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WO2013044475A1 - Procédé et système utilisant le genre cystofilobasidium de levure pour produire de la graisse - Google Patents

Procédé et système utilisant le genre cystofilobasidium de levure pour produire de la graisse Download PDF

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Publication number
WO2013044475A1
WO2013044475A1 PCT/CN2011/080337 CN2011080337W WO2013044475A1 WO 2013044475 A1 WO2013044475 A1 WO 2013044475A1 CN 2011080337 W CN2011080337 W CN 2011080337W WO 2013044475 A1 WO2013044475 A1 WO 2013044475A1
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Prior art keywords
oil
bacterium
reaction system
producing
fatty acid
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English (en)
Chinese (zh)
Inventor
吕志翼
许俼頔
吴昱昇
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Fu Jen Catholic University
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Fu Jen Catholic University
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    • 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
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • 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

Definitions

  • the present invention relates to an oil-producing microorganism, and more particularly to an oil-producing microorganism of the genus Cystofilobasidium.
  • biomass energy is roughly divided into two categories, one is alcohol and the other is biodiesel.
  • the preparation of bio-alcohol is still based on grain as raw material, but in recent years it has gradually turned into the production of alcohol by microorganisms.
  • biodiesel is partly prepared from plant raw materials or waste cooking oils and animal fats.
  • Most of the plant materials are mainly oil grains, such as soybean, corn, rapeseed, palm, jatropha seeds, sunflowers, etc.; waste cooking oil and animal fat are re-purified to extract and regenerate raw diesel.
  • neither biodiesel can meet the needs of actual use, and each has its own drawbacks.
  • Oil crops require a large range of land to be planted, crops have seasonality, and the biomass energy provided by planting per unit area is not economically viable.
  • the problem of food competition, the complicated extraction process and high cost, oil crops cannot be the raw material for continuously providing biodiesel to replace petroleum. Waste cooking oil and animal fat are more problematic, feeding and environmental pollution. Therefore, in recent years, emphasis has been placed on the production of biodiesel from micro-organisms. According to research, as long as 1-3% of the grain is planted, microbes can produce 40% of the alternative energy needed for transportation, with considerable potential and advantages.
  • Rhodotorula gram in is 36
  • the main purpose of this case is to provide a method for producing oils and fats from the yeast of the genus Cystofilobasidium for use as biodiesel to replace petroleum as an alternative energy source.
  • the present invention provides an oil production reaction system comprising: a saccharide; and a bacterium which reacts with the saccharide to produce a fat, wherein the genus of the bacterium is Cystofilobasidium.
  • the oil production reaction system further comprises a nitrogen source for use by the bacteria, wherein the nitrogen source is sodium (Na(N3 ⁇ 4)).
  • the oil produced by the active bacteria is a medium chain fatty acid of C14 ⁇ C20.
  • the oil produced by the active bacteria is an unsaturated fatty acid.
  • the oil produced by the active bacteria can be used as biodiesel.
  • the active bacterium has the 18S rDNA sequence of SEQ ID NO.
  • the active bacterium has the 26S rDNA sequence of SEQ ID NO.
  • the active bacterium has the ITS sequence of SEQ ID NO.
  • the present invention further provides an oil production reaction system comprising: a carbon source; and a bacterium that reacts with the carbon source to produce a grease, wherein the genus of the bacterium is Cystofilobasidium.
  • the active bacteria decompose the carbon source to produce the oil.
