CN111411141A - Method for producing microbial oil by co-fermenting corn straws with bacterial enzymes - Google Patents

Method for producing microbial oil by co-fermenting corn straws with bacterial enzymes Download PDF

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CN111411141A
CN111411141A CN202010451634.4A CN202010451634A CN111411141A CN 111411141 A CN111411141 A CN 111411141A CN 202010451634 A CN202010451634 A CN 202010451634A CN 111411141 A CN111411141 A CN 111411141A
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corn straws
corn
yarrowia lipolytica
mucor circinelloides
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CN111411141B (en
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张瑶
宋元达
王艳霞
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Shandong University of Technology
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    • 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
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    • 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
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • 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
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
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Abstract

The invention discloses a method for producing microbial oil by co-fermenting corn straws with bacterial enzymes, belonging to the field of microbial fermentation. The invention utilizes the synergistic interaction of mucor circinelloides, yarrowia lipolytica and cellulase to ferment corn straws, and the fermentation conditions are as follows: the addition amount of the cellulase powder is 1.5 percent, the strain ratio of the mucor circinelloides to the yarrowia lipolytica is 1:1.5, the total inoculation amount is 10 percent, the stirring speed is 200 rpm/min, the fermentation temperature is 31 ℃, the fermentation time is 54 hours, and finally 1.24g of microbial oil can be obtained in each liter of corn straw fermentation liquid. The method can directly convert lignocellulose, realizes the synchronization of cellulose-monosaccharide-grease by one step, greatly reduces the production cost and reduces the environmental pollution.

