WO2012006302A1

WO2012006302A1 - Cultivation of green algae chlorococcum pamirum for production of biofuel

Info

Publication number: WO2012006302A1
Application number: PCT/US2011/043001
Authority: WO
Inventors: Pingzhong Feng; Zhengyu Hu; Fan Lu
Original assignee: Chlor Bioenergy Inc.
Priority date: 2010-07-06
Filing date: 2011-07-06
Publication date: 2012-01-12
Also published as: CN103052715A; CN103052715B

Abstract

This invention provides methods for cultivating microalgae Chlorococcum pamirum (Chlorophyceae class) and inducing oil accumulation in the green algae. Also disclosed are methods of purifying the organisms, media used for culturing the organisms, and methods for enriching the oil content in the organisms. The oil-rich microalgae produced according to the present invention can be used as feedstock for production of biodiesel in large-scales.

Description

CULTIVATION OF GREEN ALGAE CHLOROCOCCUM PAMIRUM FOR

PRODUCTION OF BIOFUEL CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 61/361,790, filed on July 6, 2010, which is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION This invention relates to a method for purifying green algae Chlowcoccum pamirum, methods for cultivating the algae and methods for enriching its oil content. The oil-enriched algae is useful for large-scale production of biofuel.

BACKGROUND OF THE INVENTION

Sustainable energy supply and environmental protection are now being recognized as the two most critical issues facing the international communities, in particular the United States, in this century. For example, the U.S. is heavily dependent on imported oil, particularly its transportation sector that consumes two thirds of all the fossil oil used in the country. Oil import costs make up a third of the growing U.S. trade deficit and represent an increasing burden on the U.S. economy.

Although the U.S. can continue increasing its oil imports, the global oil supplies are not infinite. Even based upon an optimistic estimate of the world oil resource of approximately 2,200-3,900 billion barrels, nearly twice the proven reserve, the world supply of petroleum oil will be gone within 30 to 50 years. Demand for oil by emerging and rapidly growing economies in China, India, and elsewhere is further increasing competition and price volatility for limited global supplies.

Moreover, the U.S. is facing a confluence of problems associated with the world's heavy use of fossil energy in general, and America's heavy reliance on foreign oil imports for transportation fuels in particular. Liquid transportation fuel use drives the nation's oil import demand, and is also responsible for about half of the country's fossil fuel based greenhouse gas emissions, which is another growing concern over the impacts of national and global fossil fuel use and associated greenhouse gas emissions on climate changes.

The economic and environmental impacts associated with oil import and consumption constitute a major national security issue. The severity of the resulting impacts will depend on how much, how quickly, and how far in advance of complete exhaustion of global oil supplies it will be possible to reduce demand for transportation fuels by providing sustainable supplies from renewable, domestically-controlled alternative fuel sources while addressing greenhouse gas emissions.

Microalgae are groups of photosynthetic microorganisms that can utilize inorganic nutrients (e.g. nitrogen, phosphorus) from the environment to produce organic compounds, such as protein, pigment, and oil. Microalgae are microscopic algae that are found in freshwater and marine biosystems. They are mostly unicellular microorganisms. Microalgae are known to exhibit 10- to 20-fold higher growth rates than agricultural crop plants, and certain microalgal species can accumulate large amounts of lipids or oil (30- 60% of dry weight). As a result, the concept of using microalgae as an alternative source of feedstock for biofuel, in particular biodiesel, production was intensively studied

Although microalgae represents a promising alternative for biodiesel production in laboratory scales, no commercial production of biodiesel from microalgae is yet available. The challenge is to successfully develop and commercialize cost-effective and highly efficient technologies, systems, and processes that can realize the potential.

SUMMARY OF THE INVENTION

The present invention was designed to meet the foregoing challenge by providing methods to purify and cultivate cells of microalgae strain Chlowcoccum pamirum so as to optimize growth of this strain for optimum production of oil and lipids from the microalgae. The methods of the present invention induce and enhance accumulation of oil within the cells of Chlowcoccum pamirum. The methods disclosed are suitable for large- scale production of oil-rich microalgae.