  • the present invention further provides an oil production method which utilizes an oil-producing bacterium to produce an oily genus, the genus of which is Cystofilobasidium flick
  • the oil-producing bacteria are selected from the group consisting of Cystofilobasidium bisporidii, Cystofilobasidium capitatum, Cystofilobasidium infirmominiatum and other Cystofilobasidium species. BRIEF abstract
  • Figure 1 shows the type and oil staining results of the optical and fluorescent microscope TN01 strain (1000 times);
  • Figure 2 shows the strain type of the scanning electron microscope TN01;
  • Figure 3 is a diagram showing the relationship of 18S rDNA sequence identification
  • Figure 4 is a 26S rDNA sequence identification relationship diagram
  • Figure 5 is a diagram showing the relationship of ITS sequence identification
  • Figure 6 shows the salt concentration test and growth curve of TN01 in different salt concentration medium
  • Figure 7 shows the salt concentration test for TN01 optimal culture
  • Figure 8 shows the TN01 optimal culture temperature test
  • Figure 9 shows the simplification test of TN01 medium
  • Figure 10 shows the TN01 optimal culture pH test
  • Figure 1 1 shows the carbon source test for TN01 optimal culture
  • Figure 12 is a nitrogen source test for TN01 optimal culture
  • Figure 13 is a TN01 high glucose concentration medium test. Preferred embodiment of the invention
  • CGMCC General Microbiology Center
  • the source of the experimental strain was collected from the coastal areas of Tainan, Taiwan.
  • the anti-LgYPG medium was used for the first screening, and the colonies with different appearances were picked out and observed by an optical microscope. The results are shown in 1-2 of Fig. 1.
  • the colonies have a round appearance and a smooth surface, like wax. Glossy, pink.
  • the first screened sample was cultured in LgYPG medium at 20 ° C for 7 days for the second screening.
  • the colonies screened for the second time were stained with Nile Red and observed with a fluorescence microscope, and the results were as shown in Figs. 3-4 of Fig. 1, and the oily portions were golden yellow.
  • the oily strain was observed to be cultured in GYPG medium at 20 ° C for 7 days, and after repeated purification three times, an experimental strain was obtained.
  • LgYPG medium formulation such as Anti-LgYPG, but without streptomycin.
  • GYPG solid medium formulation such as LgYPG, but the glucose content is adjusted to 5%.
  • GYPG liquid medium formula such as GYPG, but no added vegetables.
  • TN01 is an elliptical shaped cell body
  • the surface of the bacterial cell is smooth without wrinkles or protrusions
  • the sterile silk body has a form similar to budding reproduction, so according to the type
  • the state estimation may be a yeast genus.
  • the pellet was collected after centrifugation at 14,000 rpm and reconstituted with 350 ⁇ l of 1.2 M sodium chloride.
  • the agent is dissolved in water once, and after the dissolution is completed, the pH is adjusted to 8.2 with hydrogen chloride or sodium hydroxide, and sterilized by autoclaving.
  • the medicament is dissolved in one water and sterilized by autoclaving. Polymerase chain reaction
  • This experiment is a sequence amplification of the strain identification, and three sets of primers are used.
  • the sequence and conditions are as follows: 18S sequencing primer:
  • 18SF forward
  • 18SR reverse
  • ITS1 forward
  • ITS4 reverse
  • the target used is Bio-IKB DNA Ladder (Protech)
  • the sequences were sequenced by the ClustalW tool module, analyzed by the bioanalysis software MEGA 4, and the relationship between the TN strain ⁇ 1 J and the standard strain was established using the bootstrap test and the neighbor-joining classification method, and the 18S rDNA sequence was affinities.
  • the relationship is shown in Fig. 3, the 26S rDNA D1/D2 sequence is related to Fig. 4, and the ITS sequence is related to Fig. 5, in which Rhodotorula lamellibrachiae is another oleaginous yeast, which is used as a control group. It can be seen from the figures that the TN series is the closest to the standard strain Cystofilobasidium, but there are still some differences.
  • the isolated experimental strain and the standard strain were subjected to physiological and biochemical tests of carbon sources using Yeast identification system, ID 32C (REF 32 200), purchased from bioM rieux® (http://www.biomerieux.com).
  • the experimental strain and the standard strain were cultured in a test tube containing 3 ml of GYPG liquid medium at 20 ° C.