Description

Method for producing microbial oil by co-fermenting corn straws with bacterial enzymes
Technical Field
The invention relates to a method for producing microbial oil by co-fermenting corn straws with bacterial enzymes, belonging to the field of microbial fermentation. The fermentation bacterial enzyme of the invention relates to mucor circinelloides, yarrowia lipolytica and cellulase.
Background
With the increasing shortage of fossil resources, the development of renewable oil resources from multiple channels is becoming more and more popular. The oleaginous microorganism can accumulate a large amount of triglyceride in cells, and the renewable resources are transformed by the microorganism to produce the grease, so that part of fossil resources are replaced, and the sustainable development is inevitable. In addition, a few fungi, microalgae, bacteria, etc. can synthesize functional polyunsaturated fatty acids, such as Arachidonic acid (Arachidonic acid), gamma-linolenic acid (gamma-linolenic acid), Docosahexaenoic acid (Docosahexaenoic acid), eicosapentaenoic acid (Eicosapentaenoic acid), etc., which are very beneficial to human health, and these bioactive fatty acids have been identified as important nutritional food resources. Therefore, the method develops and utilizes the oleaginous microorganisms to synthesize the grease, and has important social value and economic value.
Lignocellulose is one of the most abundant and cheap renewable raw materials in nature, for example, the annual output of crop straws in China only reaches 10 hundred million tons, and accounts for about 20-30% of the total amount of the straws all over the world. The straw is used as feed, fuel and organic fertilizer. At present, the on-site incineration treatment of the straws is generally adopted in China, so that the environment is polluted, and the health of the masses is influenced, so that how to optimize the treatment of the straws becomes a social problem, wherein the microbial fermentation is a research hotspot in recent years. The currently widely used method for producing microbial straw oil is to degrade lignocellulose in straw into small-molecular available monosaccharide (hexahydric sugar or pentahydric sugar) and then convert the small-molecular available monosaccharide into oil through microbial fermentation. In the long term, a plurality of oil-producing microorganisms and cellulase can be used for co-fermentation to directly convert lignocellulose, so that the synchronization of cellulose, monosaccharide and grease can be realized in one step, the production cost is greatly reduced, and the environmental pollution is reduced.
Mucor circinelloides is one of the main production strains of gamma-linolenic acid (G L A), and is first used for the industrial production of G L A, and then is forced to stop production due to cost problems, earlier studies show that Mucor circinelloides can absorb and utilize six-membered and five-membered sugars (L side interaction by pellet culture medium of Mucor circinelloides on corn store hydrolyate, applied Biochem Biotechnol2014, 174: 411-423) and can secrete cellulase (Genomic, and biochemical analytes of woody multicorcinellous species: evaluating cellulose catalysis in evaluating cellulose catalysis for lipid production, Plos One, 2013, 8(9): e 71068), thereby fully utilizing cellulose and hemicellulose in the straws and having outstanding advantages in reducing production cost. Yarrowia lipolytica (Yarrowia lipolytica) Is the best strain for producing the single-cell grease, can utilize a plurality of cheap substrates as carbon sources, and has great application prospect in industry. At present, the oil-producing bacteria which are widely researched and applied comprise rhodosporidium toruloides, cryptococcus, trichoderma koningii and chrysogenum, and the report of producing microbial oil by the fermentation of corn straws under the combined action of mucor circinelloides and yarrowia lipolytica is not reported. In conclusion, the microbial oil produced by co-fermenting the corn straws with the bacterial enzymes can effectively realize sustainable development of agriculture and open up a new green energy approach.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for producing microbial oil by fermenting corn straws with bacterial enzymes, which can directly convert lignocellulose, realize the synchronization of cellulose, monosaccharide and oil in one step, greatly reduce the production cost and reduce the environmental pollution.
The technical scheme of the invention is as follows:
a method for producing grease by fermenting corn straws with bacterial enzymes comprises the following steps of utilizing Mucor circinelloides WJ11 (complex genetic sequence of high lipid-producing strain of Mucor circinelloides WJ11 and complex genetic analysis with low lipid-producing strain CBS 277.49, ploS One, 2015, 10(9): e 0137543), Yarrowia lipolytica CICC1778 (Metabolic fluoride analysis of lipid biosynthesis in the maize straw Yarrowia slope using 13C-lamellar glucose and gas chromatography-mass spectrometry, PloS One, 2016, 11(7): 0159187) and treating corn straws with cellulase:
(1) pretreatment of corn straws: drying the corn stalks in an oven at 80 ℃ until the quality is unchanged, grinding, sieving, and taking the corn stalks between 50 meshes and 100 meshes for testing.
