CN110438172B - Method for producing grease by co-utilizing starch and lignocellulose raw materials - Google Patents

Method for producing grease by co-utilizing starch and lignocellulose raw materials Download PDF

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CN110438172B
CN110438172B CN201910716485.7A CN201910716485A CN110438172B CN 110438172 B CN110438172 B CN 110438172B CN 201910716485 A CN201910716485 A CN 201910716485A CN 110438172 B CN110438172 B CN 110438172B
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starch
oil
lignocellulose
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producing
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CN110438172A (en
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龚志伟
苟庆玲
赵觅
周文婷
刘义
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Jiangxi Carbon Gold Technology Co ltd
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Wuhan University of Science and Engineering WUSE
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • 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
    • 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

Abstract

The invention is suitable for the technical field of oil-producing microorganism culture, and provides a method for producing oil by co-utilizing starch and lignocellulose raw materials. The method is simple and efficient to operate, well solves the outstanding problems of low oil content and oil yield and complex process flow in the current lignocellulose raw material fermentation process, obviously reduces the production cost of microbial oil, improves the oil yield and the oil yield, and has obvious comprehensive technical advantages.

Description

Method for producing grease by co-utilizing starch and lignocellulose raw materials
Technical Field
The invention belongs to the technical field of culture of oleaginous microorganisms, and particularly relates to a method for producing grease by co-utilizing starch and lignocellulose raw materials.
Background
In nature, some oleaginous yeasts can synthesize and store a large amount of oil in vivo under specific conditions by using various cheap carbohydrates as carbon sources, and all yeasts which can accumulate oil in cells and exceed 20% of dry cell weight (w/w, wherein w/w represents mass ratio, the same below) are called oleaginous yeasts. Some oleaginous yeast accumulate oil even in excess of 70% of their dry cell weight. The main component of the oil and fat produced by the oil-producing yeast is triglyceride, the fatty acid composition is similar to that of commercial animal and vegetable oil and fat, and C is16-C18Long chain fatty acids are predominant. Compared with animal and vegetable oil, the microbial oil has short production period, is not limited by seasons and climate, has wide raw material source, basically does not occupy additional cultivated land resources, is easy to realize mass production, and is a novel oil resource with great potential. The microbial oil not only can be used as a substitute of edible oil or other functional oil, but also can provide a raw material for the sustainable development of the biodiesel industry.
Lignocellulosic feedstocks are the most abundant resource in nature, with abundant carbohydrate resources. Wherein, the annual output of various crop straws including corn straws, rice straws, wheat straws, cotton straws, rape straws and the like in China exceeds 8 hundred million tons, and the crop straws become wastes which are not useful but need to be treated. For a long time, most of the waste materials are burnt on the spot except for a small amount of waste materials used for gaskets and livestock feeding, and a part of the waste materials used for composting, which not only wastes resources, but also causes serious environmental pollution. The chemical components of lignocellulose are mainly cellulose, hemicellulose and lignin. The cellulose and hemicellulose therein may be converted to glucose and other fermentable sugars (xylose, arabinose, mannose, etc.) by enzymatic hydrolysis, which may be further bioconverted to liquid biofuels and bio-based chemicals. The lignocellulose hydrolysate is used for culturing the oil-producing microorganisms, so that the raw material cost can be greatly reduced, and the raw material for preparing the microbial oil in a large scale is ensured. However, the current cultivation of oleaginous microorganisms using straw hydrolysate has the outstanding problem of low oil yield. To induce an excessive accumulation of oil in oleaginous yeast, it is generally necessary to lack essential components or culture conditions for the growth and propagation of oleaginous microorganisms, such as nitrogen, phosphorus, sulfur, iron, zinc, and oxygen, in the culture medium. Currently, nitrogen limitation is the most common strategy to achieve large oil accumulation by oleaginous microorganisms. In the process of oil fermentation of oil-producing yeast, with the exhaustion of a nitrogen source, the growth of the strain is inhibited, and carbohydrates are mainly guided to the accumulation of the oil. The lignocellulose raw material generally contains a certain amount of nitrogen source, and in addition, a certain amount of nitrogen source is introduced by adding hydrolytic enzyme in the degradation process, so that the C/N ratio in the hydrolytic solution is lower. The denitrification process not only increases the process flow, but also causes the loss of carbohydrate. The oil-producing microorganisms in the hydrolysate mainly proliferate thallus, and the oil yield are low. In addition, the production of microbial oil from lignocellulose raw material needs a plurality of process links such as pretreatment, cellulase production, enzyme hydrolysis, oil fermentation and oil extraction, and the process flow is complex, and the production cost is very high.