  • OD 6 QQ measured concentration OD value less than 0.2 is no reaction (-), 0.2-0.3 is weak signal (W), 0.4-0.6 is strong signal (+), greater than 0.6 is the highest signal (++).
  • the results are shown in Table 2.
  • the carbon source types are shown in Table 3. Compared with the standard strains, TN 01 is the closest to the C. bisporidii bacteria. The difference carbon sources are lactic acid (LAT) and strontium. Base-D-glucopyranose (MDG), isomaltulose (PLE), sodium glucuronate (GRT), and other standard strains are quite different.
  • the fatty acid extraction was carried out for each experimental group, and the dried bacterial powder was obtained by freeze-drying, and the dried bacterial powder was used for the experiment. Since the fatty acid is in the form of triglyceride in the living body, and the three free fatty acids are combined with glycerol in the form of esterification, it is necessary to carry out the transesterification reaction, and the esterification of the sterol and the fatty acid is repeated to form A single fatty acid oxime ester can be analyzed.
  • the chemical equation for the transesterification reaction is as follows: CH2 OCORi st CH 2 OH Ri ⁇ OOCHs
  • the selection of the internal standard will generally choose not to appear in the sample to be tested, and the structure, chemical properties, and similar boiling point substances are used as internal standards, and most of the organisms are fatty acids with even carbon chain lengths. Odd carbons have very few fatty acids, so nonadecanioc acid is used as an internal standard.
  • the extraction steps are as follows:
  • the GC model is a column with a lower polarity of DB-1, a length of 60 meters and an inner diameter of 0.25 mm.
  • the inner membrane of the column is a polydithiosiloxane [-oxo-di-n-silicon] -] , thickness 0.25 ⁇ ; oven starting temperature 60 ° C, heating to 280 ° C, syringe heating temperature 250 ° C; nitrogen flow rate 1.2 ml / min, hydrogen flow rate 30 ml / min, air flow rate At 300 ml/min, the sample was injected at ⁇ , and the sample was detected using a flame ionization detector at a set temperature of 300 °C. The results were integrated using the GC kit software and the fatty acid content was estimated using the standard.
  • the estimation formula is:
  • the amount of diluted seawater added to the medium was 0%, 1%, 25%, and 50%, respectively, and 5 liters of the fermentation tank was cultured. 50 ml of the bacterial liquid was collected every 24 hours, and the dry weight was measured after lyophilization, and the total number of cells and the number of culture days were estimated.
  • Figure 6 is a standard curve for testing the growth of each condition salt concentration TN 01. The rapid growth period of the cells is from the first day to the fifth day, and the gradation is started on the sixth day. Therefore, the time for the subsequent optimal culture to collect the cells is set as On day 6, and on days 7-10, the proliferation rate of the strain became slower, but it still showed an upward trend.
  • the optimal concentration of seawater with a salt concentration of 1% is the worst, 25% seawater concentration, followed by 0% and 50%.
  • the dry weight of the cells in the growth conditions of 1% seawater concentration reached 13.09 g/L, and 0%, 25%, and 50% were 11.84 g/L, 10.73 g/L, and 11.31 g/L, respectively.
  • the cells on the sixth day were collected for measurement of dry weight and fatty acid analysis, and the results are shown in Fig. 7.
  • 0%, 1%, 25%, 50% seawater culture concentration of bacteria (50 ml) dry weight were 0.31 g, 0.39 g, 0.266 g, 0.297 g, respectively, total fatty acid content was 50.91%, 51.24%, 44.31% 41.76%, comparing the results of the growth curve with the results of the fatty acid analysis, the seawater concentration of the subsequent optimization experiments was 1%.