(2) Seed culture: respectively inoculating the strains of Mucor circinelloides WJ11 and yarrowia lipolytica CICC1778 stored in a glycerin pipe with the temperature of-80 ℃ into a seed culture medium, culturing by using a rotary constant-temperature speed regulating shaker, controlling the rotating speed to be 200 rpm/min, the culture temperature to be 28 ℃, the initial pH value to be 6.0, and culturing for 24 hours to obtain a seed solution.
(3) And (2) fermentation culture, namely weighing 5 g of the pretreated corn straw powder, putting the corn straw powder into a triangular flask containing 100 m L basic nutrient solution, inoculating the cultured seed solution according to the strain proportion of mucor circinelloides and yarrowia lipolytica of 1:1.5 under the condition that the addition amount of the cellulase powder (the protein content is 1 mg/m L) is 1.5 percent, wherein the total inoculation amount is 10 percent, the stirring speed is 200 rpm/min, the fermentation temperature is 31 ℃, and the fermentation time is 54 hours.
The seed culture medium comprises 20 g/L g of glucose, 20 g/L g of peptone and 10 g/L g of yeast extract.
The basic nutrient solution comprises 5 g/L g of glucose and 2 g/L g of ammonium tartrate2PO47 g/L，Na2HPO42 g/L，MgSO4·7H2O1.5 g/L, Yeast 1.5 g/L2·2H2O 0.1 g/L，FeCl3·6H2O 8mg/L，ZnSO4·7H2O 1 mg/L，CuSO4·5H2O 0.1 mg/L，Co(NO3)2·6H2O 0.1 mg/L，MnSO4·5H2O0.1 mg/L。
The invention has the beneficial effects that:
the invention provides a method for producing microbial oil by co-fermenting corn straws with bacterial enzymes, which utilizes the mutual synergistic effect of mucor circinelloides, yarrowia lipolytica and cellulase, and 1.24g of microbial oil can be obtained in each liter of corn straw fermentation liquid. The method can directly convert lignocellulose, realizes the synchronization of cellulose-monosaccharide-grease by one step, greatly reduces the production cost and reduces the environmental pollution.
Detailed Description
Preparation of strains:
the laboratory deposits the strains Mucor circinelloides WJ11 (Complete genome sequence of a high-level-producing string of Mucor circinelloides WJ11 and complex genome analysis with a low-level-producing string CBS 277.49, ploS One, 2015, 10(9): e 0137543) and Yarrowia lipolytica CICC1778 (metabolism flash analysis of a lipid biosynthem and the surface tissue culture using 13C-layered glucose and platelet-mass spectrometry, PloS One, 2016, 11(7): e 0159187).
Pretreatment of corn straws:
drying the corn stalks in an oven at 80 ℃ until the quality is unchanged, grinding, sieving, and taking the corn stalks between 50 meshes and 100 meshes for testing.
Seed liquid culture:
the Mucor circinelloides WJ11 and yarrowia lipolytica CICC1778 strains preserved by a glycerin pipe with the temperature of-80 ℃ are respectively inoculated into a seed culture medium, the formula of the seed culture medium is 20 g/L of glucose, 20 g/L of peptone and 10 g/L of yeast extract, a rotary constant-temperature speed-regulating shaker is used for culturing, the rotating speed is controlled to be 200 rpm/min, the culture temperature is 28 ℃, the initial pH value is 6.0, and the seed liquid is obtained after 24 hours of culture time.
Fermentation culture:
weighing 5 g of the pretreated corn straw powder, putting the corn straw powder into a triangular flask containing 100 m L basic nutrient solution, wherein the basic nutrient solution comprises 5 g/L of glucose and 2 g/L of ammonium tartrate2PO47 g/L，Na2HPO42 g/L，MgSO4·7H2O1.5 g/L, Yeast 1.5 g/L2·2H2O 0.1 g/L，FeCl3·6H2O 8 mg/L，ZnSO4·7H2O 1 mg/L，CuSO4·5H2O 0.1 mg/L，Co(NO3)2·6H2O 0.1 mg/L，MnSO4·5H2O0.1 mg/L, under the condition that the addition amount of the cellulase powder (the protein content is 1 mg/m L) is 1.5 percent, inoculating the cultured seed liquid according to the strain proportion of mucor circinelloides and yarrowia lipolytica of 1:1.5, wherein the total inoculation amount is 10 percent, the stirring speed is 200 rpm/min, the fermentation temperature is 31 ℃, and the fermentation time is 54 hours.
And (3) measuring the oil content:
treating the mixed fermentation liquor by an ultrasonic crusher for 30 min to promote the cells of mucor circinelloides and yarrowia lipolytica to be crushed and release the oil and fat in the cells, separating the fermentation mixed liquor by a centrifugal machine, pouring the upper layer liquid into a measuring cylinder with a plug, adding diethyl ether (30 m L) -petroleum ether (30 m L), fully shaking and mixing uniformly, standing for 30 min, extracting the oil and fat in the water phase, sucking the oil layer out to a test tube by a suction tube (dried to constant weight, and measuring the mass to be the same as the mass of the oil and fat in the water phase)m 1) Care was taken not to wick into the underlying water layer as much as possible. Then evaporating the ether-petroleum ether to dryness in a water bath (90 ℃), putting the dried ether-petroleum ether into a constant-temperature drying oven (105 ℃) to be dried to constant weight, and measuring the mass to bem 2Obtaining the quality of the oil by using a difference methodm。
The formula:m=m 2-m 1(g)
then: the oil content of each liter of fermentation liquor is as follows:M=1000×m/250 (g)
the result shows that 1.24g of microbial oil can be obtained in each liter of corn straw fermentation liquor.