More specifically, this invention provides growth media and culture conditions that are used for optimizing the growth of the Chlowcoccum pamirum strain and for inducing oil accumulation in the cells of Chlowcoccum pamirum. In one aspect the present invention provides a method of increasing oil accumulation in microalgae of Chlowcoccum pamirum strain, comprising incubating the microalgae in a culture medium.

In another aspect the present invention provides a culture medium used for production of oil-rich microalgae, the culture medium comprising micronutrients and inorganic compounds selected from NaN0₃, MgS0₄, Na₂C0₃, CaCl₂, Na₂EDTA, and K₂HP0₄, and the like.

In one embodiment, the culture medium contains about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄3H₂0, and about 0.2-2 mL/L of A5 micronutrients (nitrogen-deficient) .

In another embodiment, the culture medium further contains about 0.25-2.5 g/L NaN0₃.

In another embodiment, the culture medium contains about 0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, and about 0.2-2 mL/L of A5 micronutrients (pho sphorus -deficient) .

In another embodiment, the culture medium further contains NaCl at a concentration of at least 0.1% w/v.

In another aspect the present invention provides a method of making feedstock for production of biodiesel, comprising: (a) incubating microalgae Chlowcoccum pamirum in a culture medium; (b) inducing oil enrichment in the microalgae; (b) isolating the oil- enriched microalgae from the culture medium; and (c) extracting lipids or oil from the oil- enriched microalgae.

In another aspect the present invention provides use of microalgae Chlowcoccum pamirum in production of oil-rich microalgae.

In another aspect the present invention provides use of Chlowcoccum pamirum in production of oil or lipids.

In another aspect the present invention provides use of the methods described herein for producing oil-rich microalgae useful as feedstock for biodiesel production. In another aspect the present invention provides a microalgae cell collection comprising oil-enriched microalgae Chlorococcum pamirum, wherein said oil-enriched microalgae cells comprise oil at a level higher than the oil level in the corresponding microalgae cells at their natural state prior to incubation.

These and other aspects of the present invention will be better appreciated by reference to the following drawings, detailed description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and IB illustrate the effects of nitrogen concentrations on the growth of Chlorococcum pamirum measured as changes in (A) optical density at 550 nm; (B) dry weight.

FIGs. 2A and 2B illustrate the effects of phosphate concentrations on the growth of Chlorococcum pamirum measured as changes in (A) optical density at 550 nm; (B) dry weight.

FIGs. 3A and 3B illustrate the effects of nitrogen and/or phosphate deficiency on the growth (A. measured as changes in dry weight) and on the oil content (B. measured as percentage of dry weight) of Chlorococcum pamirum.

DETAILED DESCRIPTION OF THE INVENTION

Chlorococcum pamirum is a known strain of microalgae from the class Chlorophyceae and has been deposited in the University of Texas Culture Collection of Algae at the University of Texas at Austin under UTEX deposit number 2498. The present invention involves methods and compositions for purifying and culturing Chlorococcum pamirum and for enhancing the accumulation of oil in the cells of Chlorococcum pamirum.

In one aspect the present invention provides a method of increasing oil accumulation in microalgae Chlorococcum pamirum, the method comprising incubating the microalgae in a culture medium and inducing oil accumulation in the microalgae.

In one embodiment of this aspect, said incubating comprises maintaining the microalgae cells in an agar plate containing a culture medium and cultivating the microalgae cells in a container containing a culture medium. In another embodiment of this aspect, said incubating comprises maintaining the microalgae cells in an agar plate containing a culture medium; cultivating the microalgae cells in a container containing a growth medium; and further cultivating the microalgae in a photobioreactor.

In another embodiment of this aspect, said incubating comprises illuminating the microalgae cells with a light in at least one of the steps as described above.

In another embodiment of this aspect, said incubating comprises illuminating the microalgae cells with a light throughout every step.

In another embodiment of this aspect, the light used for illuminating is a fluorescent light, normal daylight, or sunlight.

