CN116479064A - Method for producing grease by open fermentation of high-sugar high-inhibitor biomass hydrolysate - Google Patents
Method for producing grease by open fermentation of high-sugar high-inhibitor biomass hydrolysate Download PDFInfo
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- CN116479064A CN116479064A CN202310419753.5A CN202310419753A CN116479064A CN 116479064 A CN116479064 A CN 116479064A CN 202310419753 A CN202310419753 A CN 202310419753A CN 116479064 A CN116479064 A CN 116479064A
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- hydrolysate
- biomass
- fermentation
- inhibitor
- pretreatment
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/36—Adaptation or attenuation of cells
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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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- C12P7/6436—Fatty acid esters
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention provides a method for producing grease by utilizing high-sugar high-inhibitor biomass hydrolysate through open fermentation, which specifically comprises the following steps: performing laboratory directed evolution on oleaginous yeast to improve the tolerance of the strain to the biomass-based inhibitor; pretreating the biomass raw material with high solid content and directly performing enzymatic hydrolysis to prepare high-sugar high-inhibitor hydrolysate; the oleaginous yeast directly utilizes the hydrolysate to produce microbial oil through non-sterilization fermentation. The method adopts a laboratory evolution method, obviously strengthens the tolerance of oleaginous yeast to biomass hydrolysate inhibitors, and can directly utilize the high-sugar high-inhibitor biomass hydrolysate to ferment under non-sterilization condition to produce grease. The strain-based hydrolysate has stronger tolerance, the hydrolysate does not need to be sterilized, and the energy consumption, the process cost and the equipment cost for preparing the microbial oil are reduced. In addition, the method can utilize cheap biomass to produce biodiesel, and has remarkable economic benefit and environmental protection benefit.
Description
Technical Field
The invention relates to a method for producing grease by using high-sugar high-inhibitor biomass hydrolysate and application thereof, belonging to the technical field of biology.
Background
Energy is one of the dynamics of the continuous development of human society. Although the reserves of fossil energy are rich, the fossil energy has a series of defects of uneven regional distribution, great development difficulty, unfriendly environment and the like. As the world demand for energy continues to increase, there is a need to develop new energy sources to replace fossil energy sources to address energy crisis.
The microbial oil is oil produced by oleaginous microorganism under certain condition, and its main component is triglyceride. The composition of fatty acid in triglyceride is similar to vegetable oil and animal oil, and is usually C1.-C1. The long chain fatty acid of (2) is mainly palmitic acid, stearic acid, oleic acid and linoleic acid. The microbial oil can be converted into fatty acid methyl ester, namely biodiesel through one-step transesterification. In addition, through a synthetic biological means, the synthesis of fatty acid with specific length and structure by cells can be regulated, so that a product with higher added value is obtained. Some oleaginous yeasts such as Rhodotorula toruloides, yarrowia lipolytica and Cryptococcus curvatus can accumulate about 70% of the dry weight of the cells, and have the advantages of short production period, no limitation of seasons, no occupation of cultivated land area, wide raw material sources and the like. Because the components of the microbial oil are highly similar to the animal and vegetable oil, the microbial oil can be used for producing biodiesel and has great significance for the long-term development of biodiesel.
At present, the production cost of microbial oil is still high, and the high cost of raw materials is one of the main factors. The lignocellulose resources in China are rich, the annual yield of the straw in rural areas is up to about 8 hundred million tons according to statistics, but the energy utilization rate is not high. Lignocellulose mainly comprises cellulose, hemicellulose and lignin, wherein the cellulose and the hemicellulose are hydrolyzed to obtain monosaccharides such as glucose, xylose, arabinose, galactose, mannose and the like, and the lignin is partially degraded into water-soluble phenolic compounds in the treatment process. Higher concentrations of biomass hydrolysate are advantageous for high grease yields, but contain significant amounts of hydrolysis byproducts, which are generally classified into three categories depending on the structure of the byproducts: weak acids including formic acid, acetic acid, ferulic acid, levulinic acid, and the like; furans, including furfural, furfuryl alcohol, hydroxymethylfurfural, and the like; phenols, including parahydroxybenzaldehyde/acid/alcohol, vanillin/acid/alcohol, syringaldehyde/acid/alcohol, and the like. These byproducts inhibit bacterial growth and oil synthesis, for example, furfural and hydroxymethylfurfural directly inhibit the activities of alcohol dehydrogenase, pyruvate dehydrogenase and aldehyde dehydrogenase, and the presence of weak acids reduces intracellular pH, thus reducing cell replication activity and the like. In addition, the sugar concentration in the high-concentration biomass hydrolysate is correspondingly increased, which can bring about the change of the osmotic pressure of the culture medium and influence the activity of cells. Many oleaginous yeasts have been found to be bio-based inhibitor tolerant. However, at higher inhibitor concentrations, oleaginous yeast detoxification and growth efficiencies are not high. Therefore, starting from the fermentation strain, the stress resistance of the strain to the hydrolysate byproducts is improved by means of laboratory evolution, and the production efficiency of producing microbial oil by utilizing the hydrolysate can be improved.