  • Culture temperature range test
  • TN 01 was cultured in 50 ml of GYPG medium at three different temperatures. After 6 days of culture, the cells were collected, and the dry weight and fatty acid analysis results were as shown in Fig. 8. The best biomass and the best fatty acid were cultured at 20 °C. Percentage, while growing at 28 ° C poor growth, culture at 37 ° C almost impossible to grow, and the culture is very different at 20 ° C. The dry weights of the cells cultured at 20 ° C, 28 ° C, and 37 ° C were 0.317 g, 0.027 g, and 0.023 g, respectively, and the total fatty acid contents were 53.1%, 4.40%, and 0.63%, respectively. Simplified medium
  • TN 01 growth and fatty acid accumulation were tested using different simplified media.
  • TN 01 was cultured in 50 ml of experimental medium. After 6 days of culture, the dried liquid was collected and the dry weight was measured and the total fatty acid content was analyzed.
  • GY, GYP, GYPG medium cultured dry weight of 0.1750 g, 0.2450 g, 0.3010 g, total fatty acid content of 53.96%, 52.35%, 47.84%, respectively, cultured in GYPG medium has the highest dry weight However, the fatty acid is the lowest; the culture in GY medium is the opposite, so the dry weight is considered to be very different, and the subsequent experimental basal medium is still tested in GYPG medium.
  • Growth pH test
  • the pH experiment was set to a range of 3 to 11 to investigate the effects of pH on the cells and the most suitable culture conditions for growth.
  • the following buffers were added according to the conditions of the GYPG medium:
  • TN 01 was cultured at each pH value, and after 6 days of culture, the bacterial liquid (50 ml) was collected, and the dry weight was measured after drying. The result was as shown in Fig. 10.
  • the dry weight of the pH 3-11 was 0.0423 g and 0.025 g, respectively.
  • 0.412 g, 0.45 g, 0.479 g, 0.596 g, 0.369 g, 0.168 g, O.lg, pH optimum for the growth of the cells is pH 8.
  • Types of carbon sources glucose, fructose, lactose, maltose, sucrose, glycerin, sorbitol, starch, acetate.
  • the results of dry weight and total fatty acid content are shown in Figure 11.
  • the carbon source available for the best biomass of TN 01 is starch, with a dry weight of 0.382 g, followed by sucrose, and a dry weight of 0.364 g, while the total fatty acid is the best.
  • the most cultivated carbon source is sucrose, the content is 70%, followed by glucose, and the content is 56%.
  • the biomass cultured in lactose and acetate is the worst and the fatty acid content is the least.
  • Nitrogen source is a necessary nutrient source for microbial growth. Studies indicate that different nitrogen sources affect the accumulation of microbial oils. Therefore, the types of organic nitrogen sources and inorganic nitrogen sources are designed. Observing the growth of bacteria and accumulation of oil, it is expected to find The best source of nitrogen. The ingredients of this practical medium are as follows:
  • Types of nitrogen sources bismuth, tryptone protein, soybean meal, ammonium nitrate, ammonium sulfate, ammonium chloride, sodium ammonium, urea, sodium thiosulfate.
  • sodium thiosulfate has no nitrogen source and is regarded as a control group.
  • the results of determining the dry weight and total fatty acid content are shown in Fig. 12.
  • the best nitrogen source for bacterial biomass is urea.
  • the dry weight was 0.318 g, followed by soybean meal, sputum and trypsin.
  • the dry weights were 0.304 g, 0.296 g and 0.293 g, respectively.
  • the accumulation of fatty acids was the best control group sodium thiosulfate (ST), which was 66.6%.
  • SA sodium ammonium
  • Carbon is the basic unit of fatty acid formation, and fermentation-fed-fatch is used to adjust the accumulation of oil. Therefore, a high-sugar concentration test is designed to see the effect of high-concentration sugar on the growth of bacteria and the fatty acid. There is an increase.
  • the experimental group was based on GYPG liquid medium, and the glucose addition amounts were 50 g/L, 100 g/L, 150 g/L, and 200 g/L, respectively.
