Nothing Special   »   [go: up one dir, main page]

US20090211150A1 - Method for producing biodiesel using high-cell-density cultivation of microalga Chlorella protothecoides in bioreactor - Google Patents

Method for producing biodiesel using high-cell-density cultivation of microalga Chlorella protothecoides in bioreactor Download PDF

Info

Publication number
US20090211150A1
US20090211150A1 US12/080,666 US8066608A US2009211150A1 US 20090211150 A1 US20090211150 A1 US 20090211150A1 US 8066608 A US8066608 A US 8066608A US 2009211150 A1 US2009211150 A1 US 2009211150A1
Authority
US
United States
Prior art keywords
bioreactor
cell
feeding
cells
density
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/080,666
Inventor
Qingyu Wu
Wenguang Zhou
Wei Xiong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tsinghua University
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Assigned to TSINGHUA UNIVERSITY reassignment TSINGHUA UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WU, QINGYU, XIONG, WEI, ZHOU, WENGUANG
Publication of US20090211150A1 publication Critical patent/US20090211150A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/003—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with alcohols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P30/00—Technologies relating to oil refining and petrochemical industry
    • Y02P30/20—Technologies relating to oil refining and petrochemical industry using bio-feedstock

Definitions

  • the present invention relates to area of regenerable biofuel, in particular to a method for producing biodiesel from microalga oil by high-cell-density heterotrophic cultivation of a specific strain of green alga Chlorella protothecoides rich in lipids coupled with biodiesel production by transeterification reaction.
  • Biodiesel is one such fuel composed of monoalkyl esters of long chain fatty acids transesterified from vegetable oil or animal fat. Biodiesel has been used in many areas, especially in the United States and Europe. The high cost of biodiesel, which is mainly due to the high cost of oil feedstock, is the main obstacle to its broader commercialization. Therefore, there have been many research efforts to minimize oil feedstock costs.
  • soybean, rapeseed and sunflower seeds along with several other oil/food crops are the major sources of feedstock for biodiesel production. Due to their low oil yield potential and high demands on land, water, and fertilizer, which create competition with food/feed industries, the conventional oil/food crop-based biodiesel production system cannot meet the growing demand on sustainable feedstock for biodiesel production.
  • Microalgae are a group of photosynthetic microorganisms. The concept of ‘algae for fuels’ has been evaluated over the past years. However, no technology of biodiesel production by microalgae is economically feasible because rapidly growing cells contain low amounts of lipids and algal cells accumulating high lipid content exhibit little growth.
  • the present invention provides a novel method to produce oil feedstock for biodiesel production from the heterotrophic microalga C. Protothecoides by high-cell-density cultivation in a bioreactor.
  • the present invention comprises the steps of screening a specific strain with characteristics of high yield of biomass and high oil content; inoculating the strain in a first bioreactor for algal-seed-cells cultivating; feeding a first solution containing carbohydrates into the first bioreactor; transferring the cultivated algal-seed-cells into a second bioreactor for high-cell-density cultivation; feeding a second solution containing carbohydrates into the second bioreactor; harvesting the high density cultivated algal cells from the second bioreactor; drying the high density cultivated algal cells; extracting oil from dried algal cells; and producing the biodiesel by reaction of transesterification using the extracted oil as feedstock.
  • the high-cell-density cultivation of the present invention further comprises heterotrophic cultivation of C. Protothecoides with high oil content, preferably between 20% to 61% dry cell weight.
  • the present invention further comprises a screening step using centrifuge to select cells with high oil content.
  • the cells in the supernatant (upper layer) of centrifuge tubes, which have high oil content and low relative gravity, are inoculated and spread in agar plates to form a single algal colony.
  • the larger colonies with higher cell dividing rate are selectively picked up and inoculated into a flask containing growth medium, including but not limiting to components of basal medium, for further culture
  • the cultivated cell density in said first bioreactor or said second bioreactor of the present invention is controlled between 15 to 108 (dry weight) g/L.
  • the screening step of the present invention further comprises to select a large single algal colony with a high growth rate from agar plates.
  • the present invention comprises a specific strain of microalga C. protothecoides, Chlorella. Protothecoides sp 0710, with characteristics of high growth rate and high yielding oil.
  • the present invention further comprises a batch feeding operation for feeding the first solution containing carbohydrates into the first bioreactor.
  • the present invention further comprises a continuous feeding operation for feeding the second solution containing carbohydrates into the second bioreactor.
  • the carbohydrates solutions include but not limit to glucose or other monosaccharides, and/or disaccharides, or polysaccharides, preferably with the concentration of glucose or other monosaccharides, disaccharides, polysaccharides, or hydrolysates of corn starch, wheat flour, cassaya starch and tapioca starch, or juices of sorghum or broomcom, in the first or the second bioreactor, preferably with concentration of the carbohydrates solutions being controlled between 0.01 and 100 gL ⁇ 1 .
  • the amount of inoculum in the first bioreactor or the second bioreactor is controlled between 0.01% and 50% by volume.
  • Another preferred embodiment of the present invention further comprises agitating the medium in the first or the second reactor, preferably with a speed of agitation maintained between 5 and 1000 rpm.
  • Yet another preferred embodiment of the present invention further comprises aerating in the first or the second bioreactor, wherein the dissolved oxygen in cultivation medium in the first or the second bioreactor is maintained at least 5% of saturation, preferably at least 20% of saturation.
  • Another preferred embodiment of the present invention further comprises feeding organic nitrogen into the first and the second bioreactor, wherein the organic nitrogen includes but not limit to glycine, yeast powder, yeast extract, or corn syrup.
  • pH value in the first or the second bioreactor is preferably maintained between 6.0 and 8.0.
  • Yet another preferred embodiment of the present invention further comprises adding a base to reduce acidity and regulate the pH of the culture medium in the first or the second bioreactor, preferably KOH or other bases.
  • the temperature in the first or the second bioreactor is preferably controlled between 20° C. to 45° C.
  • the cell density of C. protothecoides in the second bioreactor is preferably between 20 to 108 g (dry weight)/L with the oil content preferably between 40% to 61% of dry cell weight.
  • the present invention further comprises a process of enzyme-catalyzed transesterification, preferably using catalyzation by immobilized lipase from candida sp. 99-125.
  • the enzyme-catalyzed transesterification further comprises adding said lipase and methanol into said feedstock.
  • FIG. 1 is a process flow schematic for biodiesel production from high-cell-density heterotrophic cultivation of microalga C. protothecoides.
  • FIG. 2 is a graph of cell growth and glucose consumption in high-cell-density heterotrophic cultivation of C. protothecoides in a 5 L bioreactor.
  • FIG. 3 shows the relative fluorescence peak in Nile Red dyed cells under fluorescence microscope.
  • Relative fluorescence in Nile Red dyed cells under fluorescence (a) Test sample from high-cell-density heterotrophic cultivation of the specific strain of C. protothecoides ; (b) Control sample from former heterotrophic cultivation of the original strain.
  • FIG. 4 is a process flow schematic for biodiesel production by immobilized lipase.
  • FIG. 5 shows product composition for transesterification during the reaction time.
  • Biodiesel refers to a diesel-equivalent processed fuel derived from biological sources (such as vegetable oils, animal fats or microbial lipids) which can be used in unmodified diesel-engine vehicles.
  • a fatty acid mono-alkyl ester biodiesel comprises fatty acids such as oleic, stearic, linoleic or palmitic acids as well as any sort of combinations of theses fatty acids.
  • Alga, algae, algal Any of various chiefly aquatic, eukaryotic, photosynthetic organisms, ranging in size from single-celled forms to the giant kelp. Algae are once considered to be plants but are now classified separately because they lack true roots, stems, leaves, and embryos.
  • Microalgae Unicellular aquatic algae.
  • Heterotrophic requiring organic compounds of carbon and nitrogen for nourishment.
  • Transesterification a process of exchanging the alkoxy group of an ester compound by another alcohol.
  • Monoalkyl esters of long chain fatty acids, fatty acid methyl esters a class of organic compounds formed from methanol and fatty acid by removal of water.
  • Bioreactor a vessel or instrument including fermenter for cell growth or a chemical process involving organisms or biochemically active substances derived from such organisms.
  • Heterotrophic cultivation or fermentation both of terms refer to highly oxygenated and aerobic growth of microbial cells through consuming glucose or other organic carbon sources.
  • Yeast fermentation refers to the conversion process of sugar to alcohol using yeast under anaerobic conditions.
  • Fuel ethanol an alcohol fuel that's derived mainly from grain through the process of microbial fermentation.
  • Oil, lipid a class of hydrophobic, naturally-occurring molecules, which is characterized as their long hydrocarbon chain structure.
  • oil and lipid are used to refer to fatty-acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids) as well as other fat-soluble sterol-containing metabolites such as cholesterol.
  • Nile Red a lipophilic dye which is intensely fluorescent with a strong yellow-gold emission when in a lipid-rich environment.
  • Pyrolysis is the chemical decomposition of organic materials by heating in the absence of oxygen or any other reagents except possibly steam.
  • Single algal colony a single cluster of alga in agar plate culture that is produced from a single starting algal cell.
  • Incubator An apparatus in which environmental conditions, such as temperature and humidity, can be controlled, used for growing microbial cultures or providing suitable conditions for a chemical or biological reaction.
  • Organic carbon a class of carbon compounds that had its origin in living material such as sugar or lipid. It is distinguished from carbon dioxide which belongs to inorganic carbon.
  • Organic nitrogen We use the term “organic nitrogen” to describe a nitrogen compound that had its origin in living material such as the nitrogen in protein and urea.
  • Broomcorn A variety of sorghum ( Sorghum bicolor ) having a stiff, erect, much-branched flower cluster, the stalks of which are often used to make brooms.
