CN107557400B - Method for improving oil production of grease yeast cultured by corn straw hydrolysate - Google Patents
Method for improving oil production of grease yeast cultured by corn straw hydrolysate Download PDFInfo
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Abstract
A method for improving oil production of grease yeast cultured by corn straw hydrolysate belongs to the technical field of microorganism application. The invention aims to provide a method for improving the oil yield of grease yeast cultured by corn straw hydrolysate, which is used for putting grease yeast immobilized by bacterial cellulose into corn straw hydrolysate, weakening the toxicity and the inhibitive performance of the hydrolysate on the grease yeast, and improving the microbial oil yield of the grease yeast. The method comprises the following steps: the method comprises the following steps of preparing bacterial cellulose, preparing bacterial cellulose immobilized Cryptococcus curvatus, preparing corn straw hydrolysate, and culturing the bacterial cellulose immobilized Cryptococcus curvatus in the corn straw hydrolysate. The invention provides technical support for the stable utilization of the corn straws and can achieve the win-win effect of economic benefit and environmental benefit.
Description
Technical Field
The invention belongs to the technical field of microorganism application.
Background
In China, the amount of the crop straws such as corn is over 7 hundred million tons each year, and the local burning wastes resources and causes serious environmental pollution. The main components of the corn stalks are cellulose, hemicellulose and lignin, and the cellulose and the hemicellulose can be converted into monosaccharide or oligosaccharide which can be utilized by microorganisms through hydrolysis. The grease yeast can utilize nutrient substances such as monosaccharide and the like in the corn straw hydrolysate to produce grease to obtain microbial grease, which provides a new development space for sustainable development of energy and environment. However, due to the complex structure of cellulose and hemicellulose in the corn stalks, the corn stalk hydrolysate contains not only saccharides available for microorganisms but also toxic inhibitors which have great influence on the growth of microorganisms and the production of oil, and the yield of microbial oil is influenced. It is reported that the immobilized yeast cells can improve the amount of the pre-obtained product, but the microbial oil produced by the grease yeast belongs to the intracellular product of the yeast cells, if a common immobilized material is adopted, the extraction and the obtaining of the microbial oil in the cells of the subsequent oil-producing yeast can be influenced, and the common immobilized material does not have the function of resisting a toxic inhibitor in corn hydrolysate.
Disclosure of Invention
The invention aims to provide a method for improving the oil yield of grease yeast cultured by corn straw hydrolysate, which is used for putting grease yeast immobilized by bacterial cellulose into corn straw hydrolysate, weakening the toxicity and the inhibitive performance of the hydrolysate on the grease yeast, and improving the microbial oil yield of the grease yeast.
The method comprises the following steps:
(1) preparing bacterial cellulose:
A. and (3) recovery culture of freeze-dried bacteria of acetobacter xylinum: wiping an ampoule bottle with absorbent cotton soaked in 90% (v/v) alcohol by volume fraction under aseptic condition, heating the top end of the ampoule bottle by using an alcohol lamp, then dropping sterile water to the top end of the heated ampoule bottle to crack the glass at the opening of the ampoule bottle, and knocking off the top end of the cracked ampoule bottle by using tweezers; the number of the ampoule bottle is CGMCC NO: 1.1812 Acetobacter xylinumGluconacetobacterxylinumDripping 0.6mL of acetobacter xylinum enrichment medium into the freeze-dried mycelia, and lightly shaking to obtain an acetobacter xylinum freeze-dried mycelia suspension; the acetobacter xylinum enrichment culture medium comprises the following components in parts by weight: glucose 60.0, peptone 15.0, citric acid 1.0, Na2HPO4·12H2O 0.8，KH2PO4 2.5，MgSO4·7H20.5O, 1L deionized water and 7.0 pH; sucking 100uL of the acetobacter xylinum freeze-dried thallus suspension, and uniformly coating the suspension on an acetobacter xylinum solid culture medium by using a coating rod, wherein the components in parts by weight of the acetobacter xylinum solid culture medium are respectivelyComprises the following steps: glucose 60.0, peptone 15.0, citric acid 1.0, Na2HPO4·12H2O 0.8，KH2PO4 2.5，MgSO4·7H20.5O, 20.0 agar, 1L deionized water and 7.0 pH; culturing the solid culture medium coated with acetobacter xylinum in a constant-temperature electric heating incubator at 28 ℃ for 48 hours to obtain recovered and cultured acetobacter xylinum, and storing in a refrigerator at 4 ℃ for later use;
B. preparing an acetobacter xylinum seed solution: and B, selecting a strain of the acetobacter xylinum obtained in the step A by using an inoculating loop, transferring the strain into a 500mL conical flask filled with 100mL of acetobacter xylinum seed culture medium, wherein the acetobacter xylinum seed culture medium comprises the following components in parts by weight: glucose 30.0, peptone 5.0, citric acid 3.0, Na2HPO4·12H2O3.0，MgSO4·7H20.20 percent of O, 1L of deionized water and 7.0 percent of pH; oscillating for 36h in a constant temperature oscillator at 28 ℃ and 180r/min to obtain acetobacter xylinum seed liquid;
