that are produced by fermentation.
As used herein, the term "dry weight basis" means the mass of a composition when at least some of the liquid portion has been removed. For example, to determine the dry weight of fermented yeast cell biomass, one ml of culture is centrifuged at 10,000 rpm.for 10 minutes. The biomass pellet is washed twice with distilled water and dried at 40°C to constant mass (usually 24h). In this example, the biomass is determined gravimetrically, and is provided in units of gram per milliliter, grams per liter, etc.
As used herein the term "oil yield on a dry weight basis" means the amount of oil produced by a unit of yeast cell biomass measured on a dry weight bases. For example, if the yeast cell biomass on a dry weight basis is 32.8 g/L, and 7 g of oil are extracted from this biomass, the oil yield is 21 % on a dry weight basis.
As used herein, the term "isolated," when used in conjunction with a yeast strain, refers to a yeast cell that is substantially or essentially free from non-yeast components that normally accompany or interact with the yeast cell as found in its naturally occurring environment.

Thus, an isolated yeast cell is one that is no longer in its natural environment, and is, for . example, growing or preserved in culture, for example in a laboratory.
II. GENERAL
Methods and compositions are presented herein that relate to high cell density production of Cryptococcus curvatus, followed by oil extraction and transesterification of the extracted oil into fatty acid methyl esters.
Cryptococcus curvatus may be cultured in any medium typically used for culturing yeast. The nutritive requirements may include nitrogen sources such as peptone, tryptone, yeast extract, beef extract, corn steep liquor, ammonium chloride, sodium nitrate and ammonium sulphate, and carbon sources such as glucose, galactose, starch, arabinose, glycerol, mannitol, sucrose and fructose. Culturing may be performed under a range of conditions that do not adversely affect growth, or under condition to obtain the growth rate or growth pattern desired (e.g., under oil production conditions). For example, in some embodiments, Cryptococcus curvatus is cultured or fermented under oil producing conditions. By way of example but not by way of limitation, cultures may be grown under aerobic conditions, within a pH range of from about 5.0 to about 7.0, with an inoculum density of 1-5% and an inoculum age of 6 to 24 hours.
Also disclosed herein are methods for the scale up of oleaginous yeast cell mass production and oil extraction processes.
Also disclosed herein are methods for biodiesel production. In some embodiments, such methods include transesterification of the oil accumulated by an oleaginous yeast strain.
III. EMBODIMENTS
Disclosed herein are new and economical processes for oil extraction from oleaginous yeast, and for the production of biodiesel using the oil of oleaginous yeast. In some embodiments, the oleaginous yeast comprises Cryptococcus curvatus.
In some embodiments, method are provided to maximize growth conditions for oleaginous yeasts. In some embodiments, growth conditions are directed to the fermentation of oleaginous yeasts for the production and extraction of oil produced by the yeast, and/or for the

manufacture of biodiesel using oil produced by the yeast. Thus, in some embodiments, growth conditions are also "oil producing conditions." In some embodiments, growth conditions or oil producing conditions are determined by evaluating one or more of the following parameters: 1) biomass of the fermented yeast; 2) optical density ("0,D.") of the fermented yeast; 3) cost per unit of the fermented yeast biomass; 4) cost per unit of the oil extracted from fermented yeast; and 5) cost per unit of biodiesel manufactured from oil produced by the yeast. In some embodiments, growth conditions (oil producing conditions) are maximized for Cryptococcus curvatus, such that one or more of a desired biomass of fermented Cryptococcus curvatus, optical density ("O.D") of the fermented Cryptococcus curvatus; cost per unit of the fermented Cryptococcus. curvatus biomass; cost per unit of the oil extracted from fermented Cryptococcus curvatus; and cost per unit of biodiesel manufactured from oil produced by Cryptococcus curvatus is achieved.
In some embodiments, methods are provided for maximizing growth conditions (as well as oil producing conditions) for oleaginous yeasts by providing certain nutritional and physiological parameters. In some embodiments, such maximizing result in improved yields of one or more of yeast biomass, oil production, cost of a unit of fermented biomass, cost of a unit of oil produced by the fermented yeast, or cost of a unit of biodiesel manufactured from oil produced by the yeast. By way of example and not by way of limitation, physiological and nutritional parameters include carbon source, carbon source concentration, nitrogen source, nitrogen source concentration, the size of the fermentation batch and the mode of fermentation, such as batch or fed batch fermentation.
In some embodiments, methods are provided for maximizing growth conditions (as well as oil producing conditions) for oleaginous yeast by providing certain nutritional and physiological parameters and economizing the production of yeast biomass, oil and/or biodiesel. Thus, in some embodiments, inexpensive raw materials are used in the growth and fermentation media to maximize growth conditions (oil production conditions). By way of example, but not by way of limitation, inexpensive raw materials include corn steep liquor (CSL), crude glycerol and de-oiled yeast autolysate, for example de-oiled Cryptococcus yeast autolysate. In some embodiments, medium for growth of oleaginous yeast includes crude glycerol at about 1% w/v, corn steep liquor at about 2% w/v, yeast autolysate at about 0.5% w/v, wherein the growth