The mode of producing microbial oil by using lignocellulose raw material by using oil producing yeast can be divided into step-by-Step Hydrolysis Fermentation (SHF), Synchronous Saccharification Fermentation (SSF), biological processing (CBP) and the like according to the degree of process integration. SHF, enzyme hydrolysis and oil fermentation are carried out separately, so that the enzyme hydrolysis and the oil fermentation can be carried out under the optimal condition, the viscosity of the material is reduced by full saccharification, and the transportation, fermentation and stirring are facilitated; the defects are that the needed equipment is various, the operation is complex, the enzymatic hydrolysate inhibits the activity of the cellulase in the later stage of saccharification, the enzymatic hydrolysis efficiency is reduced, the production period is long, and the total production efficiency is low. SSF is to carry out the enzymatic hydrolysis and the grease fermentation of lignocellulose in one reactor at the same time, and the product of the enzymatic hydrolysis can be consumed by microorganisms in time, so that the accumulation of the product can not be caused, and the equipment investment is obviously reduced. The disadvantages are that hydrolysis and fermentation are carried out under suboptimal conditions, enzymatic hydrolysis becomes the rate-limiting step in the early stage of fermentation, sugar supply produced by enzymatic hydrolysis is seriously insufficient, and strain fermentation is inhibited. To solve this problem, a prehydrolysis process is usually introduced before SSF, i.e., prehydrolysis is performed for a period of time at the optimum temperature and pH of cellulase to produce fermentable sugars at a certain concentration, then the temperature and pH are adjusted to conditions suitable for cell growth, and oleaginous yeast is inoculated for simultaneous saccharification and fermentation. This method combines the advantages of SHF and SSF, but is complex to operate. In addition, although the technology simplifies the process flow, the technology still does not solve the outstanding problems of low oil yield and low yield in the process of preparing oil from lignocellulose. CBP highly integrates enzyme production, lignocellulose enzyme hydrolysis and grease fermentation, and can obviously reduce process cost and equipment cost. However, oleaginous yeasts with CBP potential are very rare.
In order to utilize lignocellulose resources more efficiently, a simpler and economic method needs to be found, the process flow is simplified through synchronous saccharification and fermentation, the oil yield and the oil yield are obviously improved, and the technical economy of microbial oil is improved.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a method for producing oil by co-using starch and lignocellulose raw materials, which aims to solve the problems of complex process flow, low oil yield and low oil yield of the existing synchronous saccharification and fermentation method.
The method for producing the grease by utilizing the starch and the lignocellulose raw materials comprises the following steps:
step S1: pretreating a lignocellulose raw material;
step S2: adding starch and the lignocellulose raw material pretreated in the step S1 into a reactor respectively, adding a proper amount of nutrient substances, adjusting the pH value in the reactor, and sterilizing to obtain a starch-lignocellulose mixed culture medium;
step S3: culturing the grease yeast of Sdaffia in a seed culture medium to obtain an oil-producing microorganism seed solution;
step S4: inoculating the oil-producing microorganism seed liquid obtained in the step S3 into the starch-lignocellulose mixed culture medium obtained in the step S2, adding a certain amount of cellulase, performing aeration culture at a certain temperature for a period of time, stopping fermentation when the total concentration of carbon sources in the culture medium is lower than 5g/L, collecting a solid phase containing the oil-producing yeast through solid-liquid separation, and extracting oil from the collected solid phase.
Specifically, in step S2, the solid-liquid mass ratio of the starch is 1% to 5%, the carbon source contained in the starch accounts for 5% to 50% of the total carbon source in the mixed culture medium, the solid-liquid mass ratio of the lignocellulose raw material is 5% to 15%, the carbon source contained in the lignocellulose accounts for 50% to 95% of the total carbon source in the mixed culture medium, and the carbon-nitrogen molar ratio C/N in the mixed culture medium is controlled to be 60% to 300.