The fermentation process usually adopts a closed system, and needs to be sterilized at high temperature and sterile air is introduced in the fermentation process to ensure that the fermentation process is not influenced by mixed bacteria. The high-temperature sterilization process has the characteristics of high energy consumption, high operation cost, time consumption and the like, and the preparation of the sterile air also needs a certain cost. Taking methanol as a substrate, a plant with a annual yield of 10 ten thousand tons of single-cell proteins as an example, the energy, water, and sterile air and other auxiliary costs in high-temperature sterilization account for about 10% of the production costs. The high concentration biomass hydrolysate itself contains a large amount of inhibitors, other microorganisms are difficult to grow, and oleaginous yeast is tolerant, so that oil production can be performed. Therefore, the establishment of an open fermentation system can completely meet the production requirements of microbial oil, and meanwhile, the operation process can be simplified, the production cost can be reduced, the energy consumption can be reduced, and the economy of producing the microbial oil can be improved.
Laboratory evolution of microorganisms is a means of breeding superior traits, which refers to the process of continuous culture under specific selection pressure to enrich for beneficial mutations, thereby altering the microbial traits. The microbial laboratory evolution utilizes the short growth cycle and the rapid growth speed of microorganisms, and can enrich a plurality of mutations in a short time. In addition, the method is also simple in laboratory operation and easy in phenotype acquisition compared with the common engineering technical means in microorganisms, and complex genetic engineering construction is not needed. By utilizing a laboratory evolution method, strains which can tolerate the grease produced by the high-concentration biomass hydrolysate can be quickly bred, and compared with microorganisms in the environment, the strain can grow in the high-concentration biomass hydrolysate and become a dominant strain. Therefore, an open fermentation system is established, the step of sterilization in the grease production process is omitted, the energy consumption and the production cost are reduced, the development of biodiesel industry in China is facilitated, and a certain foundation is provided for the resource utilization of biomass and the sustainable development of agriculture.
Disclosure of Invention
According to one aspect of the application, a method for producing grease by using high-sugar high-inhibitor biomass hydrolysate is provided, and a novel method for preparing microbial grease by taking lignocellulose biomass as a raw material is provided. Compared with the traditional microbial oil preparation method, the method establishes an open fermentation system, can be directly used for fermentation, does not need to carry out a high-temperature sterilization process, can simplify process steps, and reduces energy consumption. Meanwhile, the method can improve the utilization efficiency of biomass and reduce the production cost of biodiesel or other high-added-value products. The method can be suitable for different types of biomass materials and different types of pretreatment modes, and has universality.
The method for producing grease by using the high-sugar high-inhibitor biomass hydrolysate is characterized by comprising the following steps of: the method comprises the steps of directionally evolving an oleaginous yeast laboratory, carrying out high-solid-load pretreatment and high-solid-load enzymatic hydrolysis on biomass raw materials, and establishing an open fermentation system to produce microbial grease and biodiesel.
Optionally, the laboratory directed evolution uses biomass hydrolysate as an evolution condition, and three or more biomass hydrolysates with different concentrations are selected for laboratory evolution to obtain the strain which is tolerant to the high-sugar high-inhibitor biomass hydrolysate.
Optionally, the biomass pretreatment and enzymatic hydrolysis solids loading lower limit is 15% (w/v, solids to liquid ratio). Optionally, the biomass pretreatment and enzymatic hydrolysis solids loading is up to 30% (w/v).
Optionally, the biomass hydrolysate has a total reducing sugar concentration lower limit of 70g/L. Optionally, the upper limit of the total reducing sugar concentration of the biomass hydrolysate is 150g/L.
Optionally, the raw material is at least one selected from corn stalk, wheat stalk and straw.
Optionally, the biomass pretreatment method is selected from at least one of ball milling, extrusion, acid pretreatment, alkali pretreatment, dry pretreatment, steam pretreatment.
Optionally, the enzymatic hydrolysis comprises adding an enzyme to the feedstock; the enzyme is at least one of cellulase and xylanase.
Optionally, the oil fermentation process comprises preparing fermentation seed liquid and fermenting and producing oil culture.
Optionally, the seed culture medium comprises 20g/L glucose, 10g/L yeast powder and 10g/L peptone; the culture method comprises the following steps: sterilizing the seed culture medium, inoculating the microorganism, and shake culturing at 25-32deg.C and 150-250rpm for 20-24 hr.