medium has a pH of about 5.5. In some embodiments, growth conditions (oil production conditions) are provided for Cryptococcus curvatus using inexpensive raw materials such as corn steep liquor, crude glycerol and de-oiled yeast autolysate.
In some embodiments, the growth condition (oil producing condition) for oleaginous yeast are performed to accommodate large scale fermentation and/or high density fermentation. In some embodiments, oil production is done at a smaller scale volume (e.g., 5 liters, "L"); in other embodiments, oil production is done at a larger scale volume (e.g., 25 L or more). In some embodiments, oil production is performed in a smaller fermentation volume and the parameters are then scaled-up for use in a larger volume fermentor. In some embodiments, the nutritional and physiological parameters are additionally provided in the larger volume fermentor after scale-up from the small volume. In some embodiments, inexpensive raw materials such as corn steep liquor and crude glycerol are used. In some embodiments, the oleaginous yeast is Cryptococcus curvatus. Thus, in some embodiments, methods are provided to allow oleaginous yeast production to high density in fermentors using inexpensive raw materials including crude glycerol and corn steep liquor. In some embodiments, the oleaginous yeast is Cryptococcus curvatus.
In some embodiments, methods are provided for oil extraction from oleaginous yeast. In some embodiments, oleaginous yeast is fermented under oil producing conditions, and oil is extracted from the fermented cells. In some embodiments, extraction includes lysing the fermented yeast cells (e.g., by homogenization or freezing) and extracting the oil using a polar organic solvent. By way of example, but not by way of limitation, exemplary polar organic solvents include dichloromethane, chloroform and n-hexane. In some embodiments, extraction -methods include a combination of methanol chloroform, a combination of methanol :DCM or a combination of n-hexane: isopropyl alcohol ("IPA").
In some embodiments, the growth medium for fermentation includes inexpensive raw materials such as raw glycerol and corn steep liquor. In some embodiments, fermentation is performed to high density in a large volume (e.g., 25 L or greater). Thus, in some embodiments, methods are provide for oil extraction from oleaginous yeast which is cost effective, commercially viable and feasible on a large scale. In some embodiments, the oleaginous yeast is

Cryptococcus curvatus.
In some embodiments, methods are provided for the production of biodiesel (e.g., biodiesel comprising fatty acid methy esters, "FAMEs") from oleaginous yeasts. In some embodiments, methods are provided for biodiesel production using oil extracted from the yeast Cryptococcus curvatus. In some embodiments, the methods include transesterifiaction of oil extracted from the oleaginous yeast. In other embodiments, the methods include the direct transesterification of oleaginous yeast biomass to make biodiesel.
In some embodiments, methods are provided for oil extraction and biodiesel production from oleaginous yeast, such as Cryptococcus curvatus, which is cost effective, commercially viable and feasible on a large scale.
In some embodiment, methods and compositions are provided to maximize the production of yeast biomass by high cell density fermentation.
In some embodiments, methods and compositions are provided to further reduce the cost of yeast production in high density in fermentors by recycling the yeast cells after extraction of oil.
In some embodiments, methods and compositions are provided to scale up high cell density yeast fermentation in 25 L (or larger) bioreactor.
In some embodiments, methods and compositions are provided to provide a process for efficient extraction of the oil from the yeast.
In some embodiments, methods and compositions are provided to optimize the process of extraction of the oil from the yeast using varying proportion of solvents, homogenization cycles, extraction time, etc.
In some embodiments, methods and compositions are provided to provide a process that is cost effective, commercially viable and feasible on a large scale.
In some embodiments, methods and compositions are provided for maximizing the production of yeast in high density in fermentor using cheap raw materials, such as crude glycerol and