Specifically, in step S2, the nutrient includes one or more of a nitrogen source, a phosphorus source, and a sulfur source, the addition amount of the nitrogen source accounts for 0.01% to 0.1% of the total mass of the mixed culture medium, the addition amount of the phosphorus source accounts for 0.01% to 0.05% of the total mass of the mixed culture medium, the addition amount of the sulfur source accounts for 0.01% to 0.05% of the total mass of the mixed culture medium, and the pH in the reactor is adjusted to 4.0 to 7.0.
Specifically, in step S4, the volume of the oleaginous microorganism seed solution accounts for 2% -20% of the volume of the mixed culture medium, the addition amount of the cellulase is 5-25FPU/g lignocellulose, and the culture temperature is controlled between 30 ℃ and 34 ℃.
Specifically, in step S1, the lignocellulosic feedstock is a cheap biomass feedstock containing cellulose, hemicellulose and lignin, and includes one or more of corn stover, corn cobs, wheat straw, rice straw, cotton stalks, sorghum stalks, and rape stalks.
Specifically, in step S1, the pre-treatment of the lignocellulosic feedstock comprises one or more of physical, chemical, physicochemical, and biological treatment.
Specifically, in step S2, the starch includes one or more of tapioca starch, corn starch, sweet potato starch, and potato starch.
Specifically, in step S4, the extracted fat or oil is one or more kinds of fat or oil of long-chain fatty acids and derivatives thereof.
The invention has the beneficial effects that:
1. in the invention, starch and lignocellulose raw materials are utilized together, so that the coupling of starch integrated biological processing (high integration of production of amylase and fermentation of fermentable sugar and oil-producing yeast oil prepared by hydrolyzing starch with enzyme) and lignocellulose synchronous saccharification oil fermentation is realized, and compared with the method for producing microbial oil by respectively using starch and lignocellulose as raw materials, the process cost and the equipment cost are obviously reduced;
2. the grease yeast of the invention can directly utilize starch without adding amylase and saccharifying enzyme, in the initial fermentation stage, the grease yeast of the invention can generate amylase to hydrolyze starch rapidly, more fermentable sugar is provided for the oil-producing yeast to be utilized, the grease fermentation efficiency in the initial fermentation stage is improved, the pre-hydrolysis process of lignocellulose SSF is not needed by adopting the mode of common utilization of starch and lignocellulose, and the process flow is simplified obviously;
3. the starch raw material and the lignocellulose hydrolysate are mixed to prepare a starch-lignocellulose mixed culture medium, and the C/N ratio is adjusted to a reasonable level, so that the carbon source in the starch and the lignocellulose is mainly guided to oil synthesis, and the mass accumulation of the oil is realized;
the method adopted by the invention is very easy to realize, the process flow is obviously simplified, the equipment cost is reduced, the production efficiency, the oil yield and the oil yield of the oil are improved, the technical economy of microbial oil production is improved, and the method has wide application prospect.
Drawings
FIG. 1 is a process flow diagram of the method for producing oil by co-utilizing starch and lignocellulose raw materials according to the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a method for producing grease by utilizing starch and lignocellulose raw materials, which comprises the following steps:
step S1: pretreating the lignocellulosic feedstock.
In the step, the lignocellulose raw material is a cheap biomass raw material containing cellulose, hemicellulose and lignin, and comprises one or more of corn stalks, corncobs, wheat stalks, straws, cotton stalks, sorghum stalks and rape stalks, and the pretreatment method adopted by the lignocellulose raw material comprises one or more of a physical method, a chemical method, a physicochemical method and a biological method.
Step S2: and (3) preparing a starch-lignocellulose mixed culture medium.
Adding starch and the lignocellulose raw material pretreated in the step S1 into a reactor respectively, adding a proper amount of nutrient substances, adjusting the pH value in the reactor, and sterilizing to obtain the starch-lignocellulose mixed culture medium.
In the step, the starch comprises one or more of cassava starch, corn starch, sweet potato starch and potato starch, wherein the solid-liquid mass ratio of the starch is 1% -5%, a carbon source contained in the starch accounts for 5% -50% of the total carbon source mass in the mixed culture medium, the solid-liquid mass ratio of the lignocellulose raw material is 5% -15%, a carbon source contained in the lignocellulose accounts for 50% -95% of the total carbon source mass in the mixed culture medium, and the carbon-nitrogen molar ratio C/N in the mixed culture medium is controlled to be 60-300.