C. preparing bacterial cellulose: inoculating the acetobacter xylinum seed liquid obtained in the step B into a square container filled with 100mL of acetobacter xylinum fermentation culture medium according to the inoculation amount of 10% (v/v) by volume ratio, wherein the size of the square container is as follows: the bottom side length is 8cm multiplied by 8cm, the height is 8cm, and the acetobacter xylinum fermentation medium comprises the following components in parts by weight: 100.0 percent of glucose, 20.0 percent of peptone, 3.0 percent of citric acid and Na2HPO4·12H2O 2.5，KH2PO4 3.5，MgSO4·7H20.5O, 1L deionized water and 7.0 pH; then, standing the square container in a constant-temperature electric heating incubator at 28 ℃ for culturing for 6 days to obtain bacterial cellulose on the surface of the acetobacter xylinum fermentation medium;
D. extracting and processing bacterial cellulose: taking out the bacterial cellulose obtained in the step C by using a pair of tweezers, washing the bacterial cellulose for 3 times by using deionized water, then soaking the bacterial cellulose into 100mL of NaOH solution with the concentration of 0.05mol/L, carrying out water bath at 60 ℃ for 120min, removing thalli and culture medium remained on the surface of the bacterial cellulose, then repeatedly washing the bacterial cellulose by using the deionized water until the bacterial cellulose is neutral, cutting the bacterial cellulose into small blocks of 4cm multiplied by 4cm, sterilizing the bacterial cellulose in a sterilizing pot at 121 ℃ for 15min, and then storing the bacterial cellulose in a refrigerator at 4 ℃ for later use;
(2) preparation of bacterial cellulose immobilized Cryptococcus curvatus
A. Preparing a Cryptococcus curvatus seed solution: cryptococcus curvatus with strain number ATCC 20509 to be preserved on slantCryptococcus curvatusThe seed culture medium is inoculated into a seed culture medium, and the seed culture medium comprises the following components in percentage by mass: 3% of glucose, 1.5% of yeast extract powder and 1.5% of peptone; performing shake culture in a constant temperature shaking table at 28 deg.C and 160r/min for 20h to obtain Cryptococcus sinuatus seed solution;
B. bacterial cellulose immobilized Cryptococcus curvatus: sucking 1-2 mL of the Cryptococcus sinutus seed liquid obtained in the step A in the step (2) by using an injector, uniformly injecting the Cryptococcus sinutus seed liquid into the small block of bacterial cellulose with the size of 4cm multiplied by 4cm obtained in the step D in the step (1), and washing the bacterial cellulose injected with the Cryptococcus sinutus seed liquid for 3 times by using deionized water to obtain bacterial cellulose immobilized Cryptococcus sinutus;
(3) preparation of corn stalk hydrolysate
A. Preparing corn straw powder: cleaning corn straws with deionized water, putting the cleaned corn straws into a constant-temperature drying oven at 80 ℃ for drying to constant weight, cutting the corn straws into 3cm small sections, and crushing the small sections into powder by a crusher, wherein the diameter of the powder particles of the corn straws is 0.3 mm;
B. preparing corn stalk hydrolysate: weighing 30g of the corn straw powder obtained in the step A in the step (3), placing the corn straw powder into a 1L triangular flask, adding 300mL of dilute sulfuric acid with the volume fraction of 2.0% (v/v) according to the solid-to-liquid ratio of 1:10 (w/v), fully mixing uniformly, covering the triangular flask with a sealing film, and hydrolyzing for 60min at 121 ℃ in a sterilizing pot; cooling the obtained hydrolysate to room temperature, performing vacuum filtration by using four layers of gauze, discarding corn straw residues on the gauze, and taking the obtained rough-extracted filtrate; carrying out vacuum filtration on the obtained crude filtrate by using filter paper, discarding corn straw residues on the filter paper, and taking the obtained fine filtrate; using 10% (w/v) of Ca (OH) by mass percent to the fine suction filtration liquid2Adjusting pH to 10, standing for 1 hr, vacuum filtering with filter paper, discardingTaking the obtained fine filtrate from the residue on the paper; adjusting the pH value of the fine filtrate to 6 by using 2mol/L sulfuric acid, and performing suction filtration by using filter paper to obtain final filtrate; sterilizing the final filtrate in a sterilizing pot at 121 deg.C for 15min to obtain corn stalk hydrolysate, and storing in a refrigerator at 4 deg.C;
(4) culture of bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate
A. Preparation of cellulase solution: dissolving 7.5g of cellulase R-10 in 500mL of disodium hydrogen phosphate-citric acid solution with the concentration of 0.05mol/L and the pH value of 5.0 to obtain a cellulase solution with the mass fraction of 1.5% (w/v), and placing the cellulase solution in a refrigerator at 4 ℃ for later use;
B. culturing the bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate: and (3) inoculating 120 g-150 g of the bacterial cellulose immobilized cryptococcus curvatus obtained in the step (B) in the step (2) into 500mL of the corn straw hydrolysate obtained in the step (3), monitoring the change of reducing sugar in the hydrolysate every 24h, and completing the culture of the bacterial cellulose immobilized cryptococcus curvatus in the corn straw hydrolysate when the reducing sugar in the hydrolysate is completely consumed.