corn steep liquor. In some embodiments, fed batch fermentation is used.
In some embodiments, methods and compositions are provided for producing yeast biomass using crude glycerol and corn steep liquor, without using malt extract and/or peptone.
In some embodiments, methods and compositions are provided for producing yeast biomass by recycling the yeast cells after extraction of oil.
In some embodiments, methods and compositions are provided for providing yeast biomass in large scale fermentors of up to 25 L or 27 L scale.
In some embodiments methods and compositions are provided for the extraction of oil from yeast by high pressure homogenization.
In some embodiments methods and compositions are provided for the process of extracting oil from homogenized yeast by varying the proportion of solvents, homogenization cycles and extraction time.
In some embodiments, methods and compositions are provided for maximizing the production of oil from yeast.
IV. EXAMPLES
The following examples are included to demonstrate embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art will, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1: Growth conditions of organism
The yeast strain Cryptococcus curvatus used in the present study was grown in 500 ml flask containing 200 ml of the MGYP sterilized medium containing (g/L): glucose 10; peptone 5;

yeast extract 5; malt extract 3; pH 5.5 at 28°C. After 24 hours of incubation time the 20% v/v inoculum was added to fermentation medium.
Example 2: Determination of yeast dried mass
The dry weight of the fermented yeast was determined as follows. One mL of the fermentation culture was centrifuged at 13,000 rpm for 10 minutes. The biomass pellet was washed twice with distilled water and dried at 40°C to constant mass (usually 24 h). The biomass was determined gravimetrically.
Example 3: Fed batch fermentation using crude glycerol derived from biodiesel process.
Fed batch fermentation was carried out in a bioreactor (Sartorius B-plus, Germany) with a working volume of 3.0 L. To grow in high cell density, Cryptococcus curvatus was grown in medium containing (w/v) crude glycerol 1%, corn steep liquor 2% , bakers yeast autolysate 0.5% and malt extract 0.2%. The fermentor was inoculated with 20% (v/v) inoculum raised as in Example 1. The fermentor was run in fed batch mode at pH 5.5 at 28°C. The pH was controlled during the run by adding 3N sodium hydroxide and 3N sulphuric acid as necessary. The fermentation parameters included agitation at 700 rpm with 1 vvm of aeration. Residual glycerol was monitored periodically and feed of crude glycerol was added when the glycerol was consumed. A total of 7% (v/v) glycerol was added in the fermentor as feed. A maximum O.D. of 236.55 and dry weight of 79 g/L was obtained in 113 h of fermentation (Figure 1).
Example 4: Fed batch fermentation without malt extract
In order to reduce the medium cost, fed batch fermentation was carried out as in Example 3 but without malt extract (nor peptone, included in Example 1), with a working volume of 4.0 L. All other fermentation parameters were as described in Example 3. The fermentor was run for 112 h and an O.D. of 200.4 was obtained with dry weight of 56.5 g/L (Figure 2).
Example 5: Scale up of fed batch fermentation without malt extract
Scale up of fed batch fermentation was carried out 25 L scale in a Bioflow-5000 fermentor (New Brunswick). All other fermentation parameters were as described in Example 4 except the agitation was carried out at 380 rpm as per tip speed of the impeller. A maximum O.D. of

174 was obtained in 136 h with dry weight of 44,6 g/L. There was no further increase in biomass after 136 h (Figure 3).
Example 6: Fed batch fermentation using de-oiled Cryptococcus yeast as a nitrogen source
To make the process more economically viable, baker's yeast autolysate in the medium was replaced by de-oiled Cryptococcus curvatus yeast cells collected from previous fermentation and oil extraction batches. All other media components and fermentation conditions were same as in Example 4. The fermentor was run for 112 h and a maximum O.D. of 186 with dry weight of 54.2 g/L (Figure 4) was obtained.
Example 7: Yeast oil extraction with chloroform and methanol
Oil extraction of Cryptococcus curvatus was carried out using methanol: chloroform at a ratio 1:2. Wet cells (1000 g) fermented as described in example 6 were obtained by centrifugation and mixed with 5 L of methanol and 10 L of chloroform and stirred overnight at room temperature. Water (10 L) was added to the mixture to completely separate the chloroform layer. Oil was recovered from the chloroform layer by evaporation of the solvent in the rotavapor. The oil yield was 30% with respect to the dry weight of the cells.
Example 8: Yeast oil extraction with dichloromethane (DCM) and methanol
Oil extraction of Cryptococcus curvatus was carried out by using methanol:DCM at a ratio of 1:2. Wet cells (1000 g) fermented as described in example 6 were obtained by centrifugation and mixed with 5 L of methanol and 10 L of chloroform and stirred overnight at room temperature. Water (10 L) was added to the mixture to completely separate the DCM layer which separates immediately on agitation. Oil was recovered from the DCM layer by evaporation of the solvent in the rotavapor. The oil yield was 30% with respect to dry weight of the cells.
Example 9: Yeast oil extraction with n-hexane
Centrifuged Wet cells of Cryptococcus curvatus (100 g) prepared by the method described in example 6 were treated with 1 L of n-hexane, stirred overnight at room temperature and the n-

