WO2013044475A1 - Method and system utilizing cystofilobasidium genus of yeast to produce grease - Google Patents

Abstract

Method and system utilizing Cystofilobasidium genus of yeast to produce grease are provided. The system includes: reacting the yeast and the carbohydrate to produce grease, wherein the genus of the yeast is Cystofilobasidium.

Description

 Method and system for producing oil by using yeast of Cvstofilobasidium

 The present invention relates to an oil-producing microorganism, and more particularly to an oil-producing microorganism of the genus Cystofilobasidium.

Background technique

 At present, 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. There have been many studies reporting that anaerobic microbes have a high potential for the production of alcohol; 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. However, 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.

 The concept of using microbes to produce oil is not discovered in recent years. In addition to microalgae and bacteria, there are also oil-producing microorganisms in fungi, most of which are yeasts and molds, and some species produce both long-chain saturated fatty acids, units or The properties of polyunsaturated fatty acids, which produce more than 40% of the fatty acids, can accumulate up to 70% through medium restriction and formulation, all with considerable oil production potential. Table 1 summarizes common fungal oil-producing microorganisms (Beopoulos, et al, 2009; Gill, Hall, & Ratledge, 1977; Ratledge, 1993, 2002, 2004; Sergeeva la, Galanina, Andrianova, & Feofilova, 2008; Zhao, et Al., 2010).

Table 1. Oil-producing fungi and oil production Fungal dry weight% (w/w)

Aspergillus terreus 64

Cryptococcus curvatus 58

Cryptococcus albidus 65

 Candida sp. 107 42

Cunn inghamella japonica >43.8

 Lipomyces starkeyi 63

Penicillium spmulosum 64

Rhodosporidium toruloides 56.5

 Rhodotorula glutinis 72

Rhodotorula gram in is 36

 Rhizopus arrhizus 57

Schizochytrium spp. 30-50

Thraustochytrium spp. 30-50

Trichosporon pullulans 65

 Yarrowia lipolytica 36 SUMMARY OF THE INVENTION

 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.

 According to the above concept, 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. Wherein the active bacterium has the 18S rDNA sequence of SEQ ID NO.

 Wherein the active bacterium has the 26S rDNA sequence of SEQ ID NO.

 Wherein the active bacterium has the ITS sequence of SEQ ID NO.

 According to the above concept, 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.

 Wherein the active bacteria decompose the carbon source to produce the oil.

 According to the above concept, 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 „

 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

 The strains described below were deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on June 20, 2011. The address of the depository is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number of the strain in the depository is CGMCC N0.4968

Type observation

 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.

a Anti-LgYPG solid medium

b. LgYPG培养基配方如 Anti-LgYPG, 但不加入链霉素。 c. GYPG固态培养基配方如 LgYPG, 但葡萄糖含量调整为 5%。

b. LgYPG medium formulation such as Anti-LgYPG, but without streptomycin. c. GYPG solid medium formulation such as LgYPG, but the glucose content is adjusted to 5%.

d. GYPG liquid medium formula such as GYPG, but no added vegetables.

 After several times of purification and separation, the oil-producing indica organisms were selected and labeled as ΤΝ01~ ΤΝ09, and confirmed to be pure bacteria. Because the selected cockroaches series are very similar, in the subsequent experiments, TN01 was randomly selected as the most. The subject of adaptation culture.

 Further observation using a scanning electron microscope, as shown in Fig. 2, TN01 is an elliptical shaped cell body, the surface of the bacterial cell is smooth without wrinkles or protrusions, and the sterile silk body has a form similar to budding reproduction, so according to the type The state estimation may be a yeast genus.

Sequence identification and genetic relationship

Deoxyribonucleic acid extraction

a. Take 1 ml (ml) of the inoculum and centrifuge, collect the cells, and wash the cells 3 times per liter (M) of sodium chloride.

b. Add 200 μl of lysis buffer and 20 μl of protease Κ to 55 ° C overnight.

c Add hexadecyltriethylammonium bromide extraction solution to 1 ml, and react at 65 °C for 30 minutes.

d. Add chloroform 400μ1 and mix evenly for 30 seconds.

e. Centrifuge the supernatant at 14,000 revolutions per minute (rpm).

f. Add 2 volumes of cetyltriethylammonium bromide precipitation solution and let stand for 60 minutes.

g. The pellet was collected after centrifugation at 14,000 rpm and reconstituted with 350 μl of 1.2 M sodium chloride.

h. Add chloroform 350μ1, mix evenly for 30 seconds, then centrifuge to remove the supernatant.

i. Add 0.8 volumes of isopropanol and place it in a -20 ° C water tank for 30 minutes to separate out the ribonucleic acid. j. After centrifugation at 14000 rpm, the supernatant was removed, and 70% alcohol was added to 500 μl to wash the deoxyribonucleic acid precipitate, and centrifuged, and the DNA was dried at room temperature after centrifugation.

k. Add 20μ1 of secondary water with pH adjusted to 8 after sterilization, and dissolve at 37 °C.