  • Sweet broomcorn refers to the broomcorn which has sweet juicy stems.
  • sweet broomcorn is processed by pressing the juice out of the stems and used as feedstock for algae cultivation.
  • Cassaya is a shrubby, tropical, perennial plant, whose roots are high in starch.
  • Soxhlet extractor A fast semi-automated solvent extraction unit for separating lipid from algal cells.
  • Heating value Amount of heat generated by a given mass of fuel when it is completely burned.
  • Monosaccharide a simple sugar such as glucose, fructose, xylose et al., having the general formula CnH2nOn, that cannot be decomposed by hydrolysis.
  • Disaccharide molecule formed by two simple sugar units linked together.
  • Polysaccharide molecule formed by more than two simple sugar units linked together.
  • Hydrolysate any compound produced by hydrolysis, the process of decompose a chemical compound by reaction with water.
  • hydrolysis refers to the catalytic conversion of starch, cellulose or other polysaccharides to glucose or other mono-saccharides.
  • One objective of the present invention is to provide a novel method of biodiesel production from heterotrophic microalga C. protothecoides by high-cell-density cultivation in bioreactor.
  • FIG. 1 a preferred embodiment of the present invention is illustrated:
  • Microalgal strain of C. protothecoides is screened for heterotrophic growth.
  • the resulted heterotrophic cultured cells are centrifuged twice in different centrifuge rate, preferably at 8000-12000 g (earth gravity) for 30 min at 4° C.
  • the cells in the supernatant (upper layer) of centrifuge tubes, which have high oil content, are inoculated and spread in agar plates to form a single algal colony, preferably at a temperature of 28 ⁇ 0.5° C. for 240 to 360 hours.
  • the above specific strain of C. protothecoides is inoculated into a shaking flask, containing culture medium for algal-seed-cells cultivation, which serves as the first bioreactor, in an incubator.
  • the components of basal culture medium are:
  • a preferred A5 trace mineral solution comprises:
  • Algal cell yield can be determined using various methods, including but not limiting to light intensity measurement of the cell suspension, such as OD540 nm of cell suspension.
  • Preferable conditions such as glucose concentration, different nitrogen sources in the basal medium, temperatures, and flask shaking rate during algal-seed-cells cultivation in shaking flasks are determined by real-time light intensity measurement of the cell suspension.
  • a preferable glucose concentration in a range of 5 to 30 g/L glucose is added in the basal medium.
  • a preferable yeast extract in a range of 1 to 10 g/L yeast extract is added in the basal medium.
  • a temperature in incubator is set between 20-45° C., preferably at 28° C.
  • the flask shaking rate is controlled between 50 to 300 rpm, preferably at 200 rpm.
  • a weak light at 5 mol m ⁇ 2 s ⁇ 1 is also adopted.
  • the cells are harvested till the culture of algal seed cell enters into late-exponential-phase.
  • the cell harvesting time before reaching the late-exponential-phase is approximately at 168 hours.
  • the late-exponential-phase algal-seed-cells in the flask are transferred to a second bioreactor, which is larger that the shaking flak, preferably between 5 L to 11,000 L, for high-cell-density cultivation by process control and optimization.
  • Glucose and yeast extract solutions are added into the basal culture medium initiate in the second bioreactor, preferably with 5 to 50 g/L glucose and 1 to 10 g/L yeast extract, further preferably with 30 g/L glucose and 4 g/L yeast extract.
  • Parameters such as the amount of inoculum, substrate (organic carbon and nitrogen) feeding, oxygen supply, stirring rate, temperature, pH, and time of cell harvest, and adjusted to optimize cell growths in the second bioreactor.
  • dissolved oxygen (DO) in the fermentation suspension for high-cell-density cultivation of heterotrophic algal cells in the bioreactor can be used to monitor the growth conditions, such as organic carbon sources in the reactor, biomass and accumulation of lips.
  • On-line monitoring of the DO parameter is preferably used to monitor the growth conditions, and to adjust agitation speed and aerating rate.
  • the glucose added into the basal culture medium can be substituted by fructose, sucrose, corn powder hydrolysate, cassaya powder hydrolysate, wheat starch hydrolysate, juice of broomcorn, juice of sugar cane and waste water containing sugar from the food and beverage industry.
  • the growth of the cells in the second reactor is terminated.
  • the growth duration in said second bioreactor lasts about 213 hours.
  • dry biomass of the algal cell suspension from bioreactor is separated using a separation process, including but not limiting to filtration or centrifuge.
  • the separated dry biomass may be in a form of powder or other solid forms.
  • Lipids (oil) in heterotrophic C. protothecoides cell powder are subsequently extracted.
  • Techniques for extraction comprises the Soxhlet method, wherein N-hexane is used as the standard Soxhlet solvent for extracting oil from heterotrophic cells of C. protothecoides . Extraction is achieved by washing the cells repeatedly with pure solvent until no lipid is left in cells. Then the solvent in the extract is removed under reduced pressure.
  • the heterotrophic algal fermentation based biodiesel is produced from the feedstock of microalgal oil by transeterification reaction.
  • Transeterification can be catalyzed using strong acids, such as sulfate acid as a catalyst, or a lipase as a catalyst.
  • strong acids such as sulfate acid as a catalyst
  • a lipase as a catalyst.
  • a lipase extracted from Candidia sp. 99-125, triacylglycerol acylhydrolase, EC 3.1.1.3, is a preferred catalyst.
  • Catalyst activity of the triacylglycerol acylhydrolase is about 12,000 U/g.
  • Preferable conditions for transeterification are: pH value of 7.0, 60% immobilized lipase (w/w, 12,000 U/g) relative to the microalgal oil mass, 10% water content (w/w) relative to the microalgal oil mass, reaction temperature 38° C., 160 rpm, adding methanol to the microalgal oil three times to reach 3:1 methanol to the microalgal oil mass ratio.
  • the process flow schematic for enzymatic biodiesel production is shown in FIG. 4 .
  • the original strain of C. protothecoides came from the Culture Collection of Alga at the University of Texas (Austin, Tex.), which has been kept in Department of Biological Science and Biotechnology in Tsinghua University since 1990.
  • the compositions of the culture medium for this strain are:
  • the specific strain with characteristics of high cell growth rate and high oil content is obtained as Chlorella.
  • Protothecoides sp 0710 is in golden color, cell dry weight approximately 108 g/l with oil/lip content at about 61% of cell dry weight.
  • Table 1 illustrates compositions of biodiesel produced by the Chlorella.
  • Protothecoides cells are obtained by oriented selection and screening as described in step (1) above.
  • the selected strain is inoculated in a first bioreactor, comprising 500 ml aeration flasks, for algal-seed-cells cultivating at 28° C. and 200 rpm in batch cultures. 30 g/L glucose and 4 g/L yeast extract are added in the basal medium. After 168 hours of cultivation, cell density reaches about 15 g/L and cell growth reaches an end of exponential-phase grow. Weak light (5 ⁇ mol/m 2 s) is adopted during cultivation.
  • High-cell-density growth is performed in a 5 L stirred bioreactor (MINIFORS, Switzerland) with turbine impellers.
  • the C. Protothecoides cell in exponential phase is inoculated in the basal medium with 30 g/L glucose and 4 g/L yeast extract and an inoculation ratio of 25% (V/V).
  • protothecoides are set as follows: temperature at 28 ⁇ 0.5° C., aerobic rate at 180 L/h (1:1vvm), pH 6.30 ⁇ 0.1, 25% inoculum amount of seed algal cells (V/V), concentration of glucose controlled between 5 and 18 g/L, and dissolved oxygen (DO) over 20% controlled by increasing agitation and airflow, with feeding organic nitrogen (yeast extract) at 0.75 g/L/h and glucose in the same time after 128 h cultivation.
  • the strategy of high-cell-density cultivation is set up through substrate feeding and process control in the 5 L bioreactor.
  • Protothecoides are also monitored in bioreactor. After fermentation about 213 hours, dry cell density reaches 108 g/l, as illustrated in FIG. 2 .
  • the cells are harvested by centrifugation and dried by vacuum freeze dryer.
  • the algal cell suspension from bioreactor is centrifuged at 10,000 rpm, 4° C. for 2 min and the cell pellets are collected for vacuum-freeze drying.
  • the dry biomass of 108 g/L is harvested in high-cell-density heterotrophic cultivation of specific strain of C. protothecoides ( FIG. 2 ).
  • the Soxhlet method is adopted to extract total lipids in the dried C. Protothecoides cells. N-hexane is used as the standard Soxhlet solvent. Extraction is achieved by washing the sample repeatedly with pure solvent.
  • the oil extracts by the Soxhlet method are dried by distillation under a reduced pressure.
  • the oil content is about 61% of cells dry weight.
  • the transesterification is catalyzed by immobilized lipase from Candidia sp. 99-125 in shaker flasks on a constant temperature shaker, with the rotation rate of 180 rpm.
  • the reaction mixture is left to separate into two layers in a separating funnel after reaction.
  • the upper layer (biodiesel) is separated and washed with hot water (50° C.). Biodiesel product is obtained after the solvent is evaporated.
  • Variables are studied in order to determine the operation conditions that could maximize the yield of biodiesel and fulfill the specifications of the US Standard for Biodiesel (ASTM 6751) related to mono, di, and triglycerides, methanol, free glycerol, and fatty acids contents.
  • the variables are the amount of immobilized lipase related to the quantity of lipids, the quantity of methanol (methanol to lipids), the feeding frequency of methanol, the quantity of organic solvents, the water content in reaction mixtures, reaction temperature, pH value, and reaction time.
  • the conversion rate of lipids to biodiesel was estimated by gas chromatography and mass spectrometry (GC-MS), through which the relative contents of fatty acid esters, free fatty acids, mono, di, and triglycerides could be determined.
  • GC-MS gas chromatography and mass spectrometry
  • a DSQ GC system (Thermo, USA, VARIAN VF-5 ms, capillary cylinder 30 m ⁇ 0.25 mm) is used for the chromatography work and data analysis.
  • the GC was operated in constant flow mode with a flow rate of 10 mL/min.
  • the oven is initially held at 70° C. for 2 min and then elevated to 300° C. at a rate of 10° C./min, and held at this temperature for 20 min.
  • a split injector is used with a split ratio of 30:1 and a temperature of 250° C.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A method is provided to produce biodiesel from algae using a strain of microalga chlorella protothecoids, by screening a specific strain with characteristics of high yield of biomass and high oil content, cultivating the screened strain for high-cell-density growth for up to 108 grams of dry cell weight per liter of the suspension in a bioreactor using solutions containing carbohydrates as feed, harvesting and drying the high density cultivated algal cells to extract oil from the dried algal cells, and producing the biodiesel by reaction of catalyzed transesterification using the extracted oil as feedstock.