The invention discloses a method for measuring the oil yield of bacterial cellulose immobilized Cryptococcus curvatus, which comprises the following steps: taking out the cultured bacterial cellulose loaded with Cryptococcus flexuosus obtained in the step B in the step (4) by using a pair of tweezers, and washing the bacterial cellulose for 3 times by using deionized water; soaking the washed bacterial cellulose carrying cryptococcus flexus in 700mL of a beaker containing the cellulase solution obtained in the step A in the step (4), covering a bottle opening with a sealing film, putting the beaker into a constant-temperature drying oven at 50 ℃, drying for 10 hours, and degrading the bacterial cellulose; and (3) after the bacterial cellulose is degraded, filtering the bacterial cellulose by using single-layer gauze, discarding bacterial cellulose fragments on the gauze, and taking filtrate to measure the cryptococcus curvatus biomass and the microbial oil yield.
Compared with the prior art, the invention has obvious progress and positive effect:
1. the cryptococcus curvatus immobilized by bacterial cellulose is cultured in corn straw hydrolysate for the first time to obtain the microbial oil.
2. The bacterial cellulose immobilized Cryptococcus curvatus does not influence the extraction and the acquisition of the intracellular microbial oil of the subsequent oleaginous yeast.
3. The cryptococcus flexus immobilized by the bacterial cellulose can grow well in the corn straw hydrolysate, and the bacterial cellulose can effectively inhibit toxic inhibitors in the corn hydrolysate, so that the biomass and the oil yield of the oil yeast are remarkably improved by 17.24-19.69% and 17.15-20.57%, respectively.
4. The invention provides technical support for the stable utilization of the corn straws and can achieve the win-win effect of economic benefit and environmental benefit.
Detailed Description
The method comprises the following steps:
(1) preparing bacterial cellulose:
A. and (3) recovery culture of freeze-dried bacteria of acetobacter xylinum: wiping an ampoule bottle by absorbent cotton soaked in alcohol with the volume fraction of 90% (v/v) under the aseptic condition, heating the top end of the ampoule bottle by using an alcohol lamp, then dropping sterile water to the top end of the heated ampoule bottle to crack the glass at the opening of the ampoule bottle, and knocking down the top end of the cracked ampoule bottle by using tweezers; the number of the ampoule bottle is CGMCC NO: 1.1812 Acetobacter xylinumGluconacetobacterxylinumDripping 0.6mL of acetobacter xylinum enrichment medium into the freeze-dried mycelia, and lightly shaking to obtain an acetobacter xylinum freeze-dried mycelia suspension; the acetobacter xylinum enrichment culture medium comprises the following components in parts by weight: glucose 60.0, peptone 15.0, citric acid 1.0, Na2HPO4·12H2O 0.8，KH2PO4 2.5，MgSO4·7H20.5O, 1L deionized water and 7.0 pH; sucking 100uL of the acetobacter xylinum freeze-dried thallus suspension, and uniformly coating the acetobacter xylinum freeze-dried thallus suspension on an acetobacter xylinum solid culture medium by using a coating rod, wherein the acetobacter xylinum solid culture medium comprises the following components in parts by weight: glucose 60.0, peptone 15.0, citric acid 1.0, Na2HPO4·12H2O 0.8，KH2PO4 2.5，MgSO4·7H2O0.5, agar 20.0, deionized water 1L, pH 7.0; culturing the solid culture medium coated with acetobacter xylinum in a constant-temperature electric heating incubator at 28 ℃ for 48h to obtain recovered and cultured acetobacter xylinum, and storing in a refrigerator at 4 ℃ for later use.
B. Preparing an acetobacter xylinum seed solution: and B, selecting a strain of the acetobacter xylinum obtained in the step A by using an inoculating loop, transferring the strain into a 500mL conical flask filled with 100mL of acetobacter xylinum seed culture medium, wherein the acetobacter xylinum seed culture medium comprises the following components in parts by weight: glucose 30.0, peptone 5.0, citric acid 3.0, Na2HPO4·12H2O3.0，MgSO4·7H20.20 percent of O, 1L of deionized water and 7.0 percent of pH; oscillating for 36h at 28 deg.C in a constant temperature oscillator of 180r/min to obtain Acetobacter xylinum seed solution.
C. Preparing bacterial cellulose: inoculating the acetobacter xylinum seed liquid obtained in the step B into a square container filled with 100mL of acetobacter xylinum fermentation culture medium according to the inoculation amount of 10% (v/v) by volume ratio, wherein the size of the square container is as follows: the bottom side length is 8cm multiplied by 8cm, the height is 8cm, and the acetobacter xylinum fermentation medium comprises the following components in parts by weight: 100.0 percent of glucose, 20.0 percent of peptone, 3.0 percent of citric acid and Na2HPO4·12H2O 2.5，KH2PO4 3.5，MgSO4·7H20.5O, 1L deionized water and 7.0 pH; then the square container is placed statically in a constant temperature electric heating incubator at 28 ℃ for 6 days, and the bacterial cellulose is obtained on the surface of the acetobacter xylinum fermentation medium.