1. Perform colloidal electrophoresis to confirm the size of the DNA. The label used was Bio-IKB DNA Ladder (Protech). Lysis buffer

 Drug concentration

 Sodium dodecyl sulfate 2%

 Tris(hydroxyindenyl)aminodecane hydrochloride 0.25M

 Disodium edetate 0.1M

 Sodium chloride 0.1M

 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.

Cetyltriethylammonium bromide extraction solution

 Drug concentration (g/L)

 Sodium chloride 81.8

 Tris(hydroxyindenyl)aminodecane 12.1

 Disodium edetate 7.4

 Cetyltriethylammonium bromide 20

 Dissolve the agent in one water. After the dissolution is completed, adjust the pH to 8.0 with hydrogen chloride or sodium hydroxide, and sterilize in an autoclave. Cetyltriethylammonium bromide precipitation solution

 Drug concentration (g/L)

 Sodium chloride 2.3

 Cetyltriethylammonium bromide 5

 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:

 Sequence (Fell, Roeijmans, & Boekhout, 1999) Name

 18SF (forward) 5'- GCATATCAATAAGCGGAGGAAAAG - 3' 18SR (reverse) 5'- GGTCCGTGTTTCAAGACG - 3'

ITS sequencing primer:

 Name Sequence (Gadanho & Sampaio, 2002)

ITS1 (forward) 5'- TCCGTAGGTGAACCTGCGG - 3, ITS4 (reverse) 5' - TCCTCCGCTTATTGATATGC - 3'

26S D1/D2 sequencing primer:

 Name sequence (Fell, et al., 2000)

 F63 5'- GCATATCAATAAGCG GAGGAAAAG -3'

LR3 (reverse) 5'- GGTCCGTGTTTCAAGACGG -3'

Polymerase chain reaction inclusions:

 Reactant volume final concentration deoxyribonucleic acid 4 μ1

 Deoxynucleotide triphosphate 8 μΐ 2.5 mM

 10 times buffer 5 μΐ - primer (forward) 1 μΐ - primer (reverse) 1 μΐ -

Taq 0.1 μΐ 1 U Secondary water 30.9 μΐ - sum 50 μΐ - Polymerase chain reaction amplification conditions: Temperature time

 94 V 5 minutes

 94 V 1 minute

 35 times

 Cycle 57 °C for 1 minute

 72 °C 2 minutes

 72 °C 10 minutes

i. After amplification of each condition, confirm the size by electrophoresis with 1% acacia

 Ii. The target used is Bio-IKB DNA Ladder (Protech)

 Sequence alignment analysis

 After 18S rDNA, 26S rDNA D1/D2, ITS polymerase chain reaction amplification, the size of the gene was about 1200 base pairs. After BLAST alignment, the sequence of TN01 was found to be the closest to Cystofilobasidium.

The sequences were sequenced by the ClustalW tool module, analyzed by the bioanalysis software MEGA 4, and the relationship between the TN strain 歹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.

 Physiological and biochemical test

 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).

a. The experimental strain and the standard strain were cultured in a test tube containing 3 ml of GYPG liquid medium at 20 ° C.

Incubate for two days at 150 rpm.

b. Perform the experimental steps in accordance with the kit operation manual.

c. Incubate in a 20 ° C incubator for three days, remove the culture solution from the well plate and add secondary water to 1 ml to

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.

 Table 2, TN01 physiological and biochemical test

 TN 01 C. bisporidii C. capitatum C. infirmominiatum

GAL ++ ++ + +

 ACT - - - -

SAC ++ ++ ++ +

 NAG - - - -

LAT W + W +

 ARA ++ ++ ++ ++

 CEL ++ ++ ++ ++

 RAF ++ ++ ++ ++

 MAL ++ ++ ++ ++

 TER ++ ++ ++ ++

 2KG ++ ++ ++ ++

 MDG - W - -

MAN ++ ++ ++ ++

 LAC - - - +

 ION ++ ++ ++ ++

 0 - - - -

SOR ++ ++ ++ ++

 XYL ++ ++ + ++

 RIB ++ ++ - ++

 GLY ++ ++ ++ ++

 RHA ++ ++ - ++

 PLE w + - ++

 ERY - - - -

MEL + + - - GRT + ++ ++ ++

MLZ ++ ++ ++ ++

GNT ++ ++ + ++

LVT - - - -

GLU ++ ++ ++ ++

SBE ++ ++ ++ -

GLN - - - -

ESC - - - - Table 3, ID 32C carbon source types

test

 GAL D-galactose

 ACT cycloheximide

 SAC D-sucrose

 NAG N-acetylglucosamine

LAT pore acid

 ARA L-arabinose

CEL D-cellobiose

RAF D-seed sugar

 MAL D-maltose

 TER D-trehalose

 2KG 2-ketogluconate potassium

MDG thiol-D-glucopyranose

MAN D-mannitol

 LAC D-lactose (bovine source)