In the step, the nutrient substances comprise one or more of a nitrogen source, a phosphorus source and a sulfur source, the addition amount of the nitrogen source accounts for 0.01-0.1% of the total mass of the mixed culture medium, the addition amount of the phosphorus source accounts for 0.01-0.05% of the total mass of the mixed culture medium, the addition amount of the sulfur source accounts for 0.01-0.05% of the total mass of the mixed culture medium, the pH value in the reactor is adjusted to 4.0-7.0, and a high-temperature damp-heat sterilization method is adopted for culture medium sterilization.
Step S3: and (3) preparing an oil-producing yeast seed liquid.
In an aseptic operation environment, the grease yeast sidajie is inoculated in a liquid seed culture medium with rich nutrition, and is aerated and cultured for 24-48 h at the temperature of 25-30 ℃ to obtain the oil-producing yeast seed liquid, wherein the liquid seed culture medium takes glucose or starch as a main carbon source and energy source, and then a proper amount of other nutrient components such as yeast powder, peptone, corn steep liquor and the like are added.
The grease yeast of the stanza adopted in the step can be directly purchased from strain preservation organizations such as China general microbiological culture Collection center (CGMCC), China Industrial microbiological culture Collection center (CICC), American Type Culture Collection (ATCC), American agricultural research strain preservation center (NRRL), UKNCC, Dutch microbial culture Collection Center (CBS), Germany microbial culture Collection center (DSMZ) and the like or separated from the natural world, and can also use artificial or natural mutant strains with different properties from the original strains.
Step S4: starch-lignocellulose co-fermentation.
Inoculating the oil-producing yeast seed liquid into a starch-lignocellulose mixed culture medium, adding a certain amount of cellulase, performing aeration culture at a certain temperature for a period of time, stopping fermentation when the total concentration of carbon sources in the culture medium is lower than a certain value, and collecting a solid phase containing the oil-producing yeast through solid-liquid separation.
In the step, the volume of the oleaginous microorganism seed solution accounts for 2-20% of the volume of the mixed culture medium, the addition amount of the cellulase is 5-25FPU/g lignocellulose, the culture temperature is controlled between 30-34 ℃, when the total concentration of carbon sources in the culture medium is lower than 5g/L, the fermentation is stopped, solid-liquid separation is carried out to collect a solid phase containing oleaginous yeast, and the solid-liquid separation can adopt a centrifugation method or a filtration method.
Step S5: extracting microbial oil.
The solid phase obtained in the above step is a mixture of microbial cells and lignocellulose residues, oil is extracted from the mixture, the yield of microbial oil is calculated, the microbial oil is mainly glyceride of long-chain fatty acid and derivatives thereof, and the microbial oil can be made into biodiesel after transesterification, for example, biodiesel is prepared by transesterification under the condition of acid catalysis (the process is a conventional preparation process, and is not described herein any more), and in addition, unsaturated fatty acid contained in the microbial oil can also be used for preparing other products with high added values.
The present invention is illustrated by the following specific examples, which are examples of culturing typical oleaginous microorganisms from typical straw and starch feedstocks to aid in understanding the present invention, but are not intended to limit in any way the application of the present invention to other materials or oleaginous strains.
Example 1:
1. weighing 40g of corn straw sieved by a 40-mesh sieve, and adding 80mL of NaOH-CH3In OH solution, pre-treated at 80 ℃ for 1h, and then evaporated for 1h at the same temperature to remove CH3OH；
2. Preparing 20g/L cassava starch and 80g/L pretreated corn straw, controlling the C/N ratio to 199, adjusting the pH value of an oil-producing microorganism culture medium to 6.0, and sterilizing at 121 ℃ for 15min to obtain a starch-lignocellulose mixed culture medium;
3. culturing Lipomyces starkeyi AS 2.1560 (China general microbiological culture Collection center) in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and performing shake culture at 28 ℃ and 200rpm for 40h to obtain an oil-producing microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the starch-lignocellulose mixed culture medium obtained in the step 2, wherein the inoculation amount is 10% (v/v), the addition amount of cellulase is 15FPU/g straw, performing aeration culture at 30 ℃ for 96 hours, terminating fermentation, the concentration of residual glucose in fermentation liquor is 0.6g/L, collecting a solid phase containing oil-producing yeast through solid-liquid separation, obtaining 37g/L of dry bacteria and residual straw, extracting oil by a conventional method, and obtaining the yield of the oil of 16.9 g/L.