In another embodiment of this aspect, the fluorescent light has an intensity of at least 200 μιηοΐ m^" s^" .

In another embodiment of this aspect, the culture medium contains micronutrients and inorganic compounds selected from NaN0₃, MgS0₄, Na₂C0₃, CaCl₂, Na₂EDTA, and K₂HP0₄, and the like.

In another embodiment of this aspect, the culture medium contains about 0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025- 0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄3H₂0, and about 0.2-2 mL/L of A5 micronutrients, with pH in the range of 7.4-8.2.

In another embodiment of this aspect, the culture medium consists essentially of about 0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄ ^'3H₂0, and about 0.2-2 mL/L of A5 micronutrients, with pH in the range of 7.4-8.2.

In another embodiment of this aspect, said inducing oil accumulation comprises incubating the microalgae cells to a nitrogen-deficient medium. In one embodiment the nitrogen-deficient medium contains about 0.025-1.75 g/L MgS0₄ ^'7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄ ^'3H₂0, and about 0.2-2 mL/L of A5 micronutrients. In another embodiment, the nitrogen-deficient medium consists essentially of 0.025-1.75 g/L MgS0₄ ^'7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄ ^'3H₂0, and about 0.2-2 mL/L of A5 micronutrients, with pH in the range of about 7.4-8.2.

In another embodiment of this aspect, said inducing oil accumulation comprises incubating the microalgae cells in a phosphorus-deficient medium. In one embodiment, the phosphorus-deficient culture medium contains about 0.25-2.5 g/L NaN0₃, about

0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, and about 0.2-2 mL/L of A5 micronutrients. In another embodiment of this aspect, the phosphorus-deficient culture medium consists essentially of about 0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, and about 0.2-2 mL/L of A5 micronutrients, with a pH in the range of about 7.4-8.2.

In another embodiment of this aspect, said inducing oil accumulation comprises illuminating the microalgae cells Chlowcoccum pamirum with a light having an intensity that is greater than at least 200 mmol m^" s^" .

In another embodiment of this aspect, said inducing further comprises incubating the microalgae cells in a culture medium comprising NaCl at a concentration of at least

0.1% w/v. In one embodiment, such a culture medium can be obtained by adding an appropriate amount of NaCl into the culture medium.

In another aspect the present invention provides a culture medium for enhancing oil accumulation in microalgae Chlowcoccum pamirum, the culture medium comprising micronutrients and inorganic compounds selected from NaN0₃, MgS0₄, Na₂C0₃, CaCl₂,

Na₂EDTA, and K₂HP0₄, and the like.

In one embodiment of this aspect, the culture medium contains about 0.25-2.5 g/L

NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄3H₂0, and about 0.2-2 mL/L of A5 micronutrients.

In another embodiment of this aspect, the culture medium consists essentially of about 0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L

Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄3H₂0, and about 0.2-2 mL/L of A5 micronutrients. In another embodiment of this aspect, the medium culture contains about 0.025- 1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄3H₂0, and about 0.2-2 mL/L of A5 micronutrients. In another embodiment of this aspect, the culture medium consists essentially of about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025- 0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄3H₂0, and about 0.2-2 mL/L of A5 micronutrients (nitrogen-deficient).

In another embodiment of this aspect, the culture medium contains about 0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025- 0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, and about 0.2-2 mL/L of A5 micronutrients. In another embodiment of this aspect, the culture medium consists essentially of about 0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5- 3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, and about 0.2-2 mL/L of A5 micronutrients (phosphorus-deficient).

In another embodiment of this aspect, the culture medium as described in any embodiments above further contains NaCl at a concentration of at least 0.1% w/v.

In another aspect the present invention provides a microalgae cell collection comprising oil-enriched microalgae cells of Chlowcoccum pamirum strain, wherein said oil-enriched microalgae cells comprise oil at a level higher than the oil level in the corresponding microalgae cells at their natural state prior to incubation.

In one embodiment of this aspect, the cell collection is isolated from the culture medium.

In another embodiment of this aspect, the cell collection is purified.