D. Extracting and processing bacterial cellulose: and D, taking out the bacterial cellulose obtained in the step C by using a pair of tweezers, washing the bacterial cellulose for 3 times by using deionized water, immersing the bacterial cellulose into 100mL of NaOH solution with the concentration of 0.05mol/L, carrying out water bath at 60 ℃ for 120min, removing thalli and culture medium remained on the surface of the bacterial cellulose, repeatedly washing the bacterial cellulose by using the deionized water until the bacterial cellulose is neutral, cutting the bacterial cellulose into small blocks of 4cm multiplied by 4cm, sterilizing the bacterial cellulose in a sterilizing pot at 121 ℃ for 15min, and storing the bacterial cellulose in a refrigerator at 4 ℃ for later use.
(2) Preparation of bacterial cellulose immobilized Cryptococcus curvatus
A. Preparing a Cryptococcus curvatus seed solution:cryptococcus curvatus with strain number ATCC 20509 to be preserved on slantCryptococcus curvatusThe seed culture medium is inoculated into a seed culture medium, and the seed culture medium comprises the following components in percentage by mass: 3% of glucose, 1.5% of yeast extract powder and 1.5% of peptone; shaking and culturing in a constant temperature shaking table at 28 deg.C and 160r/min for 20h to obtain Cryptococcus curvatus seed solution.
B. Bacterial cellulose immobilized Cryptococcus curvatus: and (3) sucking 1-2 mL of the Cryptococcus sinuatus seed liquid obtained in the step (2) by using an injector, uniformly injecting the Cryptococcus sinuatus seed liquid into the small block of bacterial cellulose with the size of 4cm multiplied by 4cm obtained in the step (1) and D, and washing the bacterial cellulose injected with the Cryptococcus sinuatus seed liquid for 3 times by using deionized water to obtain the Cryptococcus sinuatus immobilized by the bacterial cellulose.
(3) Preparation of corn stalk hydrolysate
A. Preparing corn straw powder: the corn straws are cleaned by deionized water, then placed into a constant-temperature drying oven at 80 ℃ to be dried to constant weight, cut into 3cm small sections and then crushed into powder by a crusher, and the diameter of the powder particles of the corn straws is 0.3 mm.
B. Preparing corn stalk hydrolysate: weighing 30g of the corn straw powder obtained in the step A in the step (3), placing the corn straw powder into a 1L triangular flask, adding 300mL of dilute sulfuric acid with the volume fraction of 2.0% (v/v) according to the solid-to-liquid ratio of 1:10 (w/v), fully mixing uniformly, covering the triangular flask with a sealing film, and hydrolyzing for 60min at 121 ℃ in a sterilizing pot; cooling the obtained hydrolysate to room temperature, performing vacuum filtration by using four layers of gauze, discarding corn straw residues on the gauze, and taking the obtained rough-extracted filtrate; carrying out vacuum filtration on the obtained crude filtrate by using filter paper, discarding corn straw residues on the filter paper, and taking the obtained fine filtrate; using 10% (w/v) of Ca (OH) by mass percent to the fine suction filtration liquid2Adjusting the pH value to 10, standing for 1h, performing vacuum filtration by using filter paper, discarding residues on the filter paper, and taking the obtained fine filtrate; adjusting the pH value of the fine filtrate to 6 by using 2mol/L sulfuric acid, and performing suction filtration by using filter paper to obtain final filtrate; sterilizing the final filtrate in a sterilizing pot at 121 deg.C for 15minAnd obtaining corn straw hydrolysate, and storing the corn straw hydrolysate in a refrigerator at 4 ℃ for later use.
(4) Culture of bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate
A. Preparation of cellulase solution: 7.5g of cellulase R-10 is dissolved in 500mL of disodium hydrogen phosphate-citric acid solution with the concentration of 0.05mol/L and the pH value of 5.0 to obtain cellulase solution with the mass fraction of 1.5% (w/v), and the solution is put into a refrigerator at the temperature of 4 ℃ for standby.
B. Culturing the bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate: and (3) inoculating 120 g-150 g of the bacterial cellulose immobilized cryptococcus curvatus obtained in the step (B) in the step (2) into 500mL of the corn straw hydrolysate obtained in the step (3), monitoring the change of reducing sugar in the hydrolysate every 24h, and completing the culture of the bacterial cellulose immobilized cryptococcus curvatus in the corn straw hydrolysate when the reducing sugar in the hydrolysate is completely consumed.