Optionally, the fermentation oleaginous culture comprises: inoculating the fermentation seed liquid into high-concentration biomass hydrolysate, wherein a fermentation system is an open system, the hydrolysate is not subjected to high-temperature sterilization treatment, and sterile air is not used in the fermentation process; wherein, the volume ratio of the fermentation seed liquid to the hydrolysate is 1: 15-1:5; the temperature of the fermentation oil-producing culture is 28-32 ℃ and the pH value is 4-6; the dissolved oxygen in the hydrolysate is more than 20 percent.
Optionally, the microorganism strain is selected from at least one of oleaginous yeast Lipomyces starkeyi, rhodosporidium toruloides Rhodotorula toruloides, rhodotorula glutinis Rhodotorula glutinis, rhodosporidium throws Sporobolomyces roseus, candida Pityrosporum Trichosporon cutaneum, candida fermentata Trichosporon fermentans, cryptococcus albus Cryptococcus albidus, cryptococcus curvatus Cryptococcus curvatus.
As a specific implementation mode, the invention is realized by the following technical scheme:
(1) Laboratory evolution of strains: after the strain is activated in a seed culture medium, biomass hydrolysate with different concentrations is selected for laboratory evolution. Firstly, inoculating a strain into low-concentration biomass hydrolysate, and when the strain can adapt to the hydrolysate components and grows to a certain OD, transferring part of bacterial liquid into biomass hydrolysate with higher concentration, repeating the steps, and gradually improving the tolerance of the strain in the hydrolysate.
(2) Pretreatment of high-concentration biomass raw materials: a lignocellulose biomass material is selected for physical or chemical pretreatment, wherein the physical pretreatment method comprises ball milling and the like, and the chemical pretreatment method comprises acid pretreatment, alkali pretreatment, dry pretreatment, high-temperature pretreatment and the like.
(3) Enzymatic hydrolysis of high concentration biomass feedstock: after pretreatment is completed, adding biomass materials according to the high solid content level, carrying out enzyme hydrolysis, and after the enzyme hydrolysis is completed, carrying out solid-liquid separation and collection to obtain high-concentration biomass hydrolysate.
(4) Preparing oil fermentation seed liquid: weighing seed culture medium (glucose 20g/L, yeast powder 10g/L, peptone 10 g/L), sterilizing, cooling, inoculating oleaginous microorganism, and shake culturing at 30deg.C and 200rpm for 24 hr.
(5) Fermenting and producing oil and culturing: the oil accumulation culture medium is high-sugar high-inhibitor biomass hydrolysate, the fermentation system is an open system, no high-temperature sterilization treatment is carried out, and no sterile air is used in the fermentation process. The inoculation amount is 10% (v/v), and the seed age of the oil fermentation seed liquid is 24 hours. The fermentation temperature is 30 ℃, the pH is 5.5, and the dissolved oxygen is more than 20 percent (controlled by adjusting the stirring rotation speed and the ventilation). Stopping fermentation when the reducing sugar in the fermentation liquid is not reduced any more.
The beneficial effects that this application can produce include:
(1) According to the method, through laboratory directed evolution of oleaginous yeast, oleaginous yeast capable of tolerating high-concentration hydrolysate is obtained, and oleaginous yeast can be used for producing grease in high-concentration biomass hydrolysate, so that cheap biomass is converted into microbial grease, biodiesel or other high-added-value products.
(2) According to the method provided by the application, lignocellulose biomass is used as a raw material, three process steps of raw material pretreatment, enzyme hydrolysis and microbial oil fermentation are carried out, so that the biomass hydrolysate with high sugar and high inhibitor is obtained, and the preparation of microbial oil is realized by using the hydrolysate. The oil contains one or more medium-long chain fatty acids and derivatives thereof, and can be used for preparing biodiesel, functional oil or other high-added-value products.
(3) The method provided by the application is suitable for biomass hydrolysate obtained by different pretreatment methods, and has universality. Aiming at different biological substances and different kinds of hydrolysate obtained by different pretreatment methods, the method can obtain effective production strains and realize the production of microbial oil and biodiesel.
(4) The method establishes an open fermentation process, does not need high-temperature sterilization, can perform grease fermentation without using sterile air in the fermentation process, does not need to treat inhibitors in hydrolysate before fermentation, does not need to additionally add other nutrient substances in the fermentation process, has the technical advantages of short process flow, high raw material utilization efficiency, high grease yield, low cost and low energy consumption, can improve the technical economy of microbial grease, and has certain economic and environmental benefits.
Drawings
FIG. 1 shows the change in OD of the strain during laboratory evolution of the strain according to example 1 of the present application.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In order to illustrate the technical scheme of the invention, the following description is made by specific examples.