ION inositol

 0 no matrix

 SOR D-sorbitol

 XYL D-Xylose

RIB D-ribose GLY glycerin

 RHA L-rhamnose

 PLE isomaltose

 ERY erythritol

 MEL D-Honeyose

 GRT sodium glucuronate

 MLZ D-pine triose

 GNT potassium gluconate

 LVT levulinic acid

 GLU D-glucose

 SBE L-sorbose

 GLN Glucosamine

 Discussion on the optimum culture conditions of ESC glycoside

 First, pick a colony from the solid medium to a test tube containing 3 ml of GYPG liquid medium, shake it at 20 °C, shake it at 150 rpm for two days, then take 500 μL of the bacterial solution to the triangle containing 50 ml of GYPG liquid medium. The conical flask (model, 125 ml) was placed at 20 ° C and shaken at 150 rpm for two days as the final inoculum. Then, 500 μM of the inoculum was inoculated into the culture medium of each condition, and each culture condition was repeated three times. The number of culture days was determined by the growth curve, and the bacterial liquid was collected during the gentle growth period of the strain, and then freeze-dried after centrifugation. Fatty acid extraction

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

CH2 OCOR2 + 3HOCH3 « ^{3 3 yS} " CH ₂ OH + R2 -COOCHs

CH2 OCORs CH2 OH R3 H: OOCH3

Triglyceride Methanol Methyl esters

(parent oil) (alcohol) ^Y (biodesd) In the process of extraction, a quantitative method is needed to determine the amount of fatty acids contained in the cells, and when using Gas Chromatography (GC) to analyze samples, The detector that detects the sample is the detector that is attached to it. The purpose of adding the internal standard is to provide a quantitative basis, and the detector itself cannot provide a stable detection result. The characteristics of the chromatograph itself result in the inability to produce consistent results during the process of dissociation, so the internal standard is added for quantitative quantification of the sample. The ratio of the analyte to the internal standard is used to quantify the sample. This ratio is called RRF (Relative Response Factor), because the internal standard and the sample will show an equal increase or decrease during the detection process. Therefore, it can be more accurate. 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:

a. Scale 0.05g dry powder, put into a 10 ml glass test tube, and add chloroform: sterol (2:1) 5 ml, shake and mix.

b. Ultrasonic shattering the bacteria, shredded for 2 minutes (: shattered for 5 seconds and then stopped for 5 seconds for 4 minutes;). c After standing at room temperature for one hour, add 100 μΐ of the standard solution at a standard concentration of 10 mg / ml. d. Liquefaction 0.5 ml, centrifuge at 2500 rpm for 5 minutes, remove the supernatant.

e. Force the TUP (scientific upper phase, chloroform: secondary water: 47: 48: 3 sterol) 2.5 ml (not shaken), centrifuge at 2500 rpm for 5 minutes, remove the supernatant.

f. Repeat steps d. and e. and blow dry at room temperature with nitrogen.

g. Add 2.5 ml of sterol: benzene is 4:1 (V/V), then slowly add acetyl chloride 250 μΐ. When transesterifying the catalyst, shake and mix.

h. Cover the Teflon top cover and place in an oven at 80 °C for 4 hours.

i. After cooling at room temperature, the reaction was stopped by slowly adding 1.5 ml of 7% potassium carbonate, and centrifuged at 2500 rpm for 10 minutes. j. Take the upper benzene layer to a disposable centrifuge tube and blow dry at room temperature under nitrogen. k. Add 0.5 ml of n-hexane along the tube wall and mix by shaking.