Example 2:
1. weighing 40g of corn straw sieved by a 40-mesh sieve, and adding 80mL of NaOH-CH3In OH solution, pre-treated at 80 ℃ for 1h, and then evaporated for 1h at the same temperature to remove CH3OH；
2. Preparing 50g/L of cassava starch and 150g/L of pretreated corn straw, controlling the C/N ratio to be 300, adjusting the pH value of an oil-producing microorganism culture medium to be 6.0, and sterilizing for 15min at 121 ℃ to obtain a starch-lignocellulose mixed culture medium;
3. culturing Lipomyces starkeyi AS 2.1560 (China general microbiological culture Collection center) in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and performing shake culture at 30 ℃ and 200rpm for 40h to obtain an oil-producing microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the starch-lignocellulose mixed culture medium obtained in the step 2, wherein the inoculation amount is 15% (v/v), the addition amount of cellulase is 25FPU/g straw, performing aeration culture at 30 ℃ for 180 hours, terminating fermentation, the concentration of residual glucose in fermentation liquor is 0.6g/L, collecting a solid phase containing oil-producing yeast through solid-liquid separation, obtaining 77g/L of dry bacteria and residual straw, and extracting oil by adopting a conventional method, wherein the yield of the obtained oil is 31.2 g/L.
Example 3:
1. referring to the literature (ZHao J, Xia LM. Fuel Process Technol,2009,90: 1193-1197), 100g of straw sieved with a 20-mesh sieve was weighed, added with 0.8L of 0.5mol/L NaOH solution at a solid-to-liquid ratio of 1:8(w/w), and treated in a 80 ℃ water bath for 75 min;
2. preparing 20g/L potato starch and 80g/L pretreated straw stalk, controlling the C/N ratio to be 88, adjusting the pH value of an oil-producing microorganism culture medium to be 5.5, and sterilizing at 121 ℃ for 20min to obtain a starch-lignocellulose mixed culture medium;
3. culturing Lipomyces starkeyi NRRL 11558 (American agricultural research culture Collection) in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and performing shake culture at 28 ℃ and 200rpm for 40h to obtain an oil-producing microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the starch-lignocellulose mixed culture medium obtained in the step 2, wherein the inoculation amount is 5% (v/v), the addition amount of cellulase is 25FPU/g straw, performing aeration culture at 28 ℃ for 96 hours, terminating fermentation, the concentrations of residual glucose and reducing sugar in the fermentation liquid are 0.3g/L and 1.8g/L respectively, collecting the solid phase containing the oil-producing yeast through solid-liquid separation, obtaining 40.5g/L of dry bacteria and residual straw, and extracting oil by adopting a conventional method, wherein the yield of the obtained oil is 14.2 g/L.
Example 4:
1. referring to the literature (Ruan ZH, et al Biotechnol Bioeng,2013,110: 1039-1049), 50g of straw powder with a diameter of less than 1mm is weighed, 500mL of 2% (w/w) dilute sulfuric acid is added according to a solid-to-liquid ratio of 10% (w/w), and treated at 130 ℃ for 60 min;
2. preparing 30g/L sweet potato starch and 70g/L pretreated straw stalk, controlling the C/N ratio to be 125, adjusting the pH value of the oil-producing microorganism culture medium to be 5.0, and sterilizing at 121 ℃ for 20min to obtain a starch-lignocellulose mixed culture medium;
3. culturing Lipomyces starkeyi CBS 1807 (fungus polymorphism center of royal academy of science and art of the Netherlands) in a seed culture medium (20 g/L of glucose, 10g/L of yeast powder and 10g/L of peptone), and performing shake culture at 25 ℃ and 200rpm for 24h to obtain an oil-producing microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the starch-lignocellulose mixed culture medium obtained in the step 2, wherein the inoculation amount is 10% (v/v), the addition amount of cellulase is 20FPU/g straw, performing aeration culture at 30 ℃ for 120h, terminating fermentation, the concentrations of residual glucose and reducing sugar in the fermentation liquid are 0g/L and 1.2g/L respectively, collecting a solid phase containing oil-producing yeast through solid-liquid separation to obtain 42.3g/L of dry bacteria and residual straw, and extracting oil by a conventional method, wherein the yield of the obtained oil is 15.6 g/L.
Example 5:
1. weighing 50g of wheat straw which is sieved by a 20-mesh sieve, adding 2.5% (w/w) sulfuric acid according to a solid-to-liquid ratio of 100% (w/w), soaking for 24h, and then carrying out steam explosion treatment for 3min under water vapor at 190 ℃;
2. preparing 50g/L corn starch and 50g/L pretreated wheat straw, controlling the C/N ratio to be 245, adjusting the pH value of an oil-producing microorganism culture medium to be 4.8, and sterilizing for 20min at 121 ℃ to obtain a starch-lignocellulose mixed culture medium;
3. culturing Lipomyces starkeyi NRRL 11557 (American agricultural research culture Collection) in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and performing shake culture at 30 ℃ and 200rpm for 48h to obtain an oil-producing microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the starch-lignocellulose mixed culture medium obtained in the step 2, wherein the inoculation amount is 20% (v/v), the addition amount of cellulase is 5FPU/g straw, performing aeration culture at 30 ℃ for 120h, terminating fermentation, the concentrations of residual glucose and reducing sugar in the fermentation liquid are 0g/L and 0.6g/L respectively, collecting a solid phase containing oil-producing yeast through solid-liquid separation to obtain dry bacteria and residual straw which are 40.9g/L in total, and extracting oil by adopting a conventional method, wherein the yield of the obtained oil is 14.7 g/L.
Example 6:
1. in reference to the literature (Balan V, et al, Biotechnol Progr,2009,25: 365-;
2. preparing 40g/L cassava starch and 60g/L pretreated rape stems, controlling the C/N ratio to be 60, adjusting the pH value of an oil-producing microorganism culture medium to be 6.0, and sterilizing at 121 ℃ for 20min to obtain a starch-lignocellulose mixed culture medium;
3. culturing Lipomyces starkeyi ATCC 56304 (American type culture Collection) in seed culture medium (glucose 20g/L, yeast powder 10g/L, peptone 10g/L), and performing shake culture at 25 deg.C and 200rpm for 36h to obtain oleaginous microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the starch-lignocellulose mixed culture medium obtained in the step 2, wherein the inoculation amount is 15% (v/v), the addition amount of cellulase is 5FPU/g rape stalk, performing aeration culture at 30 ℃ for 108 hours, terminating fermentation, the concentrations of residual glucose and reducing sugar in the fermentation liquid are 0.3g/L and 1.1g/L respectively, collecting the solid phase containing the oil-producing yeast through solid-liquid separation, obtaining 41.6g/L of dry bacteria and residual straw, and extracting oil by adopting a conventional method, wherein the yield of the obtained oil is 12.5 g/L.
Example 7:
1. in reference to the literature (Zhang J, et al BioResourcer Technol,2011,102: 4480-4488), cotton stalks were washed, dried, crushed through a 2mm sieve, and subjected to a trace acid "dry" pretreatment;
2. preparing 50g/L cassava starch and 150g/L pretreated cotton stalks, controlling the C/N ratio to be 300, adjusting the pH value of an oil-producing microorganism culture medium to be 4.0, and sterilizing at 121 ℃ for 20min to obtain a starch-lignocellulose mixed culture medium;
3. culturing Lipomyces starkeyi ATCC 56304 (American type culture Collection) in seed culture medium (glucose 20g/L, yeast powder 10g/L, peptone 10g/L), and performing shake culture at 30 deg.C and 200rpm for 36h to obtain oleaginous microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the starch-lignocellulose mixed culture medium obtained in the step 2, wherein the inoculation amount is 15% (v/v), the addition amount of cellulase is 10FPU/g cotton stalk, performing aeration culture at 30 ℃ for 192 hours, terminating fermentation, the concentrations of residual glucose and reducing sugar in the fermentation liquid are 0.1g/L and 0.7g/L respectively, collecting the solid phase containing the oil-producing yeast through solid-liquid separation, obtaining 75.2g/L of dry bacteria and residual straw, and extracting oil by adopting a conventional method, wherein the yield of the obtained oil is 27.5 g/L.