Example 1
(1) Preparing high-sugar high-inhibitor hydrolysate: the corn stalk is ball milled for 12 hours by a ball mill and then stored at room temperature for standby. The preparation method of the straw hydrolysate with the solid content of 20 percent (w/v) comprises the following steps: 100g of ball-milling straw is taken and put into a 2L wide-mouth conical flask, 500mL of 12g/L NaOH solution is added, the total weight of the conical flask is recorded, and then the mixture is subjected to steam pretreatment at 121 ℃ for 1h. After the pretreatment was completed, the mixture was cooled to room temperature, the pH was adjusted to 4.8 with 4M HCl, 10mL of cellulase, 0.5g of xylanase and 500. Mu.L of 50g/L ampicillin sodium solution were added, and water was added to the weight before pretreatment. Enzymatic hydrolysis was carried out at 50℃for 48h at 200rpm and the pH was readjusted to 4.8 at 1 st and 3h of hydrolysis. After the hydrolysis is finished, transferring all samples into a centrifugal bottle, centrifuging at 8000rpm for 10min, collecting the supernatant, performing secondary centrifugation on the collected supernatant, collecting the supernatant again, and regulating the pH of the hydrolysate to 5.5 to obtain the straw hydrolysate. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Laboratory evolution of strains: a small amount of rhodosporidium R.toruloides is selected from a freezing tube at-80 ℃ and transferred to 10mL of YEPD culture medium for culture, and the culture medium is placed in a 50mL sterile centrifuge tube at 30 ℃ and 200rpm. After 48h of culture, the laboratory evolution of the strain is carried out, 10mL of 7% (w/v) hydrolysate is taken, the activated bacterial liquid is inoculated according to the inoculum size of 1:10 (v:v), 3 strains are prepared in parallel and marked as the first generation, and the strain is treated by OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment was carried out for 9 generations, and the solid content of the hydrolysis liquid of each generation was 7%, 12%, 15%, 16%, 18%, 20% (w/v) respectively, as shown in FIG. 1. The obtained strain capable of growing under 20% (w/v) was subjected to a plating separation to obtain single colonies.
(3) Production of microbial oil: inoculating R.toruloides after laboratory evolution into seed culture medium (20 g/L glucose, 10g/L yeast powder, 10g/L peptone), and shake culturing at 30deg.C for 24 hr to obtain seed solution. Inoculating the seed liquid into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquid is lower than 5g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil and high-added-value product carotenoid.
Comparative example 1
(1) Preparing high-sugar high-inhibitor hydrolysate: the corn stalk is ball milled for 12 hours by a ball mill and then stored at room temperature for standby. The preparation method of the straw hydrolysate with the solid content of 20 percent (w/w) comprises the following steps: 100g of ball-milling straw is taken and put into a 2L wide-mouth conical flask, 500mL of 12g/L NaOH solution is added, the total weight of the conical flask is recorded, and then the mixture is subjected to steam pretreatment at 121 ℃ for 1h. After the pretreatment was completed, the mixture was cooled to room temperature, the pH was adjusted to 4.8 with 4M HCl, 10mL of cellulase, 0.5g of xylanase and 500. Mu.L of 50g/L ampicillin sodium solution were added, and water was added to the weight before pretreatment. Enzymatic hydrolysis was carried out at 50℃for 48h at 200rpm and the pH was readjusted to 4.8 at 1 st and 3h of hydrolysis. After the hydrolysis is finished, transferring all samples into a centrifugal bottle, centrifuging at 8000rpm for 10min, collecting the supernatant, performing secondary centrifugation on the collected supernatant, collecting the supernatant again, and regulating the pH of the hydrolysate to 5.5 to obtain the straw hydrolysate. Sterilizing the hydrolysate at 121deg.C for 20min, and cooling.
(2) Laboratory evolution of strains: r.toruloides is selected from a-80 ℃ cryopreservation tube and transferred to 10mL of YEPD culture medium for culture, and the culture medium is placed in a 50mL sterile centrifuge tube at 30 ℃ and 200rpm. After 48h of culture, the laboratory evolution of the strain is carried out, 10mL of 20% (w/v) hydrolysate is taken, the activated bacterial liquid is inoculated according to the inoculum size of 1:10 (v:v), 3 bacteria are prepared in parallel and marked as the first generation, and the bacterial liquid is treated by OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment was carried out for 9 generations, and the solid content of the hydrolysis liquid of each generation was 7%, 12%, 15%, 16%, 18%, 20% (w/v) respectively. The obtained strain capable of growing under 20% (w/v) was subjected to a plating separation to obtain single colonies.
(3) Production of microbial oil: the R.toruloides of the rhodosporidium evolved in the laboratory is selected and inoculated in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and the culture is carried out for 24 hours at 30 ℃ in a shaking way, so as to obtain seed liquid. Inoculating the seed liquid into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquid is lower than 5g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil and high-added-value product carotenoid.