Description

    CROSS REFERENCE TO RELATED APPLICATION
  • This application claims the benefit of China Patent Application No. 200810100871.5, filed on Feb. 25, 2008, and entitled “Method for Producing Biodiesel using High-Cell-Density Cultivation Of Microalga Chlorella Protothecoides”, the entire disclosure of which is incorporated herein by reference.
    • FIELD OF THE INVENTION
  • The present invention relates to area of regenerable biofuel, in particular to a method for producing biodiesel from microalga oil by high-cell-density heterotrophic cultivation of a specific strain of green alga Chlorella protothecoides rich in lipids coupled with biodiesel production by transeterification reaction.
    • BACKGROUND OF THE INVENTION
  • Renewable carbon-neutral fuels, such as biodiesel, are considered necessary replacement of traditional petroleum fuel for environmental and economic sustainability. Biodiesel is one such fuel composed of monoalkyl esters of long chain fatty acids transesterified from vegetable oil or animal fat. Biodiesel has been used in many areas, especially in the United States and Europe. The high cost of biodiesel, which is mainly due to the high cost of oil feedstock, is the main obstacle to its broader commercialization. Therefore, there have been many research efforts to minimize oil feedstock costs.
  • Currently, soybean, rapeseed and sunflower seeds along with several other oil/food crops are the major sources of feedstock for biodiesel production. Due to their low oil yield potential and high demands on land, water, and fertilizer, which create competition with food/feed industries, the conventional oil/food crop-based biodiesel production system cannot meet the growing demand on sustainable feedstock for biodiesel production.
  • Microalgae are a group of photosynthetic microorganisms. The concept of ‘algae for fuels’ has been evaluated over the past years. However, no technology of biodiesel production by microalgae is economically feasible because rapidly growing cells contain low amounts of lipids and algal cells accumulating high lipid content exhibit little growth.
  • Wu and his coworkers first reported bio-oil and biodiesel production from the green microalga by means of heterotrophic cultivation of C. protothecoides. However, both microalgal cell yield and oil content in harvested dry cells are not high enough for its industrial application.
  • Therefore, there is a need to further increase the biomass, such as a process for high-cell-density cultivation of microalga with high oil content, to make industrial application feasible.
    • SUMMARY OF THE INVENTION
  • The present invention provides a novel method to produce oil feedstock for biodiesel production from the heterotrophic microalga C. Protothecoides by high-cell-density cultivation in a bioreactor. The present invention comprises the steps of screening a specific strain with characteristics of high yield of biomass and high oil content; inoculating the strain in a first bioreactor for algal-seed-cells cultivating; feeding a first solution containing carbohydrates into the first bioreactor; transferring the cultivated algal-seed-cells into a second bioreactor for high-cell-density cultivation; feeding a second solution containing carbohydrates into the second bioreactor; harvesting the high density cultivated algal cells from the second bioreactor; drying the high density cultivated algal cells; extracting oil from dried algal cells; and producing the biodiesel by reaction of transesterification using the extracted oil as feedstock.
  • The high-cell-density cultivation of the present invention further comprises heterotrophic cultivation of C. Protothecoides with high oil content, preferably between 20% to 61% dry cell weight.
  • The present invention further comprises a screening step using centrifuge to select cells with high oil content. The cells in the supernatant (upper layer) of centrifuge tubes, which have high oil content and low relative gravity, are inoculated and spread in agar plates to form a single algal colony. The larger colonies with higher cell dividing rate are selectively picked up and inoculated into a flask containing growth medium, including but not limiting to components of basal medium, for further culture
  • The cultivated cell density in said first bioreactor or said second bioreactor of the present invention is controlled between 15 to 108 (dry weight) g/L.
  • The screening step of the present invention further comprises to select a large single algal colony with a high growth rate from agar plates.
  • The present invention comprises a specific strain of microalga C. protothecoides, Chlorella. Protothecoides sp 0710, with characteristics of high growth rate and high yielding oil.
  • The present invention further comprises a batch feeding operation for feeding the first solution containing carbohydrates into the first bioreactor.
  • The present invention further comprises a continuous feeding operation for feeding the second solution containing carbohydrates into the second bioreactor.
  • The carbohydrates solutions include but not limit to glucose or other monosaccharides, and/or disaccharides, or polysaccharides, preferably with the concentration of glucose or other monosaccharides, disaccharides, polysaccharides, or hydrolysates of corn starch, wheat flour, cassaya starch and tapioca starch, or juices of sorghum or broomcom, in the first or the second bioreactor, preferably with concentration of the carbohydrates solutions being controlled between 0.01 and 100 gL−1.
  • In a preferred embodiment of the present invention, the amount of inoculum in the first bioreactor or the second bioreactor is controlled between 0.01% and 50% by volume.
  • Another preferred embodiment of the present invention further comprises agitating the medium in the first or the second reactor, preferably with a speed of agitation maintained between 5 and 1000 rpm.
  • Yet another preferred embodiment of the present invention further comprises aerating in the first or the second bioreactor, wherein the dissolved oxygen in cultivation medium in the first or the second bioreactor is maintained at least 5% of saturation, preferably at least 20% of saturation.
  • Another preferred embodiment of the present invention further comprises feeding organic nitrogen into the first and the second bioreactor, wherein the organic nitrogen includes but not limit to glycine, yeast powder, yeast extract, or corn syrup.
  • In a preferred embodiment of the present invention, pH value in the first or the second bioreactor is preferably maintained between 6.0 and 8.0.
  • Yet another preferred embodiment of the present invention further comprises adding a base to reduce acidity and regulate the pH of the culture medium in the first or the second bioreactor, preferably KOH or other bases.
  • In a preferred embodiment of the present invention, the temperature in the first or the second bioreactor is preferably controlled between 20° C. to 45° C.
  • Yet in another preferred embodiment of the present invention, the cell density of C. protothecoides in the second bioreactor is preferably between 20 to 108 g (dry weight)/L with the oil content preferably between 40% to 61% of dry cell weight.
  • The present invention further comprises a process of enzyme-catalyzed transesterification, preferably using catalyzation by immobilized lipase from candida sp. 99-125. The enzyme-catalyzed transesterification further comprises adding said lipase and methanol into said feedstock.
  • Other objects, advantages, and features of the present invention will become apparent from the following detailed description of the invention.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The following detailed description will be better understood when read in conjunction with the appended drawings, in which there is shown one or more of the multiple embodiments of the present invention. It should be understood, however, that the various embodiments of the present invention are not limited to the precise arrangements and instrumentalities shown in the drawings.
  • In the Drawings:
  • FIG. 1 is a process flow schematic for biodiesel production from high-cell-density heterotrophic cultivation of microalga C. protothecoides.
  • FIG. 2 is a graph of cell growth and glucose consumption in high-cell-density heterotrophic cultivation of C. protothecoides in a 5 L bioreactor.
  • FIG. 3 shows the relative fluorescence peak in Nile Red dyed cells under fluorescence microscope. Relative fluorescence in Nile Red dyed cells under fluorescence: (a) Test sample from high-cell-density heterotrophic cultivation of the specific strain of C. protothecoides; (b) Control sample from former heterotrophic cultivation of the original strain.
  • FIG. 4 is a process flow schematic for biodiesel production by immobilized lipase.
  • FIG. 5 shows product composition for transesterification during the reaction time.
    •  DEFINITIONS
  • Unless otherwise stated, the following definitions provide meanings and examples to terms used herein. Such definitions are also intended to encompass any meaning that may be contemplated or could be appreciated by a person of ordinary skill with the art.
  • Biodiesel: refers to a diesel-equivalent processed fuel derived from biological sources (such as vegetable oils, animal fats or microbial lipids) which can be used in unmodified diesel-engine vehicles.
  • The terms “mono-alkyl ester” or “monoester” and derivations thereof including, for example, mono-alkyl esters, monoesters or esters generally refer to a type of fatty acid ester biodiesel or biodiesel fuel. For example, a fatty acid mono-alkyl ester biodiesel comprises fatty acids such as oleic, stearic, linoleic or palmitic acids as well as any sort of combinations of theses fatty acids.