Example 8:
1. drying and crushing the corncobs, sieving the corncobs by a 20-mesh sieve, adding 20g/L NaOH solution according to a solid-to-liquid ratio of 1:8(w/v), treating the corncobs in a water bath kettle at 80 ℃ for 1 hour, and washing the corncobs by deionized water after the treatment is finished;
2. preparing 10g/L cassava starch and 50g/L pretreated corncobs, controlling the C/N ratio to be 300, adjusting the pH value of an oil-producing microorganism culture medium to be 4.0, and sterilizing at 121 ℃ for 20min to obtain a starch-lignocellulose mixed culture medium;
3. culturing Lipomyces starkeyi ATCC 56304 (American type culture Collection) in seed culture medium (glucose 20g/L, yeast powder 10g/L, peptone 10g/L), and performing shake culture at 30 deg.C and 200rpm for 36h to obtain oleaginous microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the starch-lignocellulose mixed culture medium obtained in the step 2, wherein the inoculation amount is 2% (v/v), the addition amount of cellulase is 10FPU/g corncob, performing aeration culture at 34 ℃ for 84 hours, terminating fermentation, the concentrations of residual glucose and reducing sugar in the fermentation liquid are 0g/L and 0.2g/L respectively, collecting a solid phase containing oil-producing yeast through solid-liquid separation, obtaining 28.5g/L of dry thalli and residual corncob, and extracting oil by adopting a conventional method, wherein the yield of the obtained oil is 10.3 g/L.
Comparative example 1:
1. weighing 40g of corn straw sieved by a 40-mesh sieve, and adding 80mL of NaOH-CH3In OH solution, pre-treated at 80 deg.C for 1h, and then evaporated at the same temperature for 1h to remove CH3OH；
2. Adjusting pH of oleaginous microorganism culture medium to 5.0, controlling C/N ratio to 300, adjusting solid-to-liquid ratio to 10% (w/w), and sterilizing at 121 deg.C for 20 min;
3. culturing Lipomyces starkeyi AS 2.1560 (China general microbiological culture Collection center) in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and performing shake culture at 28 ℃ and 200rpm for 48h to obtain an oil-producing microorganism seed solution;
4. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the oil-producing microorganism culture medium obtained in the step 2, wherein the inoculation amount is 10% (v/v), the addition amount of cellulase is 15FPU/g straw, performing aeration culture at 30 ℃ for 144h, terminating fermentation, and at the moment, no glucose or xylose can be detected in the fermentation liquid, collecting a solid phase containing oil-producing yeast through solid-liquid separation to obtain 32.3g/L of dry thalli and residual straw, and extracting oil by adopting a conventional method, wherein the yield of the obtained oil is 5.8 g/L.
Comparative example 2:
1. preparing 50g/L cassava starch, controlling the C/N ratio to be 1158, adjusting the pH value of an oil-producing microorganism culture medium to be 6.0, and sterilizing for 15min at 121 ℃ for later use;
2. culturing Lipomyces starkeyi AS 2.1560 (China general microbiological culture Collection center) in a seed culture medium (20 g/L glucose, 10g/L yeast powder and 10g/L peptone), and performing shake culture at 28 ℃ and 200rpm for 40h to obtain an oil-producing microorganism seed solution;
3. inoculating the oil-producing microorganism seed solution obtained in the step 3 into the oil-producing microorganism culture medium obtained in the step 1, wherein the inoculation amount is 10% (v/v), performing aeration culture at 30 ℃ for 120h, terminating fermentation, collecting starch with the residual sugar concentration of 14.5g/L equivalent in the fermentation liquid at the moment, performing solid-liquid separation to collect the solid phase containing the oil-producing yeast to obtain 7.0g/L of dry bacteria, and extracting oil by using a conventional method, wherein the yield of the obtained oil is 4.9 g/L.
The embodiment and the comparative example show that the microbial oil is produced by singly adopting starch or lignocellulose as raw materials, the yield of the obtained oil is lower, and the yield of the obtained oil is greatly improved by utilizing the starch and the lignocellulose as common raw materials, because the starch raw materials are lack of nitrogen sources, the C/N ratio is higher, the lignocellulose hydrolysate is rich in the nitrogen sources and the C/N ratio is lower.