The invention discloses a method for measuring the oil yield of bacterial cellulose immobilized Cryptococcus curvatus, which comprises the following steps: taking out the cultured bacterial cellulose loaded with Cryptococcus flexuosus obtained in the step B in the step (4) by using a pair of tweezers, and washing the bacterial cellulose for 3 times by using deionized water; soaking the washed bacterial cellulose carrying cryptococcus flexus in 700mL of a beaker containing the cellulase solution obtained in the step A in the step (4), covering a bottle opening with a sealing film, putting the beaker into a constant-temperature drying oven at 50 ℃, drying for 10 hours, and degrading the bacterial cellulose; and (3) after the bacterial cellulose is degraded, filtering the bacterial cellulose by using single-layer gauze, discarding bacterial cellulose fragments on the gauze, and taking filtrate to measure the cryptococcus curvatus biomass and the microbial oil yield.
The bacterial cellulose is extracellular ultramicro pure cellulose generated by microorganisms, has the characteristics of strong biological stability, strong mass transfer performance, strong poison resistance, degradability and the like, can be used as an immobilized carrier of the grease yeast, reduces the influence of a toxicity inhibitor in corn straw hydrolysate on the grease yeast, and does not influence the extraction and the acquisition of the intracellular microbial oil of the subsequent oil-producing yeast.
Example 1 preparation of bacterial cellulose
A. And (3) recovery culture of freeze-dried bacteria of acetobacter xylinum: wiping an ampoule bottle by absorbent cotton soaked in alcohol with the volume fraction of 90% (v/v) under the aseptic condition, heating the top end of the ampoule bottle by using an alcohol lamp, then dropping sterile water to the top end of the heated ampoule bottle to crack the glass at the opening of the ampoule bottle, and knocking down the top end of the cracked ampoule bottle by using tweezers; the number of the ampoule bottle is CGMCC NO: 1.1812 Acetobacter xylinumGluconacetobacterxylinumDripping 0.6mL of acetobacter xylinum enrichment culture medium into the freeze-dried bacteria, and lightly shaking to obtain an acetobacter xylinum freeze-dried bacteria suspension. The acetobacter xylinum enrichment culture medium comprises the following components: 60.0g of glucose, 15.0g of peptone, 1.0g of citric acid and Na2HPO4·12H2O 0.8g，KH2PO4 2.5g，MgSO4·7H20.5g of O, 1L of deionized water and pH 7.0. Sucking 100uL of the acetobacter xylinum freeze-dried thallus suspension, and uniformly coating the acetobacter xylinum freeze-dried thallus suspension on an acetobacter xylinum solid culture medium by using a coating rod, wherein the acetobacter xylinum solid culture medium comprises the following components: glucose 60.0g, peptone 15.0g, citric acid 1.0g, Na2HPO4·12H2O 0.8g，KH2PO4 2.5g，MgSO4·7H20.5g of O, 20.0g of agar and 1L of deionized water, and the pH value is 7.0. Culturing the solid culture medium coated with acetobacter xylinum in a constant-temperature electric heating incubator at 28 ℃ for 48h to obtain recovered and cultured acetobacter xylinum, and storing in a refrigerator at 4 ℃ for later use.
B. Preparing an acetobacter xylinum seed solution: selecting a ring of strains from the recovered and cultured acetobacter xylinum obtained in the step (1) A by using an inoculating ring, and transferring the strains into a 500mL conical flask filled with 100mL of acetobacter xylinum seed culture medium, wherein the acetobacter xylinum seed culture medium comprises the following components: glucose 30.0g, peptone 5.0g, citric acid 3.0g, Na2HPO4·12H2O 3.0g，MgSO4·7H20.20g of O, 1L of deionized water and pH 7.0. Oscillating for 36h at 28 deg.C in a constant temperature oscillator of 180r/min to obtain Acetobacter xylinum seed solution.
C. Preparing bacterial cellulose: obtained in the step (1) BInoculating the acetobacter xylinum seed liquid into a square container filled with 100mL of acetobacter xylinum fermentation culture medium according to the inoculation amount of 10% (v/v) in volume ratio, wherein the size of the square container is as follows: the bottom side length is 8cm multiplied by 8cm, the height is 8cm, and the components of the acetobacter xylinum fermentation medium are respectively as follows: 100.0g of glucose, 20.0g of peptone, 3.0g of citric acid and Na2HPO4·12H2O 2.5g，KH2PO4 3.5g，MgSO4·7H20.5g of O, 1L of deionized water and pH 7.0. Then the square container is placed statically in a constant temperature electric heating incubator at 28 ℃ for 6 days, and bacterial cellulose is obtained on the surface of the acetobacter xylinum fermentation medium.
D. Extracting and processing bacterial cellulose: and (2) taking out the bacterial cellulose obtained in the step (1) C with a pair of tweezers, washing the bacterial cellulose for 3 times with deionized water, soaking the bacterial cellulose into 100mL of NaOH solution with the concentration of 0.05mol/L, carrying out water bath at 60 ℃ for 120min, removing thalli and culture medium remained on the surface of the bacterial cellulose, repeatedly washing the bacterial cellulose with the deionized water until the bacterial cellulose is neutral, cutting the bacterial cellulose into 4cm multiplied by 4cm small blocks, sterilizing the bacterial cellulose in a sterilizing pot at 121 ℃ for 15min, and storing the bacterial cellulose in a refrigerator at 4 ℃ for later use.