Comparative example 2
(1) Preparing high-sugar high-inhibitor hydrolysate: the corn stalk is ball milled for 12 hours by a ball mill and then stored at room temperature for standby. The preparation method of the straw hydrolysate with the solid content of 20 percent (w/v) comprises the following steps: 100g of ball-milling straw is taken and put into a 2L wide-mouth conical flask, 500mL of 12g/L NaOH solution is added, the total weight of the conical flask is recorded, and then the mixture is subjected to steam pretreatment at 121 ℃ for 1h. After the pretreatment was completed, the mixture was cooled to room temperature, the pH was adjusted to 4.8 with 4M HCl, 10mL of cellulase, 0.5g of xylanase and 500. Mu.L of 50g/L ampicillin sodium solution were added, and water was added to the weight before pretreatment. Enzymatic hydrolysis was carried out at 50℃for 48h at 200rpm and the pH was readjusted to 4.8 at 1 st and 3h of hydrolysis. After the hydrolysis is finished, transferring all samples into a centrifugal bottle, centrifuging at 8000rpm for 10min, collecting the supernatant, performing secondary centrifugation on the collected supernatant, collecting the supernatant again, and regulating the pH of the hydrolysate to 5.5 to obtain the straw hydrolysate. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Production of microbial oil: selecting wild rhodosporidium R.toruloides, inoculating in seed culture medium (20 g/L glucose, 10g/L yeast powder, 10g/L peptone), and shake culturing at 30deg.C for 24 hr to obtain seed solution. Inoculating the seed liquid into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquid is lower than 5g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil and high-added-value product carotenoid.
Example 2
(1) Preparing high-sugar high-inhibitor hydrolysate: and (3) washing the wheat straw with deionized water, and drying and crushing for later use. Preparing 5% NaOH-methanol solution, adding straw raw materials into a reactor according to 50% (w/v) after NaOH is completely dissolved, and fully and uniformly mixing by stirring. The system was then sealed and reacted at 80℃for 60min. Washing for three times after the reaction is finished, and filtering and drying. The preparation method of the straw hydrolysate with the solid content of 25 percent (w/v) comprises the following steps: 125g of pretreated straw was added to a 2L Erlenmeyer flask, a suitable amount of water was added and the pH was adjusted to 4.8. And adding novelin cellulase and 50mg/L ampicillin according to the enzyme carrying amount of 15FPU/g straw, adding deionized water to 500mL of the enzymolysis system, fully mixing, carrying out enzymolysis for 72 hours in a 50 ℃ water bath shaking table, centrifuging, collecting supernatant and freezing for later use. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Laboratory evolution of strains: a small amount of rhodosporidium R.toruloides is selected from a freezing tube at-80 ℃ and transferred to 10mL of YEPD culture medium for culture, and the culture medium is placed in a 50mL sterile centrifuge tube at 30 ℃ and 200rpm. After 48h of culture, carrying out laboratory evolution of the strain, taking 10mL of 15% (w/v) hydrolysate, inoculating the activated bacterial liquid according to the inoculum size of 1:10 (v:v), making 3 parallel strains, marking the strains as the first generation, and waiting for OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment is carried out for 6 generations, the solid load of hydrolysis liquid of each generation is 15%, 17%, 20%, 22%, 24% and 25% (w/v), and the obtained strain capable of growing under the condition of 25% (w/v) is subjected to coating separation to obtain single colony.
(3) Production of microbial oil: the bacteria evolved in the laboratory are selected and inoculated in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and the seed liquid is obtained by shaking culture for 24 hours at 30 ℃. Inoculating the seed solution into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquor is lower than 10g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil.
Example 3
(1) Preparing high-sugar high-inhibitor hydrolysate: after crushed natural dry corn straw is sieved by a 40-mesh sieve, 50g of straw is weighed in a 1L triangular flask, and is dissolved by 600mL of deionized water, so that the solid content of the straw is 7.5%, 1mL of concentrated sulfuric acid is added, and the mixture is uniformly mixed and sealed. Placing into a sterilizing pot, and treating at 121deg.C for 90min. And after the completion, carrying out suction filtration on the sample to realize solid-liquid separation, wherein the obtained solid is the material pretreated by dilute acid. The preparation method of 30% solid load (w/v) straw hydrolysate comprises the following steps: 150g of pretreated straw was added to a 2L Erlenmeyer flask, a suitable amount of water was added and the pH was adjusted to 4.8. And adding novelin cellulase and 50mg/L ampicillin according to the enzyme carrying amount of 15FPU/g straw, adding deionized water to 500mL of the enzymolysis system, fully mixing, carrying out enzymolysis for 24 hours in a 50 ℃ water bath shaking table, centrifuging, collecting supernatant and freezing for later use. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Laboratory evolution of strains: a small amount of Cryptococcus curvatus Ccurvatus was picked from a-80℃cryopreservation tube and transferred to 10mL of YEPD medium for culture, the medium was placed in a 50mL sterile centrifuge tube at 30℃and 200rpm. After 48h of culture, carrying out laboratory evolution of the strain, taking 10mL of 10% (w/v) hydrolysate, inoculating the activated bacterial liquid according to the inoculum size of 1:10 (v:v), making 3 strains in parallel, marking the strains as the first generation, and waiting for OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment was carried out for 7 generations, the solid content of the hydrolysis liquid of each generation was 10%, 15%, 18%, 22%, 25%, 28%, 30% (w/v), and the obtained strain capable of growing under the condition of 30% (w/v) was subjected to coating separation to obtain single colony.