  • Alga, algae, algal: Any of various chiefly aquatic, eukaryotic, photosynthetic organisms, ranging in size from single-celled forms to the giant kelp. Algae are once considered to be plants but are now classified separately because they lack true roots, stems, leaves, and embryos.
  • Microalgae: Unicellular aquatic algae.
  • Heterotrophic: requiring organic compounds of carbon and nitrogen for nourishment.
  • Transesterification: a process of exchanging the alkoxy group of an ester compound by another alcohol.
  • Monoalkyl esters of long chain fatty acids, fatty acid methyl esters: a class of organic compounds formed from methanol and fatty acid by removal of water.
  • Bioreactor: a vessel or instrument including fermenter for cell growth or a chemical process involving organisms or biochemically active substances derived from such organisms.
  • Heterotrophic cultivation or fermentation: both of terms refer to highly oxygenated and aerobic growth of microbial cells through consuming glucose or other organic carbon sources.
  • Yeast fermentation: refers to the conversion process of sugar to alcohol using yeast under anaerobic conditions.
  • Fuel ethanol: an alcohol fuel that's derived mainly from grain through the process of microbial fermentation.
  • Oil, lipid: a class of hydrophobic, naturally-occurring molecules, which is characterized as their long hydrocarbon chain structure. Here, both oil and lipid are used to refer to fatty-acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids) as well as other fat-soluble sterol-containing metabolites such as cholesterol.
  • Nile Red: a lipophilic dye which is intensely fluorescent with a strong yellow-gold emission when in a lipid-rich environment.
  • Pyrolysis: is the chemical decomposition of organic materials by heating in the absence of oxygen or any other reagents except possibly steam.
  • Single algal colony: a single cluster of alga in agar plate culture that is produced from a single starting algal cell.
  • Incubator: An apparatus in which environmental conditions, such as temperature and humidity, can be controlled, used for growing microbial cultures or providing suitable conditions for a chemical or biological reaction.
  • Organic carbon: a class of carbon compounds that had its origin in living material such as sugar or lipid. It is distinguished from carbon dioxide which belongs to inorganic carbon.
  • Organic nitrogen: We use the term “organic nitrogen” to describe a nitrogen compound that had its origin in living material such as the nitrogen in protein and urea.
  • Broomcorn: A variety of sorghum (Sorghum bicolor) having a stiff, erect, much-branched flower cluster, the stalks of which are often used to make brooms.
  • Sweet broomcorn: refers to the broomcorn which has sweet juicy stems. In this patent, sweet broomcorn is processed by pressing the juice out of the stems and used as feedstock for algae cultivation.
  • Cassaya: Cassaya is a shrubby, tropical, perennial plant, whose roots are high in starch.
  • Soxhlet extractor: A fast semi-automated solvent extraction unit for separating lipid from algal cells.
  • Heating value: Amount of heat generated by a given mass of fuel when it is completely burned.
  • Monosaccharide: a simple sugar such as glucose, fructose, xylose et al., having the general formula CnH2nOn, that cannot be decomposed by hydrolysis.
  • Disaccharide: molecule formed by two simple sugar units linked together.
  • Polysaccharide: molecule formed by more than two simple sugar units linked together.
  • Hydrolysate: any compound produced by hydrolysis, the process of decompose a chemical compound by reaction with water. Herein, refers to the catalytic conversion of starch, cellulose or other polysaccharides to glucose or other mono-saccharides.
    • DETAILED DESCRIPTION
  • Certain terminology is used herein for convenience only and is not to be taken as a limitation on the embodiments of the present invention. It should be appreciated that the particular embodiments shown and described herein are examples of the invention and are not intended to otherwise limit the scope of the present invention in any way.
  • One objective of the present invention is to provide a novel method of biodiesel production from heterotrophic microalga C. protothecoides by high-cell-density cultivation in bioreactor.
  • Referring generally to FIG. 1, a preferred embodiment of the present invention is illustrated:
  • (1) Screening a Specific Strain with Characteristics of High-Cell-Density Growth Rate and High Oil Content
  • Microalgal strain of C. protothecoides, originally obtained from the Culture Collection of Alga at the University of Texas (Austin, Tex.), is screened for heterotrophic growth. The resulted heterotrophic cultured cells are centrifuged twice in different centrifuge rate, preferably at 8000-12000 g (earth gravity) for 30 min at 4° C. The cells in the supernatant (upper layer) of centrifuge tubes, which have high oil content, are inoculated and spread in agar plates to form a single algal colony, preferably at a temperature of 28±0.5° C. for 240 to 360 hours. Then larger colonies are selectively picked up and inoculated into flasks containing growth medium, including but not limiting to components of basal medium, for further culture. Glucose with a concentration of 30 g/L and yeast extract with a concentration of 4 g/L are added into the basal medium. The cell growth rates and cellular oil contents in different culture are then compared with each other to determine a specific strain with characteristics of the highest oil content and a high cell growth rate.
  • (2) Inoculating the Specific Strain into a First Bioreactor for Algal-Seed-Cells Cultivation
  • The above specific strain of C. protothecoides is inoculated into a shaking flask, containing culture medium for algal-seed-cells cultivation, which serves as the first bioreactor, in an incubator. The components of basal culture medium are:
      • KH2PO4: 0.7 g/L,
      • K2HPO4: 0.3 g/L,
      • MgSO4.7H2O: 0.3 g/L,
      • FeSO4.7H2O: 3 mg/L,
      • Glycine: 0.1 g/L,
      • vitamin B1: 0.01 mg/L,
      • A5 trace mineral solution 1.0 ml/L, wherein the A5 trace mineral solution comprises H3BO3, Na2MoO4.2H2O, ZnSO4.7H2O, MnCl2.4H2O, and CuSO4.5H2O.
  • A preferred A5 trace mineral solution comprises:
      • H3BO3: 2.86 g/L,
      • Na2MoO4.2H2O: 0.039 g/L,
      • ZnSO4.7H2O: 0.222 g/L,
      • MnCl2.4H2O: 1.81 g/L,
      • CuSO4.5H2O: 0.074 g/L.
  • Algal cell yield can be determined using various methods, including but not limiting to light intensity measurement of the cell suspension, such as OD540 nm of cell suspension. Preferable conditions such as glucose concentration, different nitrogen sources in the basal medium, temperatures, and flask shaking rate during algal-seed-cells cultivation in shaking flasks are determined by real-time light intensity measurement of the cell suspension.
  • A preferable glucose concentration in a range of 5 to 30 g/L glucose is added in the basal medium.
  • A preferable yeast extract in a range of 1 to 10 g/L yeast extract is added in the basal medium.
  • A temperature in incubator is set between 20-45° C., preferably at 28° C.
  • The flask shaking rate is controlled between 50 to 300 rpm, preferably at 200 rpm.
  • A weak light at 5 mol m−2s−1 is also adopted.
  • The cells are harvested till the culture of algal seed cell enters into late-exponential-phase. The cell harvesting time before reaching the late-exponential-phase is approximately at 168 hours.
  • (3) Transferring the Algal-Seed-Cells from the First Bioreactor to a Second Bioreactor for High-Cell-Density Cultivation by Process Control and Optimization
  • The late-exponential-phase algal-seed-cells in the flask are transferred to a second bioreactor, which is larger that the shaking flak, preferably between 5 L to 11,000 L, for high-cell-density cultivation by process control and optimization.
  • Glucose and yeast extract solutions are added into the basal culture medium initiate in the second bioreactor, preferably with 5 to 50 g/L glucose and 1 to 10 g/L yeast extract, further preferably with 30 g/L glucose and 4 g/L yeast extract.
  • Parameters, such as the amount of inoculum, substrate (organic carbon and nitrogen) feeding, oxygen supply, stirring rate, temperature, pH, and time of cell harvest, and adjusted to optimize cell growths in the second bioreactor.
  • Among the parameters, dissolved oxygen (DO) in the fermentation suspension for high-cell-density cultivation of heterotrophic algal cells in the bioreactor can be used to monitor the growth conditions, such as organic carbon sources in the reactor, biomass and accumulation of lips. On-line monitoring of the DO parameter is preferably used to monitor the growth conditions, and to adjust agitation speed and aerating rate.
  • The glucose added into the basal culture medium can be substituted by fructose, sucrose, corn powder hydrolysate, cassaya powder hydrolysate, wheat starch hydrolysate, juice of broomcorn, juice of sugar cane and waste water containing sugar from the food and beverage industry.
  • The conditions for high-cell-density heterotrophic cultivation of the specific strain of C. protothecoides are automatically monitored and set as follows:
      • inoculum amount of seed algal cells (V/V): 20-30%, preferably of 25%;
      • temperature at 20-45° C., preferably at 28±0.5° C.;
      • aerobic rate at 100-200 L/h (1:1vvm), preferably at 180 L/h;
      • pH 6.0 to 8.0, preferably at 6.3±0.1;
      • concentration of glucose in medium: 5 to 18 g/L;
      • continuous feeding organic nitrogen (yeast extract) at 0.5 to 1.0 g/L/h, preferably at 0.75 g/L/h;