Example 2 preparation of bacterial cellulose immobilized Cryptococcus curvatus
A. Preparing a Cryptococcus curvatus seed solution: cryptococcus curvatus with strain number ATCC 20509 to be preserved on slantCryptococcus curvatusThe seed culture medium is inoculated into a seed culture medium, and the seed culture medium comprises the following components in percentage by mass: 3% of glucose, 1.5% of yeast extract powder and 1.5% of peptone. Shaking and culturing in a constant temperature shaking table at 28 deg.C and 160r/min for 20h to obtain Cryptococcus curvatus seed solution.
B. Bacterial cellulose immobilized Cryptococcus curvatus: and (3) sucking 1mL of the Cryptococcus curvatus seed liquid obtained in the step (2) A by using an injector, uniformly injecting the Cryptococcus curvatus seed liquid into the small block of bacterial cellulose with the size of 4cm multiplied by 4cm obtained in the step (1) D, and washing the bacterial cellulose injected with the Cryptococcus curvatus seed liquid for 3 times by using deionized water to obtain the Cryptococcus curvatus immobilized by the bacterial cellulose.
Example 3 preparation of corn stover hydrolysate
A. Preparing corn straw powder: the corn straws are cleaned by deionized water, then placed in a constant-temperature drying oven at 80 ℃ to be dried to constant weight, cut into 3cm small sections and then crushed into powder by a crusher, and the diameter of the powder particles of the corn straws is 0.3 mm.
B. Preparing corn stalk hydrolysate: weighing 30g of the corn straw powder obtained in the step (3) A, placing the corn straw powder into a 1L triangular flask, adding 300mL of dilute sulfuric acid with the volume fraction of 2.0% (v/v) according to the solid-to-liquid ratio of 1:10 (w/v), fully mixing uniformly, covering the triangular flask with a sealing film, and hydrolyzing for 60min at 121 ℃ in a sterilizing pot. Cooling the obtained hydrolysate to room temperature, performing vacuum filtration by using four layers of gauze, discarding corn straw residues on the gauze, and taking the obtained rough-extracted filtrate; carrying out vacuum filtration on the obtained crude filtrate by using filter paper, discarding corn straw residues on the filter paper, and taking the obtained fine filtrate; using 10% (w/v) of Ca (OH) by mass percent to the fine suction filtration liquid2Adjusting the pH value to 10, standing for 1h, performing vacuum filtration by using filter paper, removing residues on the filter paper, and taking the obtained fine filtrate; adjusting the pH value of the fine filtrate to 6 by using 2mol/L sulfuric acid, and performing suction filtration by using filter paper to obtain final filtrate; sterilizing the final filtrate in a sterilizing pot at 121 deg.C for 15min to obtain corn stalk hydrolysate, and storing in a refrigerator at 4 deg.C for use.
Example 4 determination of oil production in corn stalk hydrolysate by bacterial cellulose immobilized Cryptococcus curvatus
A. Preparation of cellulase solution: 7.5g of cellulase R-10 is dissolved in 500mL of disodium hydrogen phosphate-citric acid solution with the concentration of 0.05mol/L and the pH value of 5.0 to obtain cellulase solution with the mass fraction of 1.5% (w/v), and the solution is put into a refrigerator at the temperature of 4 ℃ for standby.
B. Culturing the bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate: and (3) inoculating 150g of the immobilized cryptococcus curvatus obtained in the step (2) B into 500mL of the corn straw hydrolysate obtained in the step (3) B, monitoring the change of reducing sugar in the hydrolysate every 24h, and finishing the culture of the bacterial cellulose immobilized cryptococcus curvatus in the corn straw hydrolysate when the reducing sugar in the hydrolysate is completely consumed.
C. And (3) determining the oil yield of the bacterial cellulose immobilized cryptococcus curvatus: taking out the cultured bacterial cellulose loaded with Cryptococcus flexuosus obtained in the step (4) B by using a pair of tweezers, and washing the bacterial cellulose for 3 times by using deionized water; soaking the washed bacterial cellulose carrying cryptococcus flexus in a 1L beaker filled with 700mL of the cellulase solution obtained in the step (4) A, covering a bottle opening with a sealing film, putting the bottle opening into a constant-temperature drying oven at 50 ℃, drying for 10 hours, and degrading the bacterial cellulose; and (3) after the bacterial cellulose is degraded, filtering the bacterial cellulose by using single-layer gauze, discarding bacterial cellulose fragments on the gauze, and taking filtrate to measure the biomass of cryptococcus curvatus and the content of microbial oil.
The measurement results are shown in table 1, and the data in the table show that the biomass and the oil yield of the cryptococcus flexus immobilized by bacterial cellulose in the corn straw hydrolysate are respectively improved by 18.10 percent and 18.27 percent.
TABLE 1 Effect of injecting 1mL Cryptococcus curvatus seed solution and adding 150g immobilized Cryptococcus curvatus bacterial cellulose
Example 5 determination of oil production in corn stalk hydrolysate by bacterial cellulose immobilized Cryptococcus curvatus
A. Preparation of cellulase solution: 7.5g of cellulase R-10 is dissolved in 500mL of disodium hydrogen phosphate-citric acid solution with the concentration of 0.05mol/L and the pH value of 5.0 to obtain cellulase solution with the mass fraction of 1.5% (w/v), and the solution is put into a refrigerator at the temperature of 4 ℃ for standby.
B. Culturing the bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate: and (3) inoculating 120g of the immobilized cryptococcus curvatus obtained in the step (2) B into 500mL of the corn straw hydrolysate obtained in the step (3) B, monitoring the change of reducing sugar in the hydrolysate every 24h, and finishing the culture of the bacterial cellulose immobilized cryptococcus curvatus in the corn straw hydrolysate when the reducing sugar in the hydrolysate is completely consumed.
C. And (3) determining the oil yield of the bacterial cellulose immobilized cryptococcus curvatus: taking out the cultured bacterial cellulose loaded with Cryptococcus flexuosus obtained in the step (4) B by using a pair of tweezers, and washing the bacterial cellulose for 3 times by using deionized water; soaking the washed bacterial cellulose carrying cryptococcus flexus in a 1L beaker filled with 700mL of the cellulase solution obtained in the step (4) A, covering a bottle opening with a sealing film, putting the bottle opening into a constant-temperature drying oven at 50 ℃, drying for 10 hours, and degrading the bacterial cellulose; and (3) after the bacterial cellulose is degraded, filtering the bacterial cellulose by using single-layer gauze, discarding bacterial cellulose fragments on the gauze, and taking filtrate to measure the biomass of cryptococcus curvatus and the content of microbial oil.
The measurement results are shown in Table 2, and the data in the table show that the biomass and the oil yield of the cryptococcus curvatus immobilized by bacterial cellulose in the corn straw hydrolysate are respectively improved by 17.24 percent and 17.15 percent
Example 6 determination of oil production in corn stalk hydrolysate by bacterial cellulose immobilized cryptococcus flexus a preparation of cellulase solution: 7.5g of cellulase R-10 is dissolved in 500mL of disodium hydrogen phosphate-citric acid solution with the concentration of 0.05mol/L and the pH value of 5.0 to obtain cellulase solution with the mass fraction of 1.5% (w/v), and the solution is put into a refrigerator at the temperature of 4 ℃ for standby.
B. Culturing the bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate: and (3) inoculating 135g of the immobilized cryptococcus curvatus obtained in the step (2) B into 500mL of the corn straw hydrolysate obtained in the step (3) B, monitoring the change of reducing sugar in the hydrolysate every 24h, and finishing the culture of the bacterial cellulose immobilized cryptococcus curvatus in the corn straw hydrolysate when the reducing sugar in the hydrolysate is completely consumed.
C. And (3) determining the oil yield of the bacterial cellulose immobilized cryptococcus curvatus: taking out the cultured bacterial cellulose loaded with Cryptococcus flexuosus obtained in the step (4) B by using a pair of tweezers, and washing the bacterial cellulose for 3 times by using deionized water; soaking the washed bacterial cellulose carrying cryptococcus flexus in a 1L beaker filled with 700mL of the cellulase solution obtained in the step (4) A, covering a bottle opening with a sealing film, putting the bottle opening into a constant-temperature drying oven at 50 ℃, drying for 10 hours, and degrading the bacterial cellulose; and (3) after the bacterial cellulose is degraded, filtering the bacterial cellulose by using single-layer gauze, discarding bacterial cellulose fragments on the gauze, and taking filtrate to measure the biomass of cryptococcus curvatus and the content of microbial oil.
The measurement results are shown in table 3, and the data in the table show that the biomass and the oil yield of the cryptococcus flexus immobilized by the bacterial cellulose in the corn straw hydrolysate are respectively improved by 19.69 percent and 20.57 percent.