  • 0.2680 g, 0.2580 g, 0.2900 g, 0.2960 g, and fatty acid contents were 47.5%, 43.5%, 30.5%, and 24.3%, respectively.
  • the best biomass is available at a glucose concentration of 150 g and the best total fatty acid content at a concentration of 50 g. TN 01 fatty acid type
  • TN 01 was cultured in GYPG liquid medium, and the cells were collected after drying. After fatty acid extraction, in order to know the detailed type of TN 01 fatty acid, the extracted sample was sent to Xinde Biotech Co., Ltd. for GC-MS analysis. It is found that the TN 01 fatty acids are mostly low-saturation fatty acids. As shown in Table 4, most of them are C14, C16, and C18 series. These fatty acids are less susceptible to oxidation and are more suitable for biodiesel production than saturated fatty acids.
  • the heat and oxidants in the biodiesel production process may cause the cracking of saturated fatty acids, and the additional cost of adding antioxidants, and up to 50% of total fatty acids before TN 01 is optimal, so it is extremely The potential for biodiesel sources.
  • Table 4 TN 01 fatty acid species and percentage of total fatty acids Fatty acid species content (% of total fatty acids)
  • TN 01 Cystofilobasidium bisporidii BCRC 22462, Cystofilobasidium capitatum BCRC 22464 and Cystofilobasidium in firmom in iatum BCRC 22465 (BCRC: Bioresource Collection and Research Center) were cultured in the following media:

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Abstract

L'invention concerne un procédé et un système utilisant le genre Cystofilobasidium de levure pour produire de la graisse. Le système comprend : la réaction de la levure et du glucide pour produire de la graisse, le genre de la levure étant Cystofilobasidium.
PCT/CN2011/080337 2011-09-29 2011-09-29 Procédé et système utilisant le genre cystofilobasidium de levure pour produire de la graisse Ceased WO2013044475A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002300896A (ja) * 2001-01-30 2002-10-15 Toyota Motor Corp プレニルアルコールの製造方法
JP2010130912A (ja) * 2008-12-02 2010-06-17 Kaneka Corp 光学活性３−キヌクリジノールの製造方法
WO2012016177A2 (fr) * 2010-07-30 2012-02-02 Amyris, Inc. Microbes génétiquement modifiés produisant de plus hauts niveaux de composés dérivés d'acétyl-coa
US20120040419A1 (en) * 2010-08-13 2012-02-16 Fu-Jen Catholic University Method for producing oil by cystofilobasidium spp.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002300896A (ja) * 2001-01-30 2002-10-15 Toyota Motor Corp プレニルアルコールの製造方法
JP2010130912A (ja) * 2008-12-02 2010-06-17 Kaneka Corp 光学活性３−キヌクリジノールの製造方法
WO2012016177A2 (fr) * 2010-07-30 2012-02-02 Amyris, Inc. Microbes génétiquement modifiés produisant de plus hauts niveaux de composés dérivés d'acétyl-coa
US20120040419A1 (en) * 2010-08-13 2012-02-16 Fu-Jen Catholic University Method for producing oil by cystofilobasidium spp.

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 20 March 2001 (2001-03-20), FELL, J. W. ET AL.: "Cytofilobasidium bisporidii strain CBS6346 26S ribosomal RNA gene, partial sequence", accession no. F189832 *
HUI, FENGLI ET AL.: "Screening of yeast capable of fermenting xylose to lipid and its lipid component analysis", ACTA ENERGIAE SOLARIS SINICA, vol. 30, no. 1, 28 January 2009 (2009-01-28), pages 107 - 111 *
YANG, MIN ET AL.: "Breeding of microbial strains containing phenylalanine ammonia lyase (pal) for the bioconversion of trans cinnamic acid into lphenylalanine", CHINESE JOURNAL OF APPLIED AND ENVIRONMENTAL BIOLOGY, vol. 5, no. 6, 28 December 1999 (1999-12-28), pages 643 - 650 *

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