 1. After drying with nitrogen, add 250 μl of n-hexane, shake the hook, and inject the vial with the embedded tube. After sealing, use a gas chromatograph for analysis. Analysis of fatty acids by gas chromatography (GC)

 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 μπι; 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 Ιμΐ, 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:

 % single fatty acid:

 (Single fatty acid integral area X standard concentration X 100 ) ÷ Internal standard integral area Total fatty acid content %:

 [(Total fatty acid integral area - internal standard integral area) X Standard concentration 100 ] ÷ Internal standard integral area Optimum salt concentration test

 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%. After 10 days of culture, 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

 In the future, when industrial fermentation is carried out in large quantities, it is hoped that the culture can be carried out in the simplest medium to reduce the cost. Therefore, the original GYPG medium is first improved, respectively:

 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. 9, 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. In order to reduce the pH value of the metabolites during the growth of the organism, the following buffers were added according to the conditions of the GYPG medium:

 pH buffer and added concentration

 3-4 sodium acetate, 30mM

5-6 Biological Buffer (MES), 30mM 7-8 tris(hydroxyindenyl)aminodecane, 30mM

 9-11 3-(cyclohexylamine)-2-hydroxy-1-propanesulfonic acid, 30mM pH 3 and 4 using acetic acid to adjust the pH; pH 5-11 to adjust the pH with sodium hydroxide or hydrogen chloride, and the best The temperature was incubated at 150 rpm for six days.

 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. Carbon source utilization test

 Different sources of microbial growth require a carbon source. The use of carbon source species affects the growth of bacteria and the accumulation of oil. Therefore, according to the metabolic pathway of the genus Saccharomyces, various carbon sources contain monosaccharides (hexose and five carbon sugars). A carbon source with a disaccharide, a polysaccharide, and a three-carbon chain, and using glycerin as a carbon source for oils and fats, it is expected to find a carbon source that is optimal for cell growth and oil accumulation. The composition of this experimental medium is as follows:

 5% carbon source + 0.1% yeast extract + 0.1% 胨 + 0.1% gelatin

 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 utilization test

 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:

 5% glucose + 0.1% yeast extract + 0.1% nitrogen source + 0.1% gelatin

 Types of nitrogen sources: bismuth, tryptone protein, soybean meal, ammonium nitrate, ammonium sulfate, ammonium chloride, sodium ammonium, urea, sodium thiosulfate. Among them, 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%. The next is 52.3% of sodium ammonium (SA) and 51.9% of tryptone. Carbohydrate concentration test

 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.

 The results are shown in Figure 13. The dry weights of the bacteria with glucose concentrations of 50 g, 100 g, 150 g, and 200 g were

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)

 C14:0 17.34

 C16:0 20.4

 C18:0 4.69

 C18:l 40.47

C18:2 11.47 C18:3 2.41

 C20: 4 0.97

 TFA% 50.91

Fatty acid content test of TN 01 and other Cystofilobasidium strains

 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:

 The liquid culture was placed in a 20 °C:, 150 rpm shaking culture for 7 days to analyze the fatty acid content. The results are shown in Table 5. Other Cystofilobasidium species also have oil production capacity, but the oil production capacity is not as good as TN 01. Table 5. Percentage of fatty acids and total fatty acids in TN 01 and standard strains

C18:3 2.407

C18:3 2.407

 C20:0 1.14

 C24:0 0.971 1.65 Total fatty acids / biomass (%) 50.91 33.56 33.22 17.29

This case has been modified by people who are familiar with the art, and they are all modified as they are intended.

Claims

Claim  An oil-producing reaction system comprising: a saccharide; and a bacterium that reacts with the saccharide to produce a fat, wherein the genus of the bacterium is Cys tof ilobas idium. 2. The oil production reaction system according to claim 1, further comprising a nitrogen source for use by the active bacteria, wherein the nitrogen source is sodium ammonium (Na (s) ₄ ).  3. The oil-producing reaction system according to claim 1, wherein the oil produced by the active bacteria is a medium chain fatty acid of C14 to C20.  The oil-producing reaction system according to claim 3, wherein the oil produced by the active bacteria is an unsaturated fatty acid.  The oil-producing reaction system according to claim 1, wherein the oil produced by the active bacteria is used as biodiesel.  6. The oil-producing reaction system according to claim 1, wherein the bacterium has the 18S rDNA sequence of SEQ ID NO.  The oil-producing reaction system according to claim 1, wherein the bacterium has the 26S rDNA sequence of SEQ ID NO.  8. The oil-producing reaction system according to claim 1, wherein the bacterium has the ITS sequence of SEQ ID NO.  9. 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 Cys t of i lobas idium. The oil producing reaction system according to claim 9, wherein the active bacteria decompose the carbon source to produce the fat or oil. 11. An oil production method which utilizes an oil producing bacterium to produce a fat, the genus of the oil producing bacterium Cys t ofi lobas idium.  The oil-producing method according to claim 11, wherein the oil-producing bacteria are selected from the group consisting of Cys tof i lobas idium bi spor idi i , Cys tof i lobas idium capi ta tum. Cys Tof i lobas idium inf i rmominia tum and other species of Cys tof i lobas idium.