TABLE 3 Effect of injecting 1.5mL Cryptococcus curvatus seed solution and adding 135g of immobilized Cryptococcus curvatus bacterial cellulose
Claims (1)
1. A method for improving oil production of grease yeast cultured by corn stalk hydrolysate comprises the following steps:
(1) preparing bacterial cellulose:
A. restoring and culturing the freeze-dried thalli of the acetobacter xylinum;
B. preparing an acetobacter xylinum seed solution;
(2) preparing the bacterial cellulose immobilized Cryptococcus curvatus:
A. preparing Cryptococcus curvatus seed liquid;
(3) preparing corn stalk hydrolysate:
A. preparing corn straw powder;
(4) culturing the bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate:
A. preparation of cellulase solution: dissolving 7.5g of cellulase R-10 in 500mL of disodium hydrogen phosphate-citric acid solution with the concentration of 0.05mol/L and the pH value of 5.0 to obtain a cellulase solution with the mass fraction of 1.5% (w/v), and placing the cellulase solution in a refrigerator at 4 ℃ for later use;
the method is characterized in that:
after the acetobacter xylinum seed liquid is obtained in the step (1),
C. preparing bacterial cellulose: inoculating the acetobacter xylinum seed liquid obtained in the step B into a square container filled with 100mL of acetobacter xylinum fermentation culture medium according to the inoculation amount of 10% (v/v) by volume ratio, wherein the size of the square container is as follows: the bottom side length is 8cm multiplied by 8cm, the height is 8cm, and the acetobacter xylinum fermentation medium comprises the following components in parts by weight: 100.0 percent of glucose, 20.0 percent of peptone, 3.0 percent of citric acid and Na2HPO4·12H2O 2.5，KH2PO4 3.5，MgSO4·7H20.5O, 1L deionized water and 7.0 pH; then, standing the square container in a constant-temperature electric heating incubator at 28 ℃ for culturing for 6 days to obtain bacterial cellulose on the surface of the acetobacter xylinum fermentation medium;
D. extracting and processing bacterial cellulose: taking out the bacterial cellulose obtained in the step C by using a pair of tweezers, washing the bacterial cellulose for 3 times by using deionized water, then soaking the bacterial cellulose into 100mL of NaOH solution with the concentration of 0.05mol/L, carrying out water bath at 60 ℃ for 120min, removing thalli and culture medium remained on the surface of the bacterial cellulose, then repeatedly washing the bacterial cellulose by using the deionized water until the bacterial cellulose is neutral, cutting the bacterial cellulose into small blocks of 4cm multiplied by 4cm, sterilizing the bacterial cellulose in a sterilizing pot at 121 ℃ for 15min, and then storing the bacterial cellulose in a refrigerator at 4 ℃ for later use;
after the cryptococcus rhodochrous seed liquid is bent in the step (2),
B. bacterial cellulose immobilized Cryptococcus curvatus: sucking 1-2 mL of the Cryptococcus sinutus seed liquid obtained in the step A in the step (2) by using an injector, uniformly injecting the Cryptococcus sinutus seed liquid into the small block of bacterial cellulose with the size of 4cm multiplied by 4cm obtained in the step D in the step (1), and washing the bacterial cellulose injected with the Cryptococcus sinutus seed liquid for 3 times by using deionized water to obtain bacterial cellulose immobilized Cryptococcus sinutus;
after the corn straw powder is obtained in the step (3),
B. corn stalkPreparing stalk hydrolysate: weighing 30g of the corn straw powder obtained in the step A in the step (3), placing the corn straw powder into a 1L triangular flask, adding 300mL of dilute sulfuric acid with the volume fraction of 2.0% (v/v) according to the solid-to-liquid ratio of 1:10 (w/v), fully mixing uniformly, covering the triangular flask with a sealing film, and hydrolyzing for 60min at 121 ℃ in a sterilizing pot; cooling the obtained hydrolysate to room temperature, performing vacuum filtration by using four layers of gauze, discarding corn straw residues on the gauze, and taking the obtained rough-extracted filtrate; carrying out vacuum filtration on the obtained crude filtrate by using filter paper, discarding corn straw residues on the filter paper, and taking the obtained fine filtrate; using 10% (w/v) of Ca (OH) by mass percent to the fine suction filtration liquid2Adjusting the pH value to 10, standing for 1h, performing vacuum filtration by using filter paper, discarding residues on the filter paper, and taking the obtained fine filtrate; adjusting the pH value of the fine filtrate to 6 by using 2mol/L sulfuric acid, and performing suction filtration by using filter paper to obtain final filtrate; sterilizing the final filtrate in a sterilizing pot at 121 deg.C for 15min to obtain corn stalk hydrolysate, and storing in a refrigerator at 4 deg.C;
continuing in step (4)
B. Culturing the bacterial cellulose immobilized cryptococcus curvatus in corn straw hydrolysate: 120-150 g of the bacterial cellulose immobilized cryptococcus curvatus obtained in the step B in the step (2) is inoculated into 500mL of the corn straw hydrolysate obtained in the step B in the step (3), the change of reducing sugar in the hydrolysate is monitored at intervals of 24h, and when the reducing sugar in the hydrolysate is completely exhausted, the culture of the bacterial cellulose immobilized cryptococcus curvatus in the corn straw hydrolysate is completed;
C. and (3) determining the oil yield of the bacterial cellulose immobilized cryptococcus curvatus: taking out the cultured bacterial cellulose loaded with Cryptococcus flexuosus obtained in the step B in the step (4) by using a pair of tweezers, and washing the bacterial cellulose for 3 times by using deionized water; soaking the washed bacterial cellulose carrying cryptococcus flexus in 700mL of a beaker containing the cellulase solution obtained in the step A in the step (4), covering a bottle opening with a sealing film, putting the beaker into a constant-temperature drying oven at 50 ℃, drying for 10 hours, and degrading the bacterial cellulose; and (3) after the bacterial cellulose is degraded, filtering the bacterial cellulose by using single-layer gauze, discarding bacterial cellulose fragments on the gauze, and taking filtrate to measure the cryptococcus curvatus biomass and the microbial oil yield.
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