hexane layer was separated. Oil was recovered from the n-hexane layer by evaporation of the solvent in the rotavapor. The oil yield was 7% with respect to dry weight of the cells.
Example 10: Yeast oil extraction with n-hexane and isopropyl alcohol (IPA)
Oil extraction of Cryptococcus curvatus was carried out by using n-hexane:IPA at a ratio of 3:2. Centrifuged wet cells fermented as described in example 6 (500 g) were mixed with 9 L of n-hexane:IPA (3:2) and stirred overnight at room temperature. Water (1 L) was added to the mixture to separate the n-hexane layer completely which occurs immediately on agitation. Oil was recovered from the n-hexane layer by evaporation of the solvent in the rotavapor. The oil yield was 24% with respect to dry weight of the cells.
Example 11: Yeast oil extraction with n-hexane and IPA using dried celts
For the extraction of oil from yeast biomass, a 3:2 ratio of n-hexane:IPA was used. In a typical batch, 50 grams of freeze dried cells fermented as described in example 6 were mixed with 5.4 L n-hexane and 3.6 L IPA and stirred overnight at room temperature. To completely separate the n-hexane layer, 1L of water was added to the mixture and stirred. The n-hexane layer was recovered and evaporated in a rotavapor to recover oil. The extraction was repeated for four batches of 50 g each. The oil recovery was between about 10% and about 17% with respect to dry cell weight (DCW) of the yeast (Table 1).
Table 1

Weight of Cryptococcus curvatus (gm) Oil recovery
(mL) Yield oil on DCW basis %
50 5.5 11
50 5 10
50 8.5 17
50 8 16
Example 12: Yeast biomass homogenization and effect of cycles of passages on oil extraction
To boost oil production as compared to that of the previous examples, the process was

modified and cells were homogenized prior to oil extraction. The cell mass from fermented broth prepared as described in example 6 was collected, by centrifugation at 10000 rpm for 20 minutes (Sorvall evolution). The centrifuged cell mass was diluted to an O.D. of 222 (measured at 660 nm) using tap water and was subjected to homogenization at 4°C under 800 bar pressure in a homogenizer (GEA, Italy)(the process takes about 4 min for homogenization of 1 L of cells). Six passages were taken and the reduction in optical density was measured after each passage. The initial O.D. was 214 and was reduced to 135 after six passages. For the extraction of oil, 200 ml of homogenized cells were sampled after each passage. The cells were mixed with 400 ml of n-hexane and stirred overnight on a magnetic stirrer at room temperature. The bottom aqueous layer was removed and 200 ml IPA was added to the cell-hexane mixture. The top n-hexane layer was recovered and evaporated to recover the oil (Table 2). No significant increase in oil recovery was seen beyond 2 passages.
Table 2: Oil recovery from cells at various steps of passages of homogenization.

Number of Homogenization Passages Oil recovery mL
1 4.5
2 5
3 5.5
4 5.5
5 5
6 5.5
Example 13: Homogenization of the fermentation broth and oil extraction
Direct homogenization of fermented broth was performed, thereby eliminating the step of centrifugation to collect cell mass. After harvesting (collecting the fermented broth into another vessel) the cells (fermented as described in example 6), the fermented broth was subjected to homogenization at 4°C under 800 bar pressure in homogenizer to break the cells. Five passages were taken and reduction in optical density was measured at various passages. The initial optical density was 190 which was reduced to 71 after five passages. The ratio of homogenized cells to n-hexane was also varied to check its effect on extraction of oil. Table 3 represents oil recovery and it was found that oil recovery was higher if the cells:n-hexane ratio was maintained at 1:2.