      • DO over 20% controlled by increasing agitation and airflow, gradually increasing agitation speed from 100 to 600 rpm after a period of cultivation for about 88 hours, to maintain the dissolved oxygen at above 20% of saturation;
      • Continuous feeding both organic nitrogen and glucose at the same time after about 128 hours of cultivation.
  • When the cell density of C. protothecoides and/or the oil content reach desired values, preferably with the dry cell density reaching 108 g/L and the oil content reaching 61% of dry cell weight, the growth of the cells in the second reactor is terminated. The growth duration in said second bioreactor lasts about 213 hours.
  • (4) Harvesting the High Density Microalgal Cells from Bioreactor
  • After determining a sample of the high-cell-density heterotrophic cultivation to reach a desired dry biomass concentration, preferable between 20 to 108 g/L, dry biomass of the algal cell suspension from bioreactor is separated using a separation process, including but not limiting to filtration or centrifuge. The separated dry biomass may be in a form of powder or other solid forms.
  • (5) Extracting the Oil from Dried Algal Cells
  • Lipids (oil) in heterotrophic C. protothecoides cell powder are subsequently extracted. Techniques for extraction comprises the Soxhlet method, wherein N-hexane is used as the standard Soxhlet solvent for extracting oil from heterotrophic cells of C. protothecoides. Extraction is achieved by washing the cells repeatedly with pure solvent until no lipid is left in cells. Then the solvent in the extract is removed under reduced pressure.
    • (6) Producing the Biodiesel by Transesterification Reaction Using the Microalgal Oil as Feedstock
  • The heterotrophic algal fermentation based biodiesel is produced from the feedstock of microalgal oil by transeterification reaction. Transeterification can be catalyzed using strong acids, such as sulfate acid as a catalyst, or a lipase as a catalyst. A lipase extracted from Candidia sp. 99-125, triacylglycerol acylhydrolase, EC 3.1.1.3, is a preferred catalyst. Catalyst activity of the triacylglycerol acylhydrolase is about 12,000 U/g. Preferable conditions for transeterification are: pH value of 7.0, 60% immobilized lipase (w/w, 12,000 U/g) relative to the microalgal oil mass, 10% water content (w/w) relative to the microalgal oil mass, reaction temperature 38° C., 160 rpm, adding methanol to the microalgal oil three times to reach 3:1 methanol to the microalgal oil mass ratio.
  • The process flow schematic for enzymatic biodiesel production is shown in FIG. 4.
    • EXAMPLE Materials and Methods
  • The original strain of C. protothecoides came from the Culture Collection of Alga at the University of Texas (Austin, Tex.), which has been kept in Department of Biological Science and Biotechnology in Tsinghua University since 1990. The compositions of the culture medium for this strain are:
      • KH2PO4: 0.7 g/L,
      • K2HPO4: 0.3 g/L,
      • MgSO4.7H2O: 0.3 g/L,
      • FeSO4.7H2O: 3 mg/L,
      • Glycine: 0.1 g/L,
      • vitamin B1: 0.01 mg/L,
      • A5 trace mineral solution 1.0 ml/L.
  • By oriented selection and screening as described in step (1) above, the specific strain with characteristics of high cell growth rate and high oil content is obtained as Chlorella. Protothecoides sp 0710. Comparing with the original cells, the Chlorella. Protothecoides sp 0710 is in golden color, cell dry weight approximately 108 g/l with oil/lip content at about 61% of cell dry weight. Table 1 illustrates compositions of biodiesel produced by the Chlorella. Protothecoides sp 0710 and the original Chlorella. Protothecoides cells.
  • The selected strain is inoculated in a first bioreactor, comprising 500 ml aeration flasks, for algal-seed-cells cultivating at 28° C. and 200 rpm in batch cultures. 30 g/L glucose and 4 g/L yeast extract are added in the basal medium. After 168 hours of cultivation, cell density reaches about 15 g/L and cell growth reaches an end of exponential-phase grow. Weak light (5 μmol/m2s) is adopted during cultivation.
  • High-cell-density growth is performed in a 5 L stirred bioreactor (MINIFORS, Switzerland) with turbine impellers. The C. Protothecoides cell in exponential phase is inoculated in the basal medium with 30 g/L glucose and 4 g/L yeast extract and an inoculation ratio of 25% (V/V). The conditions for high-cell-density heterotrophic cultivation of our specific strain C. protothecoides are set as follows: temperature at 28±0.5° C., aerobic rate at 180 L/h (1:1vvm), pH 6.30±0.1, 25% inoculum amount of seed algal cells (V/V), concentration of glucose controlled between 5 and 18 g/L, and dissolved oxygen (DO) over 20% controlled by increasing agitation and airflow, with feeding organic nitrogen (yeast extract) at 0.75 g/L/h and glucose in the same time after 128 h cultivation. The strategy of high-cell-density cultivation is set up through substrate feeding and process control in the 5 L bioreactor. The consumptions of glucose, glycine, phosphate, and the changes of pH and DO during the growth of C. Protothecoides are also monitored in bioreactor. After fermentation about 213 hours, dry cell density reaches 108 g/l, as illustrated in FIG. 2. The cells are harvested by centrifugation and dried by vacuum freeze dryer.
  • Cell growth is measured by the absorbance of the suspension at 540 nm. Biomass concentration is calculated from OD540 by a regression equation calculated from our experiment: y=0.4155x, (R2=0.9933, P<0.05), where y (gL−1) is the cell concentration, x is the absorbance of the suspension at 540 nm.
  • After finishing high-cell-density heterotrophic cultivation of specific strain of C. protothecoides, the algal cell suspension from bioreactor is centrifuged at 10,000 rpm, 4° C. for 2 min and the cell pellets are collected for vacuum-freeze drying. The dry biomass of 108 g/L is harvested in high-cell-density heterotrophic cultivation of specific strain of C. protothecoides (FIG. 2).
  • The Soxhlet method is adopted to extract total lipids in the dried C. Protothecoides cells. N-hexane is used as the standard Soxhlet solvent. Extraction is achieved by washing the sample repeatedly with pure solvent.
  • Molecular weights of oil of the C. protothecoides are calculated from saponification and the acid values determined using the formula: M=168,300 (SV−AV)−1, where M is molecular weight of lipids, SV is the saponification value, and AV is the acid value.
  • The oil extracts by the Soxhlet method are dried by distillation under a reduced pressure. The oil content is about 61% of cells dry weight.
  • The transesterification is catalyzed by immobilized lipase from Candidia sp. 99-125 in shaker flasks on a constant temperature shaker, with the rotation rate of 180 rpm. The reaction mixture is left to separate into two layers in a separating funnel after reaction. The upper layer (biodiesel) is separated and washed with hot water (50° C.). Biodiesel product is obtained after the solvent is evaporated.
  • Variables are studied in order to determine the operation conditions that could maximize the yield of biodiesel and fulfill the specifications of the US Standard for Biodiesel (ASTM 6751) related to mono, di, and triglycerides, methanol, free glycerol, and fatty acids contents. The variables are the amount of immobilized lipase related to the quantity of lipids, the quantity of methanol (methanol to lipids), the feeding frequency of methanol, the quantity of organic solvents, the water content in reaction mixtures, reaction temperature, pH value, and reaction time. The conversion rate of lipids to biodiesel was estimated by gas chromatography and mass spectrometry (GC-MS), through which the relative contents of fatty acid esters, free fatty acids, mono, di, and triglycerides could be determined. A DSQ GC system (Thermo, USA, VARIAN VF-5 ms, capillary cylinder 30 m×0.25 mm) is used for the chromatography work and data analysis. The GC was operated in constant flow mode with a flow rate of 10 mL/min. The oven is initially held at 70° C. for 2 min and then elevated to 300° C. at a rate of 10° C./min, and held at this temperature for 20 min. A split injector is used with a split ratio of 30:1 and a temperature of 250° C.
  • The high-cell-density cultivation of heterotrophic cultivation for 213 hours under above conditions, the cell density of C. protothecoides reached up to 108 g/L, and the oil content reached 61% of dry cell weight. During transesterification, 98.15% of the oil is converted to monoalkyl esters of fatty acids (biodiesel) in 12 hours. The biodiesel production rate is 64.66 g/L in bioreactors. Nine kinds of biodiesels are detected. Oleic acid methyl ester, linoleic acid methyl ester and cetane acid methyl ester are the major components, over 80% of the total content. Main results are illustrated in Table 1.
  • TABLE 1
    Relative fatty acid
    methyl ester content (%)
    Sample from
    high-cell-
    Sample density
    Sample from cultivation in
    from former bioreactor
    seed cultivation using
    cell in in specific strain
    Components flask bioreactor screened
    Methyl tetradecanoate (C15H30O2) 0.27 1.31 2.45
    Hexadecanoic acid methyl ester 13.42 12.94 16.46
    (C17H34O2)
    Heptadecanoic acid methyl ester 2.08 0.89 1.75
    (C18H36O2)
    9,12-Octadecadienoic acid methyl 19.86 17.28 16.24
    ester
    (C19H34O2)
    9-Octadecenoic acid methyl ester 58.94 60.84 53.34
    (C19H36O2)
    Octadecanoic acid methyl ester 3.4 2.76 4.98
    (C19H38O2)
    10-Nonadecenoic acid methyl ester 1.41 0.36 2.48
    (C20H38O2)
    11-Eicosenoic acid methyl ester — 0.42 1.26
    (C21H40O2)
    Eicosanoic acid methyl ester 0.62 0.35 1.04
    (C21H42O2)