(3) Production of microbial oil: the bacteria evolved in the laboratory are selected and inoculated in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and the seed liquid is obtained by shaking culture for 24 hours at 30 ℃. Inoculating the seed solution into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquor is lower than 10g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil.
Example 4
(1) Preparing high-sugar high-inhibitor hydrolysate: the air-dried wheat straw was crushed and sieved through a 40 mesh sieve. 100g of crushed straw and 200g of ionic liquid 1-ethyl-3-methylimidazole acetate ([ Emim ] Ac) are taken out, placed in a 1L pressure-resistant bottle, placed in a baking oven at 130 ℃ for 1.5 hours, taken out, cooled to room temperature, 200mL of deionized water is slowly added into the system, and the system is centrifuged, precipitated and washed until the supernatant is colorless. Taking a pretreated sample, adding 25FPU/g cellulase and 0.05mol/L citric acid-sodium citrate buffer solution with the pH value of 4.8, and fixing the volume to 400mL. And (3) carrying out enzyme hydrolysis reaction at 50 ℃ and 100r/min to obtain 25% (w/v) wheat straw hydrolysate. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Laboratory evolution of strains: a small amount of candida utilis T.cutaneum is selected from a cryopreservation tube at-80 ℃ and transferred to 10mL of YEPD culture medium for culture, and the culture medium is placed in a 50mL sterile centrifuge tube at 30 ℃ and 200rpm. After 48h of culture, the laboratory evolution of the strain is carried out, 10mL of 10% (w/v) hydrolysate is taken, the activated bacterial liquid is inoculated according to the inoculum size of 1:10 (v:v), 3 strains are prepared in parallel and marked as the first generation, and the strain is treated by OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment is carried out for 8 generations, the solid load of hydrolysis liquid of each generation is respectively 10%, 12%, 15%, 17%, 18%, 20%, 22% and 25% (w/v), and the obtained strain capable of growing under the condition of 25% (w/v) is subjected to coating separation to obtain single colony.
(3) Production of microbial oil: the bacteria evolved in the laboratory are selected and inoculated in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and the seed liquid is obtained by shaking culture for 24 hours at 30 ℃. Inoculating the seed solution into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquor is lower than 10g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil.
Example 5
(1) Preparing high-sugar high-inhibitor hydrolysate: the straw was chopped and dried, and then pulverized with a mechanical pulverizer, and sieved to 100. Mu.m. Preparing H with mass fraction of 1% 2 SO 4 Solution, solid to liquid ratio of 1:20, soaking for 24 hours at room temperature, flushing the treated corn straw with water to pH 6.8, and then drying at 60 ℃ to constant weight. Taking 150g of pretreated straw, adding 10FPU/g of celluloseThe enzyme and 0.05mol/L citric acid-sodium citrate buffer solution with pH value of 4.8 are fixed to 500mL. And (3) performing enzyme hydrolysis reaction at 50 ℃ and 200r/min, and ending enzyme hydrolysis when the sugar concentration is not changed, namely 30% (w/v) straw stalk hydrolysate. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Laboratory evolution of strains: small amounts of candida utilis t.cutaneum were selected from-80 ℃ cryopreservation tubes and transferred to 10 mlysepd medium for culture, which was placed in 50mL sterile centrifuge tubes at 30 ℃ at 200rpm. After 48h of culture, the laboratory evolution of the strain is carried out, 10mL of 10% (w/v) hydrolysate is taken, the activated bacterial liquid is inoculated according to the inoculum size of 1:10 (v:v), 3 strains are prepared in parallel and marked as the first generation, and the strain is treated by OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment was carried out for 11 generations, the solid content of the hydrolysis liquid of each generation was 10%, 12%, 15%, 17%, 18%, 20%, 22%, 25%, 27%, 30% (w/v), and the obtained strain capable of growing under the condition of 30% (w/v) was subjected to coating separation to obtain single colony.
(3) Production of microbial oil: the bacteria evolved in the laboratory are selected and inoculated in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and the seed liquid is obtained by shaking culture for 24 hours at 30 ℃. Inoculating the seed solution into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquor is lower than 10g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil.