Table 3: Oil recovery form homogenized cells using two different ratios of cells to n-hexane.

Homogenized cell: N-hexane ratio Oil recovery (mL) Yield oil on DCW basis %
1:1 6 50
1:2 8 66
Example 14: Scale up of oil extraction in 50 L agitator vessel
The extraction process was carried out in 50 L agitator tank and the ratio of homogenized cells to n-hexane ratio was maintained at 1:2. The agitation time was varied. Homogenized cells (5 L) were mixed with 10 L n-hexane and stirred for two different agitation times including lh and 5 h at 950 rpm. The aqueous layer was removed; isopropyl alcohol ("IPA") was added to separate the organic layer which was concentrated to recover oil. Table 4 shows that there was no difference in the oil yield after 1 h or 5 h of agitation. The process can therefore be scaled up without any loss.
Table 4: Effect of agitation time on oil extraction

Agitation time (h) Oil recovery (mL) Yield oil on DCW basis %
1 110 43
5 110 43
Example 15: Validation of optimized oil extraction process
Consistency of the optimized oil extraction process was performed by testing the oil yield of six different batches. We have taken different amount of cells (from similar fermentation) which is indicated in the table now and repeated the extraction process Table 5 represents oil yield percentage in the six batches. In all batches, homogenized cells to n-hexane ratio were maintained at 1:2 and agitation was carried out for 1 h at 950 rpm. The oil yield obtained was at least 43% for all six batches, and two batches provided an oil yield of 54.34%. A comparison of fatty acid profile of Cryptococcus oil with that of Jatropha oil is also presented (Table 6). .

Jatropha oil was obtained by extracting crushed jatropha curcas seeds with petroleum ether in a Soxhlet apparatus. The oil in the seeds get extracted in the solvent which is then evaporated in a rotavapor to obtain the oil.
The oil profile shows that Cryptococcus oil contains all the major fatty acid found in jatropha oil which has been established as a source for biodiesel production. Hence, yeast oil is a potential source of biodiesel. The oil from yeast shows more saturation (29.7 % of palmitic acid as compared to 14.66 % in jatropha; 50.6 % of oleic acid vs 39.08 % in jatropha oil) which improves the stability of the oil and biodiesel.
Table 5: Oil yield from certain batches

Batch Number Cells (dry weight) g Oil recovery (naL) Yield oil on DCW basis %
1 244 110 43
2 234 115 49
3 237 102 43
4 184 82.5 44.8
5 184 100 54.34
6 184 100 54.34
Table 6: Comparison of fatty acid profile of Cryptococcus oil with Jatropha oil

Type of fatty acid Jatropha oil Cryptococcus oil
C14:0 (Myristic acid) - 0.6%
C16:0 (Palmitic acid) 14.66% 29.7%
C16:l(Palmitolic acid) 0.94% 1.0%
CI8:0 (Stearic acid) 6.86% 9.6%
C18:l (Oleic acid) 39.08% 50.6%
C18:2 (Linoleic acid) 32.48% 7.5%
C18:3 (Linolenic acid) 0.30% -
C20:0 (Arachidic acid) 0.24% -
UNKNOWN 5.44% 1.0%
Example 16: Refining of oil and transesterification
For biodiesel production, the crude yeast oil was subjected to free fatty acid measurement before the transesterification reaction. Free fatty acid levels are measured by titration with KOH. The initial free fatty acid levels were at 9.6%. To remove the impurities and free fatty acids; crude oil was subjected to neutralization process. The neutralization process included