    Components and Relative Content of Biodiesel from Heterotrophic C. protothecoedes Lipids
  • While specific embodiments have been described in detail in the foregoing detailed description and illustrated in the accompanying drawings, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and the broad inventive concepts thereof. It is understood, therefore, that the scope of the present invention is not limited to the particular examples and implementations disclosed herein, but is intended to cover modifications within the spirit and scope thereof as defined by the appended claims and any and all equivalents thereof.

Claims (20)

1. A method for producing biodiesel from algae using microalga chlorella Protothecoides in bioreactors, comprising the steps of:
screening a specific strain of said chlorella Protothecoides with characteristics of high yield of biomass and high oil content;
inoculating the strain in a first bioreactor for algal-seed-cells cultivating;
feeding a first solution containing carbohydrates into the first bioreactor;
transferring the cultivated algal-seed-cells into a second bioreactor for high-cell-density cultivation;
feeding a second solution containing carbohydrates into the second bioreactor;
harvesting the high density cultivated algal cells from the second bioreactor;
drying the high density cultivated algal cells;
extracting oil from dried algal cells; and
producing the biodiesel by reaction of transesterification using the extracted oil as feedstock.
2. The method of claim 1, wherein said high-cell-density is between 15 to 108 g/L.
3. The method of claim 1, wherein the high-cell-density cultivation further comprises heterotrophic cultivation of C. Protothecoides with a high oil content 40-61% dry cell weight.
4. The method of claim 1, wherein the screening step further comprises selecting cells with lower gravity and/or higher oil content using a centrifuge.
5. The method of claim 1, wherein said feeding the first solution containing carbohydrates into the first bioreactor and/or said feeding the second solution containing carbohydrates into the second bioreactor further comprises a batch feeding operation.
6. The method of claim 1, wherein said feeding the first solution containing carbohydrates into the first bioreactor and/or said feeding the second solution containing carbohydrates into the second bioreactor further comprises a continuous feeding operation.
7. The method of claim 1, wherein the first or the second sugar solution further comprises at least one of glucose or other monosaccharides, disaccharides, and polysaccharides.
8. The method of claim 7, wherein the concentration of said at least one of glucose or other monosaccharides, disaccharides, and polysaccharides, in the first or the second bioreactor is controlled between 0.01 and 100 g/L.
9. The method of claim 1, wherein the amount of inoculum in the first bioreactor or the second is between 0.01% and 50% by volume.
10. The method of claim 1, further comprising agitating the medium in the first or the second reactor with a preferable speed of agitation between 5 and 1,000 rpm.
11. The method of claim 1, further comprising a step of aerating in the first or the second bioreactor, wherein the dissolved oxygen (DO) in cultivation medium in the first or the second bioreactor is maintained at least 5% of DO saturation.
12. The method of claim 1, wherein the source of carbohydrates fed into the first or the second bioreactor comprises at least one of plants containing glucose, fructose, corn starch hydrolysate, cassaya starch hydrolysate, wheat starch hydrolysate, and juice of broomcorn.
13. The method of claim 1, further comprising feeding organic nitrogen into the first and the second bioreactor.
14. The method of claim 13, wherein the organic nitrogen comprises glycine, yeast powder, yeast extract, or corn syrup.
15. The method of claim 1, wherein pH value in the first or the second bioreactor is maintained between 6.0 and 8.0.
16. The method claim 1, further comprising adding a base to reduce acidity and regulate the pH of the culture medium in the first or the second bioreactor.
17. The method of claim 16, wherein the base comprises KOH.
18. The method of claim 1, wherein the temperature in the first or the second bioreactor is between 25 to 33° C.
19. The method of claim 1, wherein said transesterification further comprises catalyzation by immobilized lipase from candida sp. 99-125.
20. The method of claim 19, further comprising adding said lipase and methanol into said feedstock.
US12/080,666 2008-02-25 2008-04-04 Method for producing biodiesel using high-cell-density cultivation of microalga Chlorella protothecoides in bioreactor Abandoned US20090211150A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNA2008101008715A CN101230364A (en) 2008-02-25 2008-02-25 Method for producing biodiesel by high-density fermentation of heterotrophic chlorella
CN200810100871.5 2008-02-25

Publications (1)

Publication Number Publication Date
US20090211150A1 true US20090211150A1 (en) 2009-08-27

Family

ID=39897177

Family Applications (1)

Application Number Title Priority Date Filing Date
US12/080,666 Abandoned US20090211150A1 (en) 2008-02-25 2008-04-04 Method for producing biodiesel using high-cell-density cultivation of microalga Chlorella protothecoides in bioreactor

Country Status (3)

Country Link
US (1) US20090211150A1 (en)
CN (1) CN101230364A (en)
WO (1) WO2009105927A1 (en)

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090011480A1 (en) * 2007-06-01 2009-01-08 Solazyme, Inc. Use of Cellulosic Materials for Cultivation of Microorganisms
US20100151538A1 (en) * 2008-11-28 2010-06-17 Solazyme, Inc. Cellulosic Cultivation of Oleaginous Microorganisms
US20100170144A1 (en) * 2008-04-09 2010-07-08 Solazyme, Inc. Hydroprocessing Microalgal Oils
US20100239712A1 (en) * 2008-10-14 2010-09-23 Solazyme, Inc. Food Compositions of Microalgal Biomass
US20100297325A1 (en) * 2008-10-14 2010-11-25 Solazyme, Inc. Egg Products Containing Microalgae
US20100297295A1 (en) * 2008-10-14 2010-11-25 Solazyme, Inc. Microalgae-Based Beverages
US20100297331A1 (en) * 2008-10-14 2010-11-25 Solazyme, Inc. Reduced Fat Foods Containing High-Lipid Microalgae with Improved Sensory Properties
US20100303961A1 (en) * 2008-10-14 2010-12-02 Solazyme, Inc. Methods of Inducing Satiety
US20100303990A1 (en) * 2008-10-14 2010-12-02 Solazyme, Inc. High Protein and High Fiber Algal Food Materials
US20100303989A1 (en) * 2008-10-14 2010-12-02 Solazyme, Inc. Microalgal Flour
US20100303957A1 (en) * 2008-10-14 2010-12-02 Solazyme, Inc. Edible Oil and Processes for Its Production from Microalgae
WO2011035166A1 (en) 2009-09-18 2011-03-24 Phycoil Biotechnology International, Inc. Microalgae fermentation using controlled illumination
WO2011055229A2 (en) 2009-11-04 2011-05-12 Gne Global Natural Energy Ltd. Apparatus and method for cultivating protosythetic microorganisms and cells
WO2011159547A1 (en) * 2010-06-16 2011-12-22 General Atomics Algae biofuel carbon dioxide distribution system
WO2012087741A2 (en) * 2010-12-20 2012-06-28 Dvo, Inc. Algae bioreactor, system and process
US8278261B2 (en) 2008-04-09 2012-10-02 Solazyme, Inc. Method of making soaps from oil-bearing microbial biomass and oils
WO2012160577A3 (en) * 2011-05-26 2013-02-21 Council Of Scientific & Industrial Research Engine worthy fatty acid methyl ester (biodiesel) from naturally occurring marine microalgal mats and marine microalgae cultured in open salt pans together with value addition of co-products
WO2013048543A1 (en) * 2011-09-29 2013-04-04 Chlor Bioenergy Inc. Photobioreactor systems and methods for cultivation of photosynthetic organisms
WO2013054170A2 (en) 2011-10-12 2013-04-18 Indian Oil Corporation Ltd. A process for producing lipids suitable for biofuels
US8592188B2 (en) 2010-05-28 2013-11-26 Solazyme, Inc. Tailored oils produced from recombinant heterotrophic microorganisms
US8846352B2 (en) 2011-05-06 2014-09-30 Solazyme, Inc. Genetically engineered microorganisms that metabolize xylose
US8852885B2 (en) 2011-02-02 2014-10-07 Solazyme, Inc. Production of hydroxylated fatty acids in Prototheca moriformis
US8945908B2 (en) 2012-04-18 2015-02-03 Solazyme, Inc. Tailored oils
US9206388B1 (en) 2014-01-17 2015-12-08 Ronny Collins Process for a sustainable growth of algae in a bioreactor and for the extraction of a biofuel product
US9328351B2 (en) 2011-11-28 2016-05-03 Solazyme, Inc. Genetically engineered microbial strains including Prototheca lipid pathway genes
WO2016118509A1 (en) * 2015-01-20 2016-07-28 Bioprocess Algae Process and method for simultaneous saccharification and fermentation using microalgae
US9518277B2 (en) 2012-12-07 2016-12-13 Terravia Holdings, Inc. Genetically engineered microbial strains including Chlorella protothecoides lipid pathway genes
EP3136872A4 (en) * 2014-05-02 2017-11-15 Entech, LLC Methods of microalgae cultivation for increased resource production
US9969990B2 (en) 2014-07-10 2018-05-15 Corbion Biotech, Inc. Ketoacyl ACP synthase genes and uses thereof
US10053715B2 (en) 2013-10-04 2018-08-21 Corbion Biotech, Inc. Tailored oils
US10098371B2 (en) 2013-01-28 2018-10-16 Solazyme Roquette Nutritionals, LLC Microalgal flour
US10119947B2 (en) 2013-08-07 2018-11-06 Corbion Biotech, Inc. Protein-rich microalgal biomass compositions of optimized sensory quality
US10167489B2 (en) 2010-11-03 2019-01-01 Corbion Biotech, Inc. Microbial oils with lowered pour points, dielectric fluids produced therefrom, and related methods
US10479969B2 (en) 2010-10-11 2019-11-19 Phycoil Biotechnology International. Inc. Utilization of wastewater for microalgal cultivation
US11262342B2 (en) * 2017-07-12 2022-03-01 Accenture Global Solutions Limited Waste material analysis system
US11542456B2 (en) * 2008-10-14 2023-01-03 Corbion Biotech, Inc. Methods of microbial oil extraction and separation
CN116694474A (en) * 2023-04-10 2023-09-05 深圳元育生物科技有限公司 New exosome preparation method for skin care product and application thereof
US12116565B2 (en) 2013-06-12 2024-10-15 Solarvest BioEnergy Inc. Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products