Example 6
(1) Preparing high-sugar high-inhibitor hydrolysate: the straw was chopped and dried, and then pulverized with a mechanical pulverizer, and sieved to 100. Mu.m. Preparing NaOH solution with the mass fraction of 1%, wherein the solid-liquid ratio is 1:20, soaking for 24 hours at room temperature, flushing the treated corn straw with water to pH 6.8, and then drying at 60 ℃ to constant weight. 150g of pretreated straw is taken, 10FPU/g of cellulase and 0.05mol/L citric acid-sodium citrate buffer solution with pH value of 4.8 are added, and the volume is fixed to 500mL. And (3) performing enzyme hydrolysis reaction at 50 ℃ and 200r/min, and ending enzyme hydrolysis when the sugar concentration is not changed, namely 30% (w/v) straw stalk hydrolysate. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Laboratory evolution of strains: a small amount of candida utilis T.cutaneum is selected from a cryopreservation tube at-80 ℃ and transferred to 10mL of YEPD culture medium for culture, and the culture medium is placed in a 50mL sterile centrifuge tube at 30 ℃ and 200rpm. After 48h of culture, the laboratory evolution of the strain is carried out, 10mL of 10% (w/v) hydrolysate is taken, the activated bacterial liquid is inoculated according to the inoculum size of 1:10 (v:v), 3 strains are prepared in parallel and marked as the first generation, and the strain is treated by OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment is carried out for 9 generations, the solid load of hydrolysis liquid of each generation is respectively 10%, 14%, 17%, 18%, 20%, 22%, 25%, 27% and 30% (w/v), and the obtained strain capable of growing under the condition of 30% (w/v) is subjected to coating separation to obtain single colony.
(3) Production of microbial oil: the bacteria evolved in the laboratory are selected and inoculated in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and the seed liquid is obtained by shaking culture for 24 hours at 30 ℃. Inoculating the seed solution into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquor is lower than 10g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil.
Example 7
(1) Preparing high-sugar high-inhibitor hydrolysate: cutting corn stalk, stoving, crushing in a mechanical crusher and sieving to 40 mesh. Steam pretreatment is adopted: 150g of straw is taken, 500mL of water is added, pretreatment is carried out for 60min at 121 ℃, the straw is taken out and cooled to room temperature, 10FPU/g of cellulase is added to constant volume to 500mL, enzymatic hydrolysis reaction is carried out for 48h at 50 ℃ and 200r/min, pH of the hydrolysate is regulated to 4.8 every 12h, and supernatant is centrifugally collected after hydrolysis is finished, thus obtaining 30% (w/v) straw hydrolysate. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Laboratory evolution of strains: a small amount of oleaginous yeast L.starkey is selected from a freezing tube at-80 ℃ and transferred to 20mL of YEPD culture medium for culture, and the culture medium is placed in a 50mL shaking bottle under the culture condition of 30 ℃ and 200rpm. After 48h of culture, the laboratory evolution of the strain is carried out, 20mL of 10% (w/v) hydrolysate is taken, the activated bacterial liquid is inoculated according to the inoculum size of 1:10 (v:v), 3 strains are prepared in parallel and marked as the first generation, and the strain is treated by OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment is carried out for 9 generations, the solid load of the hydrolysis liquid of each generation is respectively 10%, 14%, 17%, 18%, 20%, 22%, 25%, 27% and 30% (w/v), the obtained strain capable of growing under the condition of 30% (w/v) is subjected to coating separation, and continuous passage is carried out, and after the stability of the strain is verified, a single colony capable of tolerating the hydrolysis liquid with high sugar and high inhibitor is obtained.
(3) Production of microbial oil: the bacteria evolved in the laboratory are selected and inoculated in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and the seed liquid is obtained by shaking culture for 24 hours at 30 ℃. Inoculating the seed solution into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquor is lower than 10g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil.
Example 8
(1) Preparing high-sugar high-inhibitor hydrolysate: the corn stalk is ball milled for 12 hours by a ball mill and then stored at room temperature for standby. The preparation method of 30% (w/v) straw hydrolysate comprises the following steps: 30g of ball-milled straw was taken in a 250mL wide-mouth conical flask, 100mL of 6% (w: w) NaOH solution was added, the total weight of the conical flask was recorded, and then steam pretreatment was performed at 121℃for 1 hour. After pretreatment, cooling to room temperature, filtering out part of liquid by a filter press, and removing part of toxic byproducts. The pH was adjusted to 4.8 with 4M HCl, 10mL of cellulase, 0.5g of xylanase, 500. Mu.L of 50g/L ampicillin sodium solution was added and the pretreatment weight was made up. Enzymatic hydrolysis was carried out at 50℃and 200rpm for 24h and the pH was readjusted to 4.8 at 1 st and 3h of hydrolysis. After the hydrolysis is completed, all samples are transferred into a centrifugal bottle, centrifuged at 8000rpm for 10min, the supernatant is collected, and the collected supernatant is ultrafiltered to obtain a hydrolysate without solid impurities. The hydrolysate is directly used for fermenting to produce grease without high-temperature sterilization.