degumming, free fatty acid removal and bleaching. Degumming is a process in which crude oil was heated at 60°C in presence of 0.1% of H3PO4 and 1% hot water ( at a temp of >90°C). After heating for 1 h, the solution was settled at room temperature for 2 h and the gum was drained away. The oil was then subjected to a free fatty acid removal process as follows. Oil was heated to 70-75°C in presence of sodium hydroxide with slow stirring. The solution was allowed to settle for 2 hours at room temperature. The soap formed by the reaction was then drained away. Two to three hot water ( at a temp of >90°C). washes were then performed to remove additional soap. The free fatty acid content, moisture and volatile matters were measured at each step of neutralization process. The moisture and volatile matter content is determined by taking 10 g (W).of oil sample in a petri dish, measuring the weight (Wl) and heating to 105 + 1°C for one hour in a hot air oven. The weight of the dish with the oil is again measured (W2) and moisture content is calculated by the formula lOOx (Wl-W2)/W. After free fatty acid removal described above, the oil was dried and subjected to a bleaching process in presence of 1% bleaching earth. Refined oil was dried under vacuum at 105 + 1°C for one hour. For bleaching, oil after neutralisation was treated with 1% SiO2 at 100 °C under vacuum for one hour. After this filtration was done to remove impurities and SiO2 and refined oil was collected. The bleached oil (refined oil) was then subjected to a trans-esterification process. Refined oil was treated with 30% sodium methylate at 60-65°C for 2 hours. The lower glycerol layer was removed and crude biodiesel was subjected to hot water washes to remove soap and impurities. Parameters of crude oil, refined oil and yeast biodiesel are given in Table 7 and 8. Table 7: Parameters of crude yeast oil and refined yeast oil.

Parameter Crude yeast oil Refined yeast oil
Free fatty acid (%) 9.5 0.26
P content (ppm) 700 5
Gum (%) 1.75 0.013
Saponification value 152 152
Unsaponifiable matter (%) 3.2 1
Moisture content (%) 1.7 0.06
Iodine value 60 60
Color 28.5 unit 22.5 unit

Table 8: Parameters of yeast biodiesel obtained after trans ester ification of refined yeast oil)

Parameters (Test method) Yeast Biodiesel
Acid value (EN14104) 0.47
Soap (ppm) 50
Moisture content (ppm) 500
Density g/ml (EN IS03 675) 878.6
Viscosity cm2/sec (EN IS03 3 04) 6.061
Iodine value (EN14111) 59
Cloud point (ASTM-D2500) +8°C
Flash point (ASTM D93) >120°C
Cu strip corrosion (ASTM D130) la
Carbon residue % (ASTM D524) 0.01
Sulphated ash % (IS1448P:04) 0.02
Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.

WE CLAIM:
1. A composition comprising:
crude glycerol,
corn steep liquor,
yeast autolysate, and
oleaginous yeast,
wherein the pH of the composition is about 5.5.
2. The composition of claim 1, comprising:
crude glycerol at about 1% (w/v), corn steep liquor at about 2% (w/v), yeast autolyaste at about 0.5% (w/v), and oleaginous yeast comprising Cryptococcus curvatus.
3. A method for isolating oil from fermented oleaginous yeast, the method comprising:
a) fermenting oleaginous yeast in a fermentation medium comprising:
crude glycerol,
corn steep liquor,
yeast autolysate, wherein the pH of the fermentation medium is about 5.5;
b) extracting the oil from the fermented oleaginous yeast.
4. The method of claim 3, wherein extracting comprises treating the fermented oleaginous yeast with one or more of the compounds or combinations selected from the group consisting of: dichloromethane; chloroform; n-hexane; a combination of methanol and chloroform; a combination of methanol and dichloromethane; and a combination of n-hexane and isopropyl alcohol.
5. The method of claim 3, wherein the fermented oleaginous yeast is collected by centrifugation prior to the extraction step.

6. The method of claim 3, wherein the fermented oleaginous yeast is homogenized prior to the extraction step.
7. The method of claim 3, wherein the fermented oleaginous yeast is dried prior to the extraction step.
8. The method of claim 3, wherein the fermented oleaginous yeast is frozen prior to the extraction step.
9. The method of claim 6,.wherein the fermented oleaginous yeast is collected by. centrifugation prior to the homogenization step.

10. The method of claim 6, wherein the fermented oleaginous yeast is homogenized without a prior centrifugation collection step.
11. The method of claim 3, wherein the oleaginous yeast comprises Cryptococcus curvatus.
12. The method of claim 10, wherein the oleaginous yeast comprises Cryptococcus curvatus.
13. The method of claim 11, wherein the extracted oil yield comprises at least about 40% of the dry weight of the fermented yeast.
14. The method of claim 11, wherein the extracted oil comprises about 50% of the dry weight of the fermented yeast.
15. The method of claim 11, wherein the fermentation medium consists of a volume of about 25 liters or greater.
16. The method of claim 3, wherein the fermentation medium comprises:

crude glycerol at about 1 % (w/v),
com steep liquor at about 2% (w/v),
yeast autolyaste at about 0.5% (w/v)s and wherein the pH of the fermentation medium
is about 5.5
17. A method for producing biodiesel, the method comprising:
a) fermenting oleaginous yeast in a fermentation medium comprising:
crude glycerol,
corn steep liquor,
yeast autolyaste, wherein the pH of the fermentation. medium is about 5.5;
b) extracting the oil from the fermented oleaginous yeast; and
c) transesterifying the extracted oil.
18. The method of claim 17, wherein:
a) the fermentation medium comprises:
crude glycerol at about 1% (w/v),
corn steep liquor at about 2% (w/v),
yeast autolyaste at about 0.5% (w/v), and wherein the pH of the fermentation medium is about 5.5;
b) extracting the oil comprises:
homogenizing the fermented oleaginous yeast without a prior centrifugation collection step; and
c) the oleaginous yeast comprises Cryptococcus curvatus.
19. A composition comprising the oil extracted by the method of claim 3, wherein the oil
comprises:
at least 0.2% myristic acid; at least 25% palmitic acid; at least 0.5% palmitoleic acid; at least 9% stearic acid; at least 45% oleic acid; and

at least 5% linoleic acid.
20. The composition of claim 19, wherein the oil comprises:
about 0.6% myristic acid;
about 29.7% palmitic acid; about 1.0% palmitoleic acid; about 9.6% stearic acid; about 50.6% oleic acid; and about 7.5% linoleic acid.
21. A composition comprising biodiesel produced by the method of claim 18, the biodiesel
having the following parameters:
acid value (EN14104) of 0.47;
soap (ppm) of 50;
moisture content (ppm) of 500;
density g/ml (ENIS03675) of 878.6;
viscosity cm2/sec (EN ISO3104) of 6.061;
cloud point (ASTM-D2500) of +8°C
flash point (ASTM D93) of > 120°C;
Cu strip corrosion (ASTM D130) of la;
carbon residue % (ASTM D524) of 0.01; and
sulphated ash % (ISI448P:04) 0.02.
22. The method of claim 11 or 17, wherein the method does not comprise fermenting the . yeast in a fermentation medium comprising malt extract.
23. The method of claim 11 or 17, wherein the method does not comprise fermenting the yeast in a fermentation medium comprising peptone or malt extract.
24. The composition of claim 1, wherein the oleaginous yeast comprises Cryptococcus curvatus, and the composition does not comprise peptone or malt extract.

25. The method of claim 3, wherein the oleaginous yeast comprises Cryptococcus curvatus, and yeast cell biomass after fermentation is at least about 40 g/L on a dry weight basis.
26. The method of claim 25, wherein the yeast cell biomass after fermentation is at least about 50 g/L on a dry weight basis.
27. A method for isolating oil from fermented oleaginous yeast, the method comprising: a) fermenting Cryptococcus curvatus in a fermentation medium comprising:
crude glycerol,
corn steep liquor,
yeast autolysate, wherein the pH of the fermentation medium is about 5.5; b) extracting the oil from the fermented Cryptococcus curvatus; wherein the method does not comprise fermenting the Cryptococcus curvatus in a fermentation medium comprising malt extract or peptone, and yeast cell biomass after fermenting is at least about 40 g/L on a dry weight basis.
28. A method for isolating oil from fermented oleaginous yeast, the method comprising:
a) fermenting Cryptococcus curvatus in a fermentation medium comprising:
crude glycerol, corn steep liquor, and previously de-oiled yeast cells;
b) extracting the oil from the fermented Cryptococcus curvatus;
wherein the method does not comprise fermenting the Cryptococcus. curvatus in fermentation medium comprising malt extract or peptone or bakers yeast autolysate, and yeast cell biomass after fermenting is at least about 40 g/L on a dry weight basis.
29. The method of claim 28, wherein the yeast cell biomass after fermenting is at least
about 50 g/L on a dry weight basis.

30. The method of claim 11,17 or 27, wherein the method does not comprise fermenting
Cryptococcus curvatus in a fermentation medium comprising peptone, tryptone, beef .
extract, malt extract, galactose, starch, arabinose, glycerol, mannitol, sucrose or
fructose.
31. A composition comprising:
crude glycerol,
corn steep liquor,
previously fermented and de-oiled yeast cells, and
oleaginous yeast.
32. A method for isolating oil from fermented oleaginous yeast, its compositions and application in biodiesel as claimed above exemplified herein substantially in the examples and figures.