Claims (3)

1. A method for producing microbial oil by fermenting corn straws with bacterial enzymes is characterized in that the corn straws are fermented by the synergistic interaction of mucor circinelloides WJ11, yarrowia lipolytica CICC1778 and cellulase, and the method comprises the following steps:
(1) the method comprises the following steps of (1) corn straw pretreatment, namely drying the corn straws in an oven at 80 ℃ until the mass of the corn straws is unchanged, grinding and sieving, and taking the corn straws between 50 meshes and 100 meshes for a test, (2) seed culture, namely respectively inoculating mucor circinelloides WJ11 and yarrowia lipolytica CICC1778 strains stored in an oil tube at-80 ℃ into a seed culture medium, and culturing by using a rotary constant-temperature speed regulating shaker at the rotation speed of 200 rpm/min and the culture temperature of 28 ℃ for 24 hours to obtain a seed solution, (3) fermentation culture, namely weighing 5 g of pretreated corn straw powder, putting the pretreated corn straw powder into a triangular flask containing 100 m L basic nutrient solution, and inoculating the cultured seed solution according to the proportion of 1:1.5 of the mucor circinelloides yarrowia lipolytica strain with the total inoculation amount of 10%, stirring at the rotation speed of 200 rpm/min and the fermentation temperature of 31 h for 54 hours under the condition that the addition amount of 1.5% of cellulase powder (protein content of 1 mg/m L).
2. The method of claim 1, wherein the seed medium comprises glucose 20 g/L, peptone 20 g/L, and yeast extract 10 g/L.
3. The method of claim 1, wherein the base nutrient solution comprises glucose 5 g/L and ammonium tartrate 2 g/L2PO47 g/L，Na2HPO42 g/L，MgSO4·7H2O1.5 g/L, Yeast 1.5 g/L2·2H2O 0.1 g/L，FeCl3·6H2O 8 mg/L，ZnSO4·7H2O 1 mg/L，CuSO4·5H2O 0.1 mg/L，Co(NO3)2·6H2O 0.1 mg/L，MnSO4·5H2O 0.1 mg/L。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112021451A (en) * 2020-08-07 2020-12-04 东北农业大学 Fermentation method for increasing content of fatty and acid soluble proteins in wheat bran

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140234919A1 (en) * 2011-07-06 2014-08-21 Washington State University Research Foundation Simultaneous saccharification and fermentation(ssf) of lignocellulosic biomass for single cell oil production by oleaginous microorganisms
CN104673848A (en) * 2014-12-18 2015-06-03 江南大学 Biotransformation method for preparing cocoa butter equivalent by adopting mutton fat as raw material
CN104946539A (en) * 2014-12-18 2015-09-30 江南大学 Mucor circinelloides strain with high grease yield and application thereof
CN108795997A (en) * 2018-06-11 2018-11-13 南京理工大学 The method for producing microbial grease with maize straw acid processing hydrolyzate

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140234919A1 (en) * 2011-07-06 2014-08-21 Washington State University Research Foundation Simultaneous saccharification and fermentation(ssf) of lignocellulosic biomass for single cell oil production by oleaginous microorganisms
CN104673848A (en) * 2014-12-18 2015-06-03 江南大学 Biotransformation method for preparing cocoa butter equivalent by adopting mutton fat as raw material
CN104946539A (en) * 2014-12-18 2015-09-30 江南大学 Mucor circinelloides strain with high grease yield and application thereof
CN108795997A (en) * 2018-06-11 2018-11-13 南京理工大学 The method for producing microbial grease with maize straw acid processing hydrolyzate

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112021451A (en) * 2020-08-07 2020-12-04 东北农业大学 Fermentation method for increasing content of fatty and acid soluble proteins in wheat bran

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