(2) Laboratory evolution of strains: r.glutinis colony of the rhodotorula shapefaciens is selected from a freezing tube at the temperature of minus 80 ℃ and transferred to 10mL of YEPD culture medium for culture, and the culture medium is placed in a 50mL sterile centrifuge tube, wherein the culture condition is 30 ℃ and 200rpm. After 48h of culture, the laboratory evolution of the strain is carried out, 10mL of 20% (w/v) hydrolysate is taken, the activated bacterial liquid is inoculated according to the inoculum size of 1:10 (v:v), 3 bacteria are prepared in parallel and marked as the first generation, and the bacterial liquid is treated by OD 600 After the value reaches about 20, taking the best-grown 1 pipe, transferring the best-grown 1 pipe into the hydrolysis liquid with another solid load, and similarly making 3 parallel pipes, and gradually increasing the solid load of the hydrolysis liquid according to the method. The experiment is carried out for 7 generations, and the solid load of the hydrolysis liquid of each generation is 15%, 17%, 20%, 22%, 25%, 28% and 30% (w/v) respectively. The obtained strain capable of growing under 30% (w/v) was subjected to a plating separation to obtain single colonies.
(3) Production of microbial oil: the R.glutinis evolved in the laboratory is selected and inoculated in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and the seed solution is obtained by shaking culture for 24 hours at 30 ℃. Inoculating the seed liquid into the hydrolysate culture medium in the step (1) with the inoculum size of 10% (v/v), conducting aeration and shaking culture at 30 ℃, ending fermentation when the content of reducing sugar in the fermentation liquid is lower than 5g/L, performing solid-liquid separation, and collecting thalli to extract microbial oil and high-added-value product carotenoid.
In summary, the invention provides a method for preparing microbial oil by taking high-sugar high-inhibitor biomass hydrolysate as a raw material. Compared with the traditional microbial oil preparation method, the method has strong universality, can be suitable for different biomass and different pretreatment methods, and simultaneously establishes an open fermentation system without high-temperature sterilization and use of sterile air, so that the process steps are simplified, the energy consumption is reduced, the utilization efficiency of biomass is improved, and the production cost of biodiesel or other high-added-value products is reduced.
The foregoing description is only a few examples of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred examples, it is not intended to limit the present application, and any person skilled in the art may make some changes or modifications to the disclosed technology without departing from the scope of the technical solution of the present application, and the technical solution is equivalent to the equivalent embodiments.
Claims (10)
1. A method for producing grease by utilizing open fermentation of high-sugar high-inhibitor biomass hydrolysate is characterized in that laboratory directed evolution is carried out on oleaginous yeast to improve the tolerance of a strain to a biomass-based inhibitor, so that the strain can grow and ferment in the biomass hydrolysate containing high-concentration inhibitor to produce microbial grease; the biomass raw material is subjected to high-solid-load pretreatment and high-solid-load enzyme hydrolysis, so that the utilization efficiency of biomass is improved; in addition, a non-sterilization fermentation oil production system is established, and the oil production yeast can directly utilize the biomass hydrolysate containing high sugar and high inhibitor under the condition of no need of sterilization, so that the fermentation process is simplified, the fermentation cost is reduced, and the economy of producing microbial oil and biodiesel by biomass is improved.
2. The method of claim 1, wherein the laboratory evolution uses biomass hydrolysate as an evolution condition to promote tolerance of the strain to biomass-based inhibitors.
3. The method of claim 1, wherein the pretreatment and enzymatic hydrolysis loadings are each 15% -30% (w/v, solids to liquid ratio).
4. The method according to claim 1, wherein the total reducing sugar concentration of the hydrolysate is 70g/L to 150g/L.
5. The method of claim 1, wherein the biomass is selected from at least one of corn stover, wheat straw, and straw.
6. The method of claim 1, wherein the biomass pretreatment method is selected from at least one of ball milling, acid pretreatment, alkali pretreatment, dry pretreatment, steam pretreatment.
7. The method of claim 1, wherein the enzymatic hydrolysis comprises adding an enzyme to the feedstock; the enzyme is at least one of cellulase and hemicellulase.
8. The method of claim 1, wherein the oil fermentation process comprises preparing a fermentation seed liquid and fermenting an oleaginous culture. Wherein the fermentation oil production culture system is an open system, and the hydrolysis liquid is not required to be sterilized at high temperature.
9. The method of claim 1, wherein the microorganism is oleaginous yeast and its evolved strain with certain inhibitor tolerance, specifically comprising at least one of oleaginous yeast Lipomyces starkeyi, rhodosporidium toruloides Rhodotorula toruloides, rhodotorula glutinis Rhodotorula glutinis, rhodosporidium throws Sporobolomyces roseus, candida pinnata Trichosporon cutaneum, candida fermentata Trichosporon fermentans, cryptococcus albus Cryptococcus albidus, cryptococcus curvatus Cryptococcus curvatus.
10. The method according to claims 1-9, characterized in that the microbial oil prepared can be applied as biodiesel, functional oil.
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