In addition, in the embodiment of the invention, the adopted grease yeast of Sdaffia can directly utilize starch without adding amylase and saccharifying enzyme additionally, in the early stage of fermentation, the lipase generated by the grease yeast of Sdalbergia can quickly hydrolyze starch, generate more fermentable sugars for the utilization of the oil-producing yeast, improve the grease fermentation efficiency in the early stage of fermentation, by adopting the mode of co-utilizing starch and lignocellulose, the prehydrolysis process of the lignocellulose SSF is not needed any more, the process flow is obviously simplified, simultaneously realizes the coupling of starch integrated biological processing (high integration of the production of amylase and the preparation of fermentable sugar and the fermentation of oil-producing yeast oil by hydrolyzing starch with enzyme) and the fermentation of lignocellulose synchronous saccharification oil, compared with the method for producing the microbial grease by respectively using starch and lignocellulose as raw materials, the method has the advantages that the process cost and the equipment cost are obviously reduced.
The invention provides a method for producing grease by utilizing starch and lignocellulose raw materials together, which has simple and efficient operation, well solves the outstanding problems of low grease content and grease yield and complex process flow in the current lignocellulose raw material fermentation process, obviously reduces the production cost of microbial grease, improves the grease yield and the grease yield, and has obvious comprehensive technical advantages.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (8)

1. A method for producing grease by co-utilizing starch and lignocellulose raw materials, which is characterized by comprising the following steps:
step S1: pretreating a lignocellulose raw material;
step S2: adding starch and the lignocellulose raw material pretreated in the step S1 into a reactor respectively, adding a proper amount of nutrient substances, adjusting the pH value in the reactor, and sterilizing to obtain a starch-lignocellulose mixed culture medium;
step S3: culturing the grease yeast of Sdaffia in a seed culture medium to obtain an oil-producing microorganism seed solution;
step S4: inoculating the oil-producing microorganism seed liquid obtained in the step S3 into the starch-lignocellulose mixed culture medium obtained in the step S2, adding a certain amount of cellulase, performing aeration culture at a certain temperature for a period of time, stopping fermentation when the total concentration of carbon sources in the culture medium is lower than 5g/L, collecting a solid phase containing oil-producing yeast through solid-liquid separation, and extracting oil from the collected solid phase;
in the step S2, the solid-liquid mass ratio of the starch is 1-5%, the carbon source contained in the starch accounts for 5-50% of the total carbon source mass in the mixed culture medium, the solid-liquid mass ratio of the lignocellulose raw material is 5-15%, the carbon source contained in the lignocellulose accounts for 50-95% of the total carbon source mass in the mixed culture medium, and the carbon-nitrogen molar ratio C/N in the mixed culture medium is controlled to be 60-300.
2. The method for producing fats and oils according to claim 1, wherein the nutrients comprise one or more of a nitrogen source, a phosphorus source, and a sulfur source, the nitrogen source is added in an amount of 0.01 to 0.1% by mass, the phosphorus source is added in an amount of 0.01 to 0.05% by mass, and the sulfur source is added in an amount of 0.01 to 0.05% by mass, based on the total mass of the mixed medium, in step S2, and the pH in the reactor is adjusted to 4.0 to 7.0.
3. The method for producing fats and oils according to claim 1, wherein the volume of said oleaginous microorganism seed solution is 2 to 20% of the volume of the mixed culture medium, the cellulase is added in an amount of 5 to 25FPU/g lignocellulose, and the culture temperature is controlled between 30 ℃ and 34 ℃ in step S4.
4. The method for producing fats and oils according to claim 1, wherein the lignocellulosic raw material is an inexpensive biomass raw material containing cellulose, hemicellulose and lignin, comprising one or more of corn stover, corn cob, wheat straw, rice straw, cotton stalk, sorghum stalk, and rape stalk in step S1.
5. The method for producing fats and oils by co-using starch and lignocellulosic feedstocks according to claim 1, wherein the pretreatment of the lignocellulosic feedstocks in step S1 comprises one or more of physical, chemical, physicochemical, and biological treatment.
6. The method for producing fats and oils by co-using starch and lignocellulosic feedstocks according to claim 1, wherein the starch comprises one or more of tapioca starch, corn starch, sweet potato starch, potato starch in step S2.
7. The method for producing an oil and fat by using starch and a lignocellulosic raw material in combination as claimed in claim 1, wherein the oleaginous microorganism is oleaginous yeast of the S3, i.e., oleaginous yeast of the S.
8. The method for producing fats and oils by using starch and lignocellulosic raw materials in combination according to claim 1, wherein the fats and oils obtained by extraction in step S4 are fats and oils of one or more long chain fatty acids and derivatives thereof.
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