Families Citing this family (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101475823B (en) * 2009-01-16 2012-05-23 清华大学 Method for preparing biodiesel from sugarcane
CN101781587B (en) * 2009-12-15 2013-04-03 张建旺 Method for preparing biodiesel by using animal fat and fresh sweet potato
BR112013002003A2 (en) * 2010-07-26 2019-09-24 Sapphire Energy Inc process for recovery of biomass oilseeds
CN103282483B (en) * 2010-09-22 2015-05-06 科学与工业研究委员会 Integrated process for the production of oil bearing chlorella variabilis for lipid extraction utilizing by-products of jatropha methyl ester (jme) production
CN103045478B (en) * 2011-10-14 2017-02-08 中国科学院烟台海岸带研究所 Method for culturing oil-producing microalgae with high density and high lipid production rate
CN102599338A (en) * 2012-03-26 2012-07-25 四川大学 Method by utilizing cassava starch hydrolyzate to prepare high-energy feed
CN102718325B (en) * 2012-07-13 2015-01-07 广西湘桂糖业集团有限公司 Method for culturing high-density oil microalgae to treat yeast industrial wastewater
CN103060199B (en) * 2013-01-29 2014-02-12 河南天冠企业集团有限公司 Semi-continuous fermentation method of heterotrophic chlorella
CN105073976A (en) 2013-03-29 2015-11-18 罗盖特兄弟公司 Microalgal biomass protein enrichment method
FR3008991B1 (en) * 2013-07-26 2016-11-04 Roquette Freres METHOD FOR FERMENTATION OF CHLORELS IN DISCONTINUOUS MODE SUPPLIED BY SEQUENTIAL AND AUTOMATED GLUCOSE FEEDS
JP2016527912A (en) * 2013-08-23 2016-09-15 ロケット フレールＲｏｑｕｅｔｔｅ Ｆｒｅｒｅｓ Industrial production of powder from lipid-rich microalgal biomass without "off-note" by controlling oxygen availability
CN103642579B (en) * 2013-11-25 2015-11-18 济南开发区星火科学技术研究院 A kind of method preparing biofuel with micro-algae
CN103952313B (en) * 2013-12-31 2016-07-06 上海交通大学 One strain algae Chlorella sorokiniana HN01 and application thereof
CN103952311B (en) * 2013-12-31 2016-03-16 上海交通大学 The beautiful glue net algae Heynigia riparia SX01 of fresh water and application thereof
CN103952312B (en) * 2013-12-31 2016-03-02 上海交通大学 One strain limnetic chlorella Chlorella sorokiniana GS03 and application thereof
CN103911217B (en) * 2014-03-14 2016-04-27 河南天冠企业集团有限公司 A kind of method of single stage method biofuel
CN103911208B (en) * 2014-03-14 2016-08-10 河南天冠企业集团有限公司 A kind of heterophytic chlorella grease extraction
CN105621802A (en) * 2016-01-19 2016-06-01 华夏亿科信息技术（北京）有限公司 Photosynthetic organism-mediated wastewater comprehensive ecological treatment system and method
WO2017137668A1 (en) 2016-02-08 2017-08-17 Roquette Freres Method for the protein enrichment of microalgal biomass
CN105755091B (en) * 2016-03-17 2019-05-14 浙江工商大学 A method of it co-cultures using general bacterium raw in rice and chlorella and improves chlorella lipid-producing
CN106281483B (en) * 2016-08-30 2018-08-24 南宁华侨投资区政孙贸易有限公司 A method of preparing biodiesel by raw material of algae
CN108315263A (en) * 2018-04-08 2018-07-24 中国科学院青岛生物能源与过程研究所 Using sorgo juice as the method for raw material culture microalgae
CN108587917B (en) * 2018-05-25 2020-12-29 杭州富阳高博信息技术服务有限公司 Method for preparing biodiesel by using chlorella pyrenoidosa cells as main raw material by using cassava residues
CN110387315A (en) * 2019-08-27 2019-10-29 济宁学院 Microalgae adsorbs the device and method of carbon dioxide preparation biodiesel
CN111763698A (en) * 2020-06-19 2020-10-13 山东天智绿业生物科技有限公司 Chlorella fermentation method for preparing and producing EPA and EPA extraction method
CN113667606B (en) * 2021-07-05 2023-11-03 南昌大学 Method for preparing protein by efficiently assimilating ammonia by taking crushed rice saccharification liquid as carbon source

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100582199C (en) * 2005-06-01 2010-01-20 清华大学 Process for preparing bio-diesel-oil by using microalgae fat
CN1837352A (en) * 2006-04-29 2006-09-27 清华大学 Method for culturing heterotrophic chlorella with high density