In another embodiment of this aspect, the oil level of the cell collection is at least 10% higher than the natural oil level.

In another embodiment of this aspect, the oil level of the cell collection is at least 20% higher than the natural oil level.

In another embodiment of this aspect, the oil level of the cell collection is at least 30% higher than the natural oil level.

In another embodiment of this aspect, the oil level of the cell collection is at least

50% higher than the natural oil level. In another embodiment of this aspect, the oil level of the cell collection is at least 100% higher than the natural oil level.

In another embodiment of this aspect, the microalgae cell collection comprises at least 30% of oil based on dry weight.

In another embodiment of this aspect, the microalgae cell collection comprises at least 40% of oil based on dry weight.

In another embodiment of this aspect, the microalgae cell collection comprises at least about 50% of oil based on dry weight.

In another embodiment of this aspect, the microalgae cell collection comprises about 50-70% of oil based on dry weight.

In another embodiment of this aspect, the microalgae cell collection comprises about 55-65% of oil based on dry weight.

As a person of ordinary skill in the art would understand, the initial cell concentration is in general not critical to the practice of the present invention. For example, without limitations, one can start with an initial cell concentration in the range of 10,000-1,000,000 cells per milliliter (mL) of culture medium as described herein.

In another aspect the present invention provides a method of making feedstock for production of biodiesel, comprising: (a) incubating microalgae of Chlowcoccum pamirum strain in a culture medium; (b) inducing oil enrichment in the microalgae; (b) isolating the oil-enriched microalgae from the culture medium; and (c) extracting lipids or oil from the oil-enriched microalgae.

In one embodiment of this aspect, said purifying comprises transferring the microalgae to agar plates containing the culture medium.

In another embodiment of this aspect, said inducing oil accumulation comprises cultivating cells of microalgae at a temperature from about 5 °C to about 35 °C.

In another embodiment of this aspect, said inducing oil accumulation comprises cultivating cells of microalgae at a temperature from about 25 °C to about 30 °C.

In another embodiment of this aspect, the method further comprises incubating microalgae cells in a nitrogen deficient medium.

In another embodiment of this aspect, the method further comprises incubating microalgae cells in a phosphorus deficient medium. In another embodiment of this aspect, said inducing oil accumulation comprises illuminating the microalgae with a light.

In another embodiment of this aspect, the light for illuminating is a fluorescent light, normal daylight or sunlight.

In another embodiment of this aspect, the method further comprises scaling up the culture of the microalgae.

In another embodiment of this aspect, the scaling up comprises: maintaining the microalgae in agar plates containing a growth medium; culturing the microalgae in a reactor containing 50-1000 mL medium; and culturing the microalgae in 5-20 L photobioreactor s .

In another aspect the present invention provides use of microalgae Chlorococcum pamirum in production of oil or lipids.

In another embodiment of this aspect, the microalgae chlorococcum pamirum is enriched with oil through incubating in a culture medium.

In another aspect the present invention provides use of an oil-enriched cell collection of microalgae Chlorococcum pamirum as feedstock for biodiesel production.

In one embodiment of this aspect, the oil level of the cell collection is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 100%, or at least 120% higher than the oil level in the Chlorococcum pamirum microalgae strain in its natural state.

In another embodiment of this aspect, the oil level of the cell collection is 30-70% of oil based on dry weight.

In another embodiment of this aspect, the oil level of the cell collection is 40-70% of oil based on dry weight.

In another embodiment of this aspect, the oil level of the cell collection is 50-65% of oil based on dry weight.

In another embodiment of this aspect, the oil level of the cell collection is 55-60% of oil based on dry weight. In another aspect the present invention provides use of an oil-enriched cell collection of Chlowcoccum pamirum strain, or an isolate variant thereof, in production of oil and/or lipids.

In another embodiment of this aspect, the oil level of the cell collection is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 100%, or at least 120% higher than the oil level in the Chlowcoccum pamiraum microalgae strain in its natural state.

In another embodiment of this aspect, the oil level of the cell collection is 55-60% of oil based on dry weight.

The term "about," as used herein, refers to a range of values within ten percent (10%) of a baseline value. Thus, for example, the phrase "about 100" refers to a range of values between 90 and 110.

When the term "about" is applied to a range, it indicates that both the upper limit and lower limit can vary up to ten percent (10%) of the base line value.

The term "a," "an," or "the," as used herein, represents both singular and plural forms. In general, when either a singular or a plural form of a noun is used, it denotes both singular and plural forms of the noun.

The term "biofuel," as used herein, refers generally to a type of fuel that is in some way derived from biomass. The term covers solid biomass, liquid fuels and various biogases. However, in the present invention, the term more pertinently refers to biodiesel.

The term "biodiesel," as used herein, refers to commonly known fatty acid esters (e.g., methyl, ethyl, propyl, etc.). The microalgae produced according to the present invention contains mainly lipids (including oil - triglycerides, free fatty acids, phospholipids, and so on). After cultivation of the microalgae, a microalgal biomass is first harvested, which is then extracted to obtain lipids (oil and other lipids). These lipids will be used as feedstock for biodiesel production. For example, the lipids extracted from the oil-rich microalgae can be readily converted to biodiesel by known chemical reactions and/or chemical engineering processes (e.g., by esterification and/or transesterification). In this disclosure, the terms "oil" and "lipids" are sometimes used interchangeably.

The biodiesel produced from the oil-rich microalgae of the present invention has wide applications, which include, but are not limited to, use in standard diesel engines or converted diesel engines of vehicles, trains, aircrafts, etc., or use as heating fuel in either domestic or commercial boilers. The biodiesel can be used alone or blended with petroleum diesel. It can also be used as a low carbon alternative to heating oil. Such uses or variants of uses are within the knowledge of a person of ordinary skill in the art, description of which is merely for illustration purpose, but not intended to be limiting.

The term "nitrogen-deficient medium" or "nitrogen-depleted medium," as used herein, refers to a culture medium that contains no added nitrogen-containing compounds in any form (e.g., nitrate, nitrite, ammonium, etc.). In one embodiment, a nitrogen- deficient medium contains 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄ ^'3H₂0, and about 0.2-2 mL/L of A5 micronutrients.

The term "phosphorus-deficient medium" or "phosphorus-depleted medium," as used herein, refers to a culture medium that contains no added phosphorus-containing compounds in any form (e.g., phosphate, monohydrogenphosphate, dihydrogenphosphate, phosphite, etc.). In one embodiment, a phosphorus-deficient medium contains about 0.25- 2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄ ^'3H₂0, and about 0.2-2 mL/L of A5 micronutrients.

The term "A5 micronutrients," as used herein, refers to a solution containing

H₃B0₃ (about 2.86 g/L), MnCl₂ (about 1.81 g/L), ZnS0₄4H₂0 (about 0.222 g/L), Na₂Mo0₄ (about 0.0177 g/L), CuS0₄5H₂0 (about 0.07 g/L), or any commercial products containing substantially similar components that can be used for substantially similar purposes.

When a hydrate form of a compound is indicated in this application, as a person of ordinary skill in the art would understand, an equivalent amount of anhydrous form or other solvate (including hydrate) forms of the same compound may be used in lieu of the hydrate form indicated, because they would be able to serve substantially similar purpose and provide substantially similar results. Therefore, the anhydrous form, hydrate forms, and other solvate forms, which can be considered to be functional equivalents, are all encompassed by the present invention.

The invention will be further illustrated by the following non-limiting examples.

EXAMPLES

EXAMPLE 1

Collection and purification of microalgae Chlorococcum pamirum

Cells were collected from the Yadkin River of Forsyth County in North Carolina using a phytoplankton net (5 μιη pore size). After washing with sterile medium, the collected cells were spread onto agar plates containing growth media (see 2) below). The plates were illuminated with fluorescent light. After one week, cells from the plates were transferred to a fresh plate using a microbiological transferring loop. After three transfers, axenic single colonies were obtained. A single colony of Chlorococcum pamirum was identified, which was selected for further cultivation and confirmation. The cells of the Chlorococcum pamirum strain are ellipsoidal in shape with a single chloroplast; the cells become spherical in stationary phase cultures.

EXAMPLE 2

Cultivation of microalgae cells of Chlorococcum pamirum species General Procedures

Chlorococcum pamirum cells at exponential phase were inoculated into columns with the inner diameter of 4.5 cm or 2.8 cm, respectively. The maximum culture volume in the corresponding columns were 450 ml and 250 ml accordingly. The columns with the inner diameter of 4.5 cm were used in the experiments of inducing lipid accumulation, while the other type of column was used in the experiments of growth characteristics of. The cultures were illuminated with continuous light intensity of 100 μιηο1·ιη^{~ »}s^~ at the temperature of 25 °C, aeration was 0.6 L/min for each column (the air was filtered through microporous filtering film, the pore size was 0.22 μιη).

Culture medium and growth conditions

The growth media of the present invention used to achieve accelerated growth of Chlorococcum pamirum organisms and to increase the oil content of the organisms contain a concentration of more than 0.1% NaCl. The medium contains the following components: 0.25-2.5 g/L NaN0₃, 0.025-1.75 g/L MgS0₄7H₂0, 0.5-3.6 g/L Na₂C0₃, 0.025-0.20 g/L CaCl₂, 0.001-0.005 g/L Na₂EDTA, 0.075-1.5 g/L K₂HP0₄3H₂0, 0.2-2 mL/L A5 micronutrients, with a final pH in the range of 7.4-8.2.

The method for cultivation of the Chlorococcum pamirum species according to the present invention includes the following steps:

A. Maintenance of the Chlorococcum pamirum strain in agar plates containing the culture medium described above;

B. Cultivation of the Chlorococcum pamirum strain in 125-mL flasks containing 50 mL of the above growth medium for at least 72 hours. The cells are then transferred to

2-liter flasks that contain 1 -liter of the medium described above.

C. The strain is then cultivated in 2-liter flasks containing 1 liter medium for at least 24 hours.

D. The Chlorococcum pamirum strain is then cultivated in 8-liter column photobioreactors for at least 120 hours. Suitable bioreactors are described in International

Patent Publication WO 2005074622, which is hereby incorporated by reference in its entirety.

The Chlorococcum pamirum cells are preferably continuously exposed to a μg light source at all stages of the culturing process. The light intensity to which the cells are exposed during cultivation is between 50-200 μιηοΐ m^"2 s^"1. The culture temperature is preferably maintained between 15°C and 30°C. EXAMPLE 3

Enhanced oil accumulation in microalgae Chlorococcum pamirum

Nitrogen-deficient conditions

Enhanced oil accumulation is induced in Chlorococcum pamirum by exposing the cells to a nitrogen-depleted medium. In operation, the Chlorococcum pamirum cells from the 8-liter photobioreactor are washed with nitrogen-deficient medium containing the following components: 0.025-1.75 g/L MgS0₄7H₂0, 0.5-3.6 g/L Na₂C0₃, 0.025-0.20 g/L CaCl₂, 0.001-0.005 g/L Na₂EDTA, 0.075-1.5 g/L K₂HP0₄3H₂0, 0.2-2 mL/L A5 micronutrient, with a final pH in the range of 7.4-8.2.

Then the cells are incubated in the same nitrogen-depleted (nitrogen free) medium.

The oil content in the cells of the Chlorococcum pamirum strain is significantly enhanced within a week to ten days of cultivation in the nitrogen deficient medium (from an initial oil content of 20-30% of algal dried weight to an oil content of 50-65% of algal dried weight).

Phosphorus-deficient conditions

In a subsequent step, the Chlorococcum pamirum cells are washed with phosphorus-deficient medium containing the following components: 0.25-2.5 g/L NaN0₃, 0.025-1.75 g/L MgS0₄7H₂0, 0.5-3.6 g/L Na₂C0₃, 0.025-0.20 g/L CaCl₂, 0.001-0.005 g/L Na₂EDTA, 0.2-2 mL/L A5 micronutrients, with a final pH in the range of 7.4-8.2.

The cells are then incubated in the phosphorus-deficient medium for ten days or longer. The oil content of Chlorococcum pamirum incubated by this method is significantly enhanced within about ten days (from an initial oil content of 20-30% of algal dried weight to 50-65% of algal dried weight).

Nitrogen-deficient growth medium in combination with light illumination

Incubating the algal cells under a higher light intensity (>200 μιηοΐ m^"2 s^"1) also induces and enhances the oil production by the Chlorococcum pamirum strain. Incubating the algal cells under a combination of nitrogen-depleted atmosphere and high light intensity (greater than 200 μιηοΐ m^"2 s^"1) can further increase the speed of the oil accumulation, but does not increase the final oil content of the cells (about 50-65% of algal dried weight). NaCl-containing growth medium

The addition of NaCl in the culture medium at a concentration higher than 0.1% NaCl also helps to induce oil accumulation in the Chlorococcum pamirum cells. Using this approach, the oil content can reach 50-65% of algal dried weight.

EXAMPLE 4

Effects of different nitrogen concentrations on the growth of microalgae

Chlorococcum pamirum

To investigate the growth characteristics of Chlorococcum pamirum, the effects of NaNC concentrations on cell growth were studied. Cells were inoculated into columns with initial cell dry weight of 0.0078 g/L (initial OD₅₅₀: 0.020). The culture medium was BG-11 with the NaN0₃ concentration set as 0 g/L, 0.1 g/L, 0.25 g/L, 1.0 g/L, 1.5 g/L, respectively. Samples were withdrawn at designated time intervals, Optical Density at 550 nm (O.D 550) and dry weight were measured. The results are shown in FIGs. 1A and IB.

EXAMPLE 5

Effects of different phosphorus concentrations on the growth of microalgae

Chlorococcum pamirum

To investigate the growth characteristics of Chlorococcum pamirum, the effects of

Κ₂ΗΡθ₄·3Η₂0 concentrations on cell growth were studied. Cells were inoculated into columns with initial cell dry weight of 0.0078 g/L (initial OD₅₅₀: 0.020). The culture medium was BG-11 with the NaN0₃ concentration set as 0 g/L, 0.01 g/L, 0.04 g/L, 0.08 g/L, 0.2 g/L, 0.4 g/L, respectively. Samples were withdrawn at designated time intervals, Optical Density at 550 nm (O.D. 550) and dry weight were measured. The results are shown in FIGs. 2A and 2B.

EXAMPLE 6

Effects of nitrogen and/or phosphorus deficiency on the growth of, and oil/lipids accumulation in, microalgae Chlorococcum pamirum In order to find out the ability of Chlorococcum pamirum to accumulate oil/lipids under photoautrophic conditions, cells grown in full BG-11 medium were inoculated into N and/or P deficiencies medium, respectively, with an initial dry weight of 0.694 g/L. Four different treatments were set up: N+P+ (full medium, control group); N-P+ (only nitrogen deficiency); N+P- (only phosphate deficiency); N-P- (both nitrogen and phosphate deficiencies). Samples were withdrawn at designated time intervals, dry weight and lipid contents (measured as percentage of dry weight) were measured. The results are shown in FIGs. 3 A and 3B.

Although the invention herein has been described with reference to particular embodiments or examples, it is to be understood that these embodiments or examples are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of increasing oil accumulation in microalgae Chlowcoccum pamirum, comprising incubating the microalgae in a culture medium and inducing oil accumulation in the microalgae.

2. The method of claim 1, wherein said incubating comprises maintaining the microalgae cells in an agar plate containing a culture medium and cultivating the microalgae cells in a container containing a culture medium.

3. The method of claim 2, further comprising cultivating the microalgae in a photobioreactor.

4. The method of claim 3, wherein said incubating comprises illuminating the microalgae cells with a light in at least one of the steps.

5. The method of claim 3, wherein said incubating comprises illuminating the microalgae cells with a light in every step.

6. The method of claim 4 or 5, wherein said light is a fluorescent light, normal daylight, or sunlight.

7. The method according to claim 6, wherein said fluorescent light has an intensity of at least 200 μιηοΐ m^" s^" .

8. The method of claim 1 or 2, wherein said culture medium comprises about

0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5-3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄3H₂0, and about 0.2-2 mL/L of A5 micronutrients.

9. The method of claim 1 or 2, wherein said culture medium consists essentially of about 0.25-2.5 g/L NaN0₃, about 0.025-1.75 g/L MgS0₄7H₂0, about 0.5- 3.6 g/L Na₂C0₃, about 0.025-0.20 g/L CaCl₂, about 0.001-0.005 g/L Na₂EDTA, about 0.075-1.5 g/L K₂HP0₄3H₂0, and about 0.2-2 mL/L of A5 micronutrients.

10. The method of claim 8, wherein said culture medium has a pH in the range of 7.4-8.2.

11. The method of claim 1, wherein said inducing comprises incubating the microalgae cells in a nitrogen-deficient medium.

12. The method of claim 1, wherein said inducing comprises incubating the microalgae cells in a phosphorus-deficient medium.

13. The method of claim 1, wherein said inducing comprises illuminating the microalgae cells with a light having an intensity of at least 200 μιηοΐ m^" s^" .

14. The method of claim 1, wherein said inducing comprises incubating the microalgae cells in a culture medium comprising NaCl at a concentration of at least 0.1% w/v.

15. An oil-enriched microalgae cell collection comprising Chlorococcum pamirum strain, wherein said cell collection is enriched with oil at a level higher than the oil level in the corresponding natural microalgae cells prior to incubation.

16. The oil-enriched microalgae cell collection of claim 15, wherein the cell collection is isolated from the culture medium.

17. The oil-enriched microalgae cell collection of claim 15, wherein the cell collection is purified.

18. The oil-enriched microalgae cell collection of claim 15, wherein the oil level of the cell collection is at least 10% higher than the natural oil level.

19. The oil-enriched microalgae cell collection of claim 15, wherein the oil level of the cell collection is at least 50% higher than the natural oil level.

20. The oil-enriched microalgae cell collection of claim 15, wherein the oil level of the cell collection is at least 100% higher than the natural oil level.

21. The oil-enriched microalgae cell collection of claim 15, comprising at least

30% of oil based on dry weight.

22. The oil-enriched microalgae cell collection of claim 15, comprising at least 40% of oil based on dry weight.

23. The oil-enriched microalgae cell collection of claim 15, comprising at least 50% of oil based on dry weight.

24. The oil-enriched microalgae cell collection of claim 15, comprising about

50-70% of oil based on dry weight.

25. The oil-enriched microalgae cell collection of claim 15, which comprises about 55-65% of oil based on dry weight.

26. A method of making feedstock for production of biodiesel, comprising: (a) incubating microalgae Chlorococcum pamirum in a culture medium; (b) inducing oil enrichment in the microalgae; (b) isolating the oil-enriched microalgae from the culture medium; and (c) extracting lipids and/or oil from the oil-enriched microalgae.

27. The method of claim 26, further comprising scaling up the culture of the microalgae in a photobioreactor.

28. The method according to claim 27, wherein said scaling up comprises: maintaining the microalgae in agar plates containing a growth medium; culturing the microalgae in a reactor containing 50-1000 mL medium; and culturing the microalgae in 5-20 L photobioreactors.

29. Use of microalgae Chlorococcum pamirum in production of oil or lipids.

30. The use of claim 29, wherein said microalgae is enriched with oil by cultivation in a culture medium.

31. Use of the method according to any of claims 1-14 for producing oil-rich microalgae useful as feedstock for biodiesel production.

32. Use of the oil-enriched microalgae cell collection according to any of claims 15-25 as feedstock for biodiesel production.

33. Use of the oil-enriched microalgae cell collection according to any of claims 15-25 in production of oil and/or lipids.