Cited By (93)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8802422B2 (en) 2007-06-01 2014-08-12 Solazyme, Inc. Renewable diesel and jet fuel from microbial sources
US20110015417A1 (en) * 2007-06-01 2011-01-20 Solazyme, Inc. Production of Oil in Microorganisms
US8790914B2 (en) 2007-06-01 2014-07-29 Solazyme, Inc. Use of cellulosic materials for cultivation of microorganisms
US20090061493A1 (en) * 2007-06-01 2009-03-05 Solazyme, Inc. Lipid Pathway Modification in Oil-Bearing Microorganisms
US8889401B2 (en) 2007-06-01 2014-11-18 Solazyme, Inc. Production of oil in microorganisms
US8889402B2 (en) 2007-06-01 2014-11-18 Solazyme, Inc. Chlorella species containing exogenous genes
US10138435B2 (en) 2007-06-01 2018-11-27 Corbion Biotech, Inc. Renewable diesel and jet fuel from microbial sources
US9434909B2 (en) 2007-06-01 2016-09-06 Solazyme, Inc. Renewable diesel and jet fuel from microbial sources
US20110190522A1 (en) * 2007-06-01 2011-08-04 Solazyme, Inc. Renewable Chemicals and Fuels From Oleaginous Yeast
US8647397B2 (en) 2007-06-01 2014-02-11 Solazyme, Inc. Lipid pathway modification in oil-bearing microorganisms
US20090047721A1 (en) * 2007-06-01 2009-02-19 Solazyme, Inc. Renewable Diesel and Jet Fuel from Microbial Sources
US20090011480A1 (en) * 2007-06-01 2009-01-08 Solazyme, Inc. Use of Cellulosic Materials for Cultivation of Microorganisms
US8476059B2 (en) 2007-06-01 2013-07-02 Solazyme, Inc. Sucrose feedstock utilization for oil-based fuel manufacturing
US8518689B2 (en) 2007-06-01 2013-08-27 Solazyme, Inc. Production of oil in microorganisms
US8512999B2 (en) 2007-06-01 2013-08-20 Solazyme, Inc. Production of oil in microorganisms
US20100323413A1 (en) * 2007-06-01 2010-12-23 Solazyme, Inc. Production of Oil in Microorganisms
US20090035842A1 (en) * 2007-06-01 2009-02-05 Solazyme, Inc. Sucrose Feedstock Utilization for Oil-Based Fuel Manufacturing
US8497116B2 (en) 2007-06-01 2013-07-30 Solazyme, Inc. Heterotrophic microalgae expressing invertase
US8822176B2 (en) 2008-04-09 2014-09-02 Solazyme, Inc. Modified lipids produced from oil-bearing microbial biomass and oils
US8450083B2 (en) 2008-04-09 2013-05-28 Solazyme, Inc. Modified lipids produced from oil-bearing microbial biomass and oils
US8822177B2 (en) 2008-04-09 2014-09-02 Solazyme, Inc. Modified lipids produced from oil-bearing microbial biomass and oils
US20100170144A1 (en) * 2008-04-09 2010-07-08 Solazyme, Inc. Hydroprocessing Microalgal Oils
US8278261B2 (en) 2008-04-09 2012-10-02 Solazyme, Inc. Method of making soaps from oil-bearing microbial biomass and oils
US20100303989A1 (en) * 2008-10-14 2010-12-02 Solazyme, Inc. Microalgal Flour
US20100303957A1 (en) * 2008-10-14 2010-12-02 Solazyme, Inc. Edible Oil and Processes for Its Production from Microalgae
US20100239712A1 (en) * 2008-10-14 2010-09-23 Solazyme, Inc. Food Compositions of Microalgal Biomass
US11542456B2 (en) * 2008-10-14 2023-01-03 Corbion Biotech, Inc. Methods of microbial oil extraction and separation
US20100297325A1 (en) * 2008-10-14 2010-11-25 Solazyme, Inc. Egg Products Containing Microalgae
US20100297295A1 (en) * 2008-10-14 2010-11-25 Solazyme, Inc. Microalgae-Based Beverages
US20100297331A1 (en) * 2008-10-14 2010-11-25 Solazyme, Inc. Reduced Fat Foods Containing High-Lipid Microalgae with Improved Sensory Properties
US20100303961A1 (en) * 2008-10-14 2010-12-02 Solazyme, Inc. Methods of Inducing Satiety
US12059006B2 (en) 2008-10-14 2024-08-13 Corbion Biotech, Inc. Microalgal flour
US20100303990A1 (en) * 2008-10-14 2010-12-02 Solazyme, Inc. High Protein and High Fiber Algal Food Materials
US20110203168A1 (en) * 2008-11-28 2011-08-25 Solazyme, Inc. Novel Triglyceride and Fuel Compositions
US8187860B2 (en) 2008-11-28 2012-05-29 Solazyme, Inc. Recombinant microalgae cells producing novel oils
US8435767B2 (en) 2008-11-28 2013-05-07 Solazyme, Inc. Renewable chemical production from novel fatty acid feedstocks
US20110165634A1 (en) * 2008-11-28 2011-07-07 Solazyme, Inc. Renewable chemical production from novel fatty acid feedstocks
US9062294B2 (en) 2008-11-28 2015-06-23 Solazyme, Inc. Renewable fuels produced from oleaginous microorganisms
US9464304B2 (en) 2008-11-28 2016-10-11 Terravia Holdings, Inc. Methods for producing a triglyceride composition from algae
US20100151538A1 (en) * 2008-11-28 2010-06-17 Solazyme, Inc. Cellulosic Cultivation of Oleaginous Microorganisms
US8951777B2 (en) 2008-11-28 2015-02-10 Solazyme, Inc. Recombinant microalgae cells producing novel oils
US20100151567A1 (en) * 2008-11-28 2010-06-17 Solazyme, Inc. Nucleic Acids Useful in the Manufacture of Oil
US8222010B2 (en) 2008-11-28 2012-07-17 Solazyme, Inc. Renewable chemical production from novel fatty acid feedstocks
US8697427B2 (en) 2008-11-28 2014-04-15 Solazyme, Inc. Recombinant microalgae cells producing novel oils
US8268610B2 (en) 2008-11-28 2012-09-18 Solazyme, Inc. Nucleic acids useful in the manufacture of oil
WO2011035166A1 (en) 2009-09-18 2011-03-24 Phycoil Biotechnology International, Inc. Microalgae fermentation using controlled illumination
JP2013505024A (en) * 2009-09-18 2013-02-14 フィコイル バイオテクノロジー インターナショナル，インコーポレイテッド Fermentation of microalgae using controlled lighting
CN102656261A (en) * 2009-09-18 2012-09-05 菲科尔生物技术国际公司 Microalgae fermentation using controlled illumination
CN102656261B (en) * 2009-09-18 2015-07-01 菲科尔生物技术国际公司 Microalgae fermentation using controlled illumination
US9932554B2 (en) 2009-09-18 2018-04-03 Phycoil Biotechnology International, Inc. Microalgae fermentation using controlled Illumination
WO2011055229A2 (en) 2009-11-04 2011-05-12 Gne Global Natural Energy Ltd. Apparatus and method for cultivating protosythetic microorganisms and cells
US10006034B2 (en) 2010-05-28 2018-06-26 Corbion Biotech, Inc. Recombinant microalgae including keto-acyl ACP synthase
US8592188B2 (en) 2010-05-28 2013-11-26 Solazyme, Inc. Tailored oils produced from recombinant heterotrophic microorganisms
WO2011159547A1 (en) * 2010-06-16 2011-12-22 General Atomics Algae biofuel carbon dioxide distribution system
CN102971429A (en) * 2010-06-16 2013-03-13 通用原子公司 Algae biofuel carbon dioxide distribution system
US10479969B2 (en) 2010-10-11 2019-11-19 Phycoil Biotechnology International. Inc. Utilization of wastewater for microalgal cultivation
US10167489B2 (en) 2010-11-03 2019-01-01 Corbion Biotech, Inc. Microbial oils with lowered pour points, dielectric fluids produced therefrom, and related methods
US10344305B2 (en) 2010-11-03 2019-07-09 Corbion Biotech, Inc. Microbial oils with lowered pour points, dielectric fluids produced therefrom, and related methods
US9005918B2 (en) 2010-12-20 2015-04-14 Dvo, Inc. Algae bioreactor, system and process
WO2012087741A2 (en) * 2010-12-20 2012-06-28 Dvo, Inc. Algae bioreactor, system and process
WO2012087741A3 (en) * 2010-12-20 2012-08-09 Dvo, Inc. Algae bioreactor, system and process
US10100341B2 (en) 2011-02-02 2018-10-16 Corbion Biotech, Inc. Tailored oils produced from recombinant oleaginous microorganisms
US8852885B2 (en) 2011-02-02 2014-10-07 Solazyme, Inc. Production of hydroxylated fatty acids in Prototheca moriformis
US9249436B2 (en) 2011-02-02 2016-02-02 Solazyme, Inc. Tailored oils produced from recombinant oleaginous microorganisms
US9499845B2 (en) 2011-05-06 2016-11-22 Terravia Holdings, Inc. Genetically engineered microorganisms that metabolize xylose
US8846352B2 (en) 2011-05-06 2014-09-30 Solazyme, Inc. Genetically engineered microorganisms that metabolize xylose
WO2012160577A3 (en) * 2011-05-26 2013-02-21 Council Of Scientific & Industrial Research Engine worthy fatty acid methyl ester (biodiesel) from naturally occurring marine microalgal mats and marine microalgae cultured in open salt pans together with value addition of co-products
WO2013048543A1 (en) * 2011-09-29 2013-04-04 Chlor Bioenergy Inc. Photobioreactor systems and methods for cultivation of photosynthetic organisms
WO2013054170A2 (en) 2011-10-12 2013-04-18 Indian Oil Corporation Ltd. A process for producing lipids suitable for biofuels
US9328351B2 (en) 2011-11-28 2016-05-03 Solazyme, Inc. Genetically engineered microbial strains including Prototheca lipid pathway genes
US9649368B2 (en) 2011-11-28 2017-05-16 Terravia Holdings, Inc. Genetically engineered microbial strains including prototheca lipid pathway genes
US10683522B2 (en) 2012-04-18 2020-06-16 Corbion Biotech, Inc. Structuring fats and methods of producing structuring fats
US9909155B2 (en) 2012-04-18 2018-03-06 Corbion Biotech, Inc. Structuring fats and methods of producing structuring fats
US11401538B2 (en) 2012-04-18 2022-08-02 Corbion Biotech, Inc. Structuring fats and methods of producing structuring fats
US9551017B2 (en) 2012-04-18 2017-01-24 Terravia Holdings, Inc. Structuring fats and methods of producing structuring fats
US9200307B2 (en) 2012-04-18 2015-12-01 Solazyme, Inc. Tailored oils
US9102973B2 (en) 2012-04-18 2015-08-11 Solazyme, Inc. Tailored oils
US9068213B2 (en) 2012-04-18 2015-06-30 Solazyme, Inc. Microorganisms expressing ketoacyl-CoA synthase and uses thereof
US10287613B2 (en) 2012-04-18 2019-05-14 Corbion Biotech, Inc. Structuring fats and methods of producing structuring fats
US8945908B2 (en) 2012-04-18 2015-02-03 Solazyme, Inc. Tailored oils
US9518277B2 (en) 2012-12-07 2016-12-13 Terravia Holdings, Inc. Genetically engineered microbial strains including Chlorella protothecoides lipid pathway genes
US10264809B2 (en) 2013-01-28 2019-04-23 Corbion Biotech, Inc. Microalgal flour
US10098371B2 (en) 2013-01-28 2018-10-16 Solazyme Roquette Nutritionals, LLC Microalgal flour
US12116565B2 (en) 2013-06-12 2024-10-15 Solarvest BioEnergy Inc. Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products
US10119947B2 (en) 2013-08-07 2018-11-06 Corbion Biotech, Inc. Protein-rich microalgal biomass compositions of optimized sensory quality
US10053715B2 (en) 2013-10-04 2018-08-21 Corbion Biotech, Inc. Tailored oils
US9206388B1 (en) 2014-01-17 2015-12-08 Ronny Collins Process for a sustainable growth of algae in a bioreactor and for the extraction of a biofuel product
EP3136872A4 (en) * 2014-05-02 2017-11-15 Entech, LLC Methods of microalgae cultivation for increased resource production
US10316299B2 (en) 2014-07-10 2019-06-11 Corbion Biotech, Inc. Ketoacyl ACP synthase genes and uses thereof
US9969990B2 (en) 2014-07-10 2018-05-15 Corbion Biotech, Inc. Ketoacyl ACP synthase genes and uses thereof
WO2016118509A1 (en) * 2015-01-20 2016-07-28 Bioprocess Algae Process and method for simultaneous saccharification and fermentation using microalgae
US11262342B2 (en) * 2017-07-12 2022-03-01 Accenture Global Solutions Limited Waste material analysis system
CN116694474A (en) * 2023-04-10 2023-09-05 深圳元育生物科技有限公司 New exosome preparation method for skin care product and application thereof

Also Published As

Publication number Publication date
CN101230364A (en) 2008-07-30
WO2009105927A1 (en) 2009-09-03

Similar Documents

Publication Publication Date Title
US20090211150A1 (en) Method for producing biodiesel using high-cell-density cultivation of microalga Chlorella protothecoides in bioreactor
Widjaja et al. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris
Li et al. Large‐scale biodiesel production from microalga Chlorella protothecoides through heterotrophic cultivation in bioreactors
Bharathiraja et al. Aquatic biomass (algae) as a future feed stock for bio-refineries: A review on cultivation, processing and products
EP2292782B1 (en) Method for producing biodiesel by two-stage culture of chlorella from autotrophy to heterotrophy
Ghasemi et al. Microalgae biofuel potentials
He et al. Cultivation of Chlorella vulgaris on wastewater containing high levels of ammonia for biodiesel production
Heredia-Arroyo et al. Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from non-sugar materials
Yousuf Fundamentals of microalgae cultivation
CN101475823B (en) Method for preparing biodiesel from sugarcane
US20120282651A1 (en) System and Method of Co-Cultivating Microalgae with Fungus
Frac et al. Microalgae for biofuels production and environmental applications: A review
JP2018520636A (en) Omega-7 fatty acid composition, and method and application for culturing yellow-green algae to produce the composition
Yi-Feng et al. Production of biodiesel from algal biomass: current perspectives and future
Sun et al. Screening of Isochrysis strains and utilization of a two-stage outdoor cultivation strategy for algal biomass and lipid production
Rismani-Yazdi et al. High-productivity lipid production using mixed trophic state cultivation of Auxenochlorella (Chlorella) protothecoides
TWI648400B (en) Micractinium sp. and uses thereof
UA121872C2 (en) Oleaginous yeast variant, method for obtaining thereof and use thereof for lipid production
Kumar et al. Plants and algae species: Promising renewable energy production source.
Haldar et al. Microalgae as a potential source of biofuels and its current advances
US9441252B2 (en) Process for producing lipids suitable for biofuels
CA2904038C (en) Production of omega-3 fatty acids from pythium species
Poonia et al. Algae: The high potential resource for biofuel production
Viswanath et al. Biodiesel production potential of wastewater microalgae Chlorella sp. under photoautotrophic and heterotrophic growth conditions
Ferreira et al. Microalgae Biomass as an Alternative to Fossil Carbons

Legal Events

Date Code Title Description
AS Assignment

Owner name: TSINGHUA UNIVERSITY, CHINA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WU, QINGYU;ZHOU, WENGUANG;XIONG, WEI;REEL/FRAME:021286/0912

Effective date: 20080611

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION