CN112941120A - Method for producing microbial oil by using VFAs and lignocellulose raw materials - Google Patents
Method for producing microbial oil by using VFAs and lignocellulose raw materials Download PDFInfo
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Abstract
The invention is suitable for the technical field of oil-producing microorganism culture, and provides a method for producing microbial oil by using VFAs and lignocellulose raw materials, which directly neutralizes the lignocellulose raw materials pretreated by the VFAs and the lignocellulose raw materials pretreated by alkali, wherein the lignocellulose raw materials contain high-concentration VFAs and can be directly used as a carbon source for fermentation to produce oil; after the pretreated lignocellulose raw material is hydrolyzed by cellulase, the obtained sugar solution can be directly utilized by oil-producing microorganisms; the method has the advantages that the VFAs are utilized to break cells, and the residual VFAs in the liquid separated after the normal hexane is used for extracting the grease can be continuously used for preprocessing the lignocellulose raw material, so that the cost is greatly reduced, and the method has remarkable economic benefit.
Description
Technical Field
The invention belongs to the technical field of oil-producing microbial culture, and particularly relates to a method for producing microbial oil by using VFAs and lignocellulose raw materials.
Background
Some microorganisms in nature can synthesize and store a large amount of fats and oils in vivo under specific conditions using various carbon sources, and any microorganism which can accumulate fats and oils in cells and exceeds the dry weight of cells by 20% (w/w, where w/w denotes a mass ratio, the same applies hereinafter) is called an oleaginous microorganism. Some oleaginous yeast accumulate oil even in excess of 80% of their dry cell weight. The microbial oil, especially the oil produced by oleaginous yeast, has triglyceride as main component and fatty acid composition similar to that of commercial animal and vegetable oil, and has C14-C22 long chain fatty acid as main component. 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.
The production cost of the current microbial oil is still higher than that of vegetable oil and animal oil due to the reasons of overhigh cost of carbon sources used in the microbial fermentation process and the like. The industrial and agricultural wastes which have wide sources, rich content, low cost and sustainable development, such as wood fiber biomass, crude glycerol, VFAs, various waste water and the like, can be considered and utilized, so that the production cost of the microbial oil can be reduced, and the problem of environmental pollution is relieved.
For Volatile Fatty Acids (VFAs), the major component is acetic acid, followed by propionic and butyric acids. In recent years, VFAs generated by anaerobic fermentation of various organic wastes such as hydrogen production wastewater by anaerobic fermentation, food residues, human or animal excrement, industrial wastewater and waste activated sludge are gradually increased, and the VFAs generated by the wastes are converted into microbial oil with high added value by oleaginous microorganisms under certain culture conditions, so that the production cost of the oil can be reduced. And the relatively shorter route of conversion of VFAs to important precursors of fats and oils relative to carbohydrates, is a better candidate for fat accumulation.
However, when the concentration of VFAs as a carbon source material is higher than 10g/L, the VFAs adversely affect the cell growth of oleaginous microorganisms, and the utilization rate and oil yield of VFAs are drastically reduced. The adverse effects of VFAs on microbial cells are mainly caused by the non-dissociated form of the acid molecule. Lipophilic non-dissociable forms of VFAs permeate into the plasma membrane and dissociate intracellularly into hydrogen ions and the corresponding anions, leading to cellular acidification and stress on cellular metabolism. Oleaginous microorganisms have a series of regulatory mechanisms to control the entry and exit of hydrogen ions into and out of cells to maintain a near neutral intracellular environment. However, this capability is limited adaptation and adjustment. Therefore, if VFAs are used as a carbon source, the effect of producing microbial oils and fats by using VFAs is still not satisfactory from the viewpoint of the present technical idea.
In addition, there is also a method for producing microbial oils and fats by lignocellulose. Lignocellulose materials are widely available and are one of the most abundant renewable resources in nature, and the chemical components of the lignocellulose materials mainly comprise cellulose, hemicellulose and lignin, and huge carbohydrate resources are reserved. The main product of the complete hydrolysis of the cellulose is glucose; hydrolysis of hemicellulose can produce a mixture of xylose, arabinose, glucose, galactose, mannose, etc. The cheap lignocellulose raw materials mainly comprise agricultural wastes represented by corn stalks, corncobs, straws and wheat straws, forestry wastes represented by trees, twigs and sawdust, industrial wastes represented by bagasse, sweet sorghum residues and the like, and the like. However, current lignocellulosic feedstocks are less technically economical to bioconvert than traditional sugar feedstocks based on the degradation resistant properties of biomass. Lignocellulose generally needs to be pretreated to destroy the degradation-resistant structure of the lignocellulose, so that the enzymolysis efficiency of the lignocellulose is improved, and fermentable sugar is obtained. Pretreatment is a common problem in lignocellulosic bioconversion. The pretreatment process usually requires the addition of chemical reagents or higher energy consumption and higher cost.
Therefore, in the current oil-producing microbial method, high concentration of VFAs as carbon source has negative influence on cell growth and oil accumulation. The lignocellulose raw material is a carbon source, and has the outstanding problems of high production cost, difficult wastewater treatment, difficult resource utilization of byproducts and the like. In order to fully utilize VFAs and lignocellulose resources, a simpler and more economic method needs to be found, the oil yield and 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 is to provide a method for producing microbial oils and fats from VFAs and lignocellulosic feedstocks, which solves the problems of low efficiency and yield of microbial oils and fats from VFAs and high cost of microbial oils and fats from lignocellulosic feedstocks.
The invention adopts the following technical scheme:
the method for producing microbial grease by using VFAs and lignocellulose raw materials comprises the following steps:
step S1, preparing VFAs solution through anaerobic fermentation;
step S2, mixing the lignocellulose raw material with a certain amount of VFAs solution uniformly, carrying out high-temperature pretreatment, and then supplementing water to obtain an acidic regenerated lignocellulose raw material;
step S3, uniformly mixing the lignocellulose raw material with a certain amount of alkali solution, carrying out high-temperature pretreatment, and then supplementing water to obtain an alkaline regenerated lignocellulose raw material;
step S4, directly neutralizing the acidic and alkaline regenerated lignocellulose raw materials, adjusting the pH value to be neutral, and then performing sterilization treatment to obtain an oil-producing microorganism culture medium;
step S5, culturing oleaginous microorganisms in a seed culture medium to obtain oleaginous microorganism seed liquid;
step S6, inoculating the oil-producing microorganism seed liquid into the oil-producing microorganism culture medium for ventilation fermentation culture until the concentration of residual VFAs in the fermentation culture liquid is lower than 5 g/L;
step S7, adjusting the pH value, adding a proper amount of lignocellulose degrading enzyme, hydrolyzing the regenerated lignocellulose raw material into fermentable biomass sugar, and continuing fermentation until the concentration of residual total sugar in the fermentation liquor is lower than 5g/L, and then stopping fermentation;
and S8, performing solid-liquid separation to collect oil-producing microbial cells, treating the oil-producing microbial cells at high temperature by using a VFAs solution, cracking the cells, extracting oil by using an extracting agent, and returning to the step S2 to pretreat lignocellulose after the concentration of the VFAs is adjusted, wherein the VFAs is remained in the residual liquid phase after extraction.
Further, the step S1 specifically includes: selecting organic wastes from different sources, placing the organic wastes in a fermentation tank, adjusting the pH value to be 4.5-6.5 and the reaction temperature to be 30-40 ℃, carrying out anaerobic fermentation, and carrying out solid-liquid separation to obtain a VFAs solution with high concentration, wherein the VFAs solution contains short-chain VFAs capable of being used by oleaginous microorganisms.
Further, the VFAs solution is a mixture of short chain fatty acids including acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and n-valeric acid, in a total concentration of 10g/L to 70g/L, with the highest acetic acid content in the mixture, followed by propionic acid and butyric acid.
Further, in step S2, uniformly mixing the lignocellulose raw material and the VFAs solution according to the solid-liquid mass ratio of 10-30%, performing high-temperature pretreatment, and supplementing water until the solid-liquid mass ratio is 5-20% to obtain an acidic regenerated lignocellulose raw material; in the step S3, the lignocellulose raw material and an alkali solution with the mass concentration of 0.5-5% are uniformly mixed according to the solid-liquid mass ratio of 10-30%, the mixture is pretreated at high temperature, water is supplemented until the solid-liquid mass ratio is 5-20%, so that an alkaline regenerated lignocellulose raw material is obtained, and the alkali solution is prepared from calcium oxide or calcium hydroxide.
Further, in the steps S2 and S3, the temperature of the high-temperature pretreatment is 80-180 ℃ and the time is 15min-4 h.
Further, in step S6, the inoculation amount of the oil-producing microorganism seed liquid is 2-20%, the inoculation amount is the volume ratio, and the oil-producing microorganism seed liquid is aerated and cultured at the temperature of 20-37 ℃; in step S7, the pH is adjusted to 4-8.
Further, in step S8, the VFAs solution is treated at a high temperature of 70-85 ℃ to lyse the bacterial cells of the oleaginous microorganisms for 0.5-2 h.
Further, the lignocellulose degrading enzyme is an enzyme capable of reducing the polymerization degree of carbohydrate polymers, and comprises one or more of cellulase, beta-glucosidase, xylanase, beta-xylosidase and pectinase, and the fermentable biomass sugar obtained by hydrolysis comprises glucose and xylose, and further comprises arabinose, galactose, mannose, cellobiose and galacturonic acid.
The invention has the beneficial effects that: the lignocellulose raw material pretreated by VFAs and the lignocellulose raw material pretreated by alkali are directly neutralized, the pH is simply adjusted and the pretreated lignocellulose raw material is used as an oil-producing microorganism culture medium after sterilization, and the lignocellulose raw material is pretreated by VFAs, so that the lignocellulose raw material has a loose structure and various pores, can adsorb a certain amount of free VFAs, the relative concentration of VFAs molecules in fermentation liquor is reduced, and the free VFAs are slowly released to continue fermentation along with the change of fermentation time, therefore, the VFAs with lower concentration in the fermentation liquor can be kept to reduce the toxicity to cells, the fermentation efficiency is greatly improved, the mass accumulation of grease is realized, the subsequent detoxification and neutralization processes are reduced, and the operation process is simpler; in addition, the VFAs and the alkali solution respectively pretreat the medium to treat the lignocellulose raw material, so that the enzyme accessibility of the lignocellulose can be obviously improved; in addition, the VFAs can also be used for cracking the somatic cells and promoting the extraction of the grease, and a large amount of the VFAs remained in the liquid phase part can be recycled, so that the utilization efficiency of the VFAs is improved.
Therefore, the method for producing the microbial oil by using the VFAs and the lignocellulose raw materials has high oil conversion efficiency and high yield, can realize the recycling of the raw materials, greatly reduces the cost and has good application prospect.
Drawings
FIG. 1 is a flow diagram of the process for producing microbial greases using VFAs and lignocellulosic feedstocks in accordance with the present 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.
As shown in FIG. 1, the present invention provides a method for producing microbial oils and fats using VFAs and lignocellulosic feedstocks, comprising the steps of:
step S1, preparing VFAs solution by anaerobic fermentation.
Selecting organic wastes from different sources to be placed in a fermentation tank, wherein the organic wastes comprise one or more of food residues, human or animal excrement, activated sludge, high-content organic wastewater and the like to be subjected to anaerobic fermentation. Adjusting the pH value to 4.5-6.5, the reaction temperature to 30-40 ℃, carrying out anaerobic fermentation, and carrying out solid-liquid separation to obtain a VFAs solution with high concentration, wherein the VFAs solution contains volatile short-chain fatty acids which can be used by oil-producing microorganisms and comprise acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and n-valeric acid, the concentration of the VFAs can reach 10g/L-70g/L, the content of acetic acid in the mixture is highest, and the mass percentage content reaches 40% -70%, and then the VFAs solution is propionic acid and butyric acid.
And step S2, uniformly mixing the lignocellulose raw material with a certain amount of VFAs solution, carrying out high-temperature pretreatment, and then supplementing water to obtain the acidic regenerated lignocellulose raw material.
Taking a certain amount of VFAs solution, uniformly mixing the lignocellulose raw material and the VFAs solution according to the solid-liquid mass ratio of 10-30%, carrying out high-temperature pretreatment at the temperature of 80-180 ℃ for 15min-4h, and supplementing water until the solid-liquid mass ratio is 5-20% to obtain the acidic regenerated lignocellulose raw material.
Lignocellulose is a biomass material whose main components are cellulose, hemicellulose and lignin. The fertilizer comprises one or more than two of agricultural biomass such as corn stalks, corncobs, straws, rice husks, wheat stalks and the like, forestry biomass such as trees, branches, sawdust and the like, industrial biomass such as bagasse, beet pulp, sweet sorghum slag and the like, weeds such as water hyacinth, barnyard grass and the like, and energy plants such as switch grass, miscanthus and the like.
And step S3, uniformly mixing the lignocellulose raw material with a certain amount of alkali solution, carrying out high-temperature pretreatment, and then supplementing water to obtain the alkaline regenerated lignocellulose raw material.
Uniformly mixing the lignocellulose raw material and aqueous alkali with the mass concentration of 0.5-5% according to the solid-liquid mass ratio of 10-30%, carrying out high-temperature pretreatment at the temperature of 80-180 ℃ for 15min-4h, and supplementing water until the solid-liquid mass ratio is 5-20% to obtain the alkaline regenerated lignocellulose raw material, wherein the aqueous alkali is prepared from calcium oxide or calcium hydroxide.
And step S4, directly neutralizing the acidic and alkaline regenerated lignocellulose raw materials, adjusting the pH value to be neutral, and then performing sterilization treatment to obtain the oleaginous microorganism culture medium.
And step S5, culturing the oleaginous microorganism in a seed culture medium to obtain oleaginous microorganism seed liquid.
The oil-producing microorganisms are eukaryotic microorganisms with the bacterial oil content of more than 20% of the dry weight of cells after fermentation, and the oil-producing microorganisms comprise but are not limited to one of the following: lipid yeast (such as Trichosporon trichosporum cutaneum and Lipomyces starkeyi), Rhodotorula (such as Rhodosporidium toruloides, Rhodotorula glutinis, and Sporobolomyces roseus), Trichosporon (such as Trichosporon cutaneum), Cryptococcus (such as Cryptococcus albicans), Yarrowia lipolytica, and molds (such as Geotrichum robustum and Mortierella isabellina). The strains can be directly purchased from strain preservation organizations such as China general microbiological culture Collection center (CGMCC), American Type Culture Collection (ATCC), China Industrial microbial culture Collection center (CICC), UK national strain Collection center (UKNCC) and the like or separated from the nature, and artificial or natural mutant strains with different properties from the original strains can also be used.
And (3) inoculating the oleaginous microorganism into a seed culture medium, setting the temperature to be 30 ℃, and carrying out shaking culture at 200rpm for 24h to obtain oleaginous microorganism seed liquid.
And step S6, inoculating the oil-producing microorganism seed liquid into the oil-producing microorganism culture medium, and performing aeration fermentation culture until the concentration of residual VFAs in the fermentation culture liquid is lower than 5 g/L.
The inoculation amount of the oil-producing microorganism seed liquid is 2-20 percent, the inoculation amount is volume ratio, and the oil-producing microorganism seed liquid is aerated and cultured at the temperature of 20-37 ℃.
And step S7, adjusting the pH value to 4-8, adding a proper amount of lignocellulose degrading enzyme, hydrolyzing the regenerated lignocellulose raw material into fermentable biomass sugar, and continuing fermentation until the concentration of residual total sugar in the fermentation liquor is lower than 5g/L, and then stopping fermentation.
The lignocellulose degrading enzyme is an enzyme capable of reducing the polymerization degree of carbohydrate polymers, and comprises one or more of cellulase, beta-glucosidase, xylanase, beta-xylosidase and pectinase, and the fermentable biomass sugar obtained by hydrolysis contains glucose and xylose, and also contains arabinose, galactose, mannose, cellobiose and galacturonic acid.
And S8, performing solid-liquid separation to collect oil-producing microbial cells, treating the oil-producing microbial cells at high temperature to crack the cells by using a VFAs solution, extracting oil by using an extracting agent, remaining VFAs in the residual liquid phase after extraction, and returning to the step S2 to pretreat lignocellulose after adjusting the concentration of the VFAs.
After the fermentation is finished, the fermentation liquor is centrifuged and washed, and thallus precipitates are collected. Drying the thallus at 105 ℃ to constant weight to obtain dry thallus, extracting by using an acid-heat-organic solvent method to obtain grease, and calculating the grease content of the thallus. Specifically, a high-concentration VFAs solution is used for treating the bacterial cells for 0.5h-2h at the high temperature of 70-85 ℃, then normal hexane is used for extracting grease, a liquid phase is recycled, VFAs remain in the liquid phase, the concentration is adjusted (for example, the concentration of the VFAs is increased through concentration), and the VFAs returns to the step S2 to be recycled for pretreating lignocellulose. The microbial oil is mainly glyceride of long-chain fatty acid and its derivative, and can be made into biodiesel after transesterification. Unsaturated fatty acid and odd-carbon fatty acid contained in the microbial oil can also be used for preparing other high-added-value products.
In the invention, the regenerated lignocellulose raw material can adsorb a certain amount of free VFAs, so that the relative concentration of VFAs molecules in the fermentation liquid is reduced, the free VFAs are slowly released along with the change of the fermentation time, the cytotoxicity of the fermentation liquid is reduced, the fermentation efficiency is greatly improved, and the large-scale accumulation of the grease is realized. The VFAs solution and the alkali solution are used as pretreatment media to treat the lignocellulose raw material, so that the enzyme accessibility of the lignocellulose can be obviously improved, and the VFAs can be used as the pretreatment media of the lignocellulose and a carbon source of oil-producing microorganisms to be converted into microbial oil; the lignocellulose raw material is hydrolyzed and fermented at the same time, so that the glucose effect is reduced. The VFAs solution can also crack cells under high temperature condition to promote oil extraction. The liquid phase fraction, with a large amount of VFAs remaining, can be recycled for reuse in the pretreatment of lignocellulose and cell lysis.
Therefore, in conclusion, the method directly neutralizes the lignocellulose raw material pretreated by VFAs and the lignocellulose raw material pretreated by alkali, reduces the toxicity of high-concentration VFAs to cells, obtains a large amount of fermentable sugar through subsequent enzyme hydrolysis, and takes the VFAs and the sugar as carbon sources to strengthen the fermentation process of the grease so as to produce the microbial grease.
The present invention is illustrated below by means of specific examples and comparative examples. The following examples, which illustrate exemplary VFAs and lignocellulosic feedstocks as materials for culturing typical oleaginous microorganisms, aid in the understanding of the present invention, but are not intended to limit in any way the application of the present invention to other materials or oleaginous strains.
Comparative example 1
1) Placing the organic wastewater in a 6L fermentation tank with a jacket, adjusting the pH value of the fermentation tank to 5.5, adjusting the reaction temperature to 37 ℃, performing anaerobic fermentation for 48h, performing solid-liquid separation to obtain a high-concentration VFAs solution, diluting the VFAs solution to 10g/L, adjusting the pH to 7.0, and sterilizing the obtained fermentation liquor as a culture medium at 121 ℃ for 15min for later use;
2) inoculating oil and fat of Trichosporon trichosporum Cutaneotrichporon oleginosum ATCC 20509 (purchased from American type culture Collection), and performing shake culture at 30 ℃ and 200rpm for 24h to obtain a seed solution;
3) inoculating the oil-producing microorganism seed solution prepared in the step 2) into the culture medium obtained in the step 1), wherein the inoculation amount is 10% (v/v), and performing aeration culture at 43 ℃ for 72 h;
4) terminating fermentation, wherein the concentration of the VFAs remained in the fermentation liquid is 1.8g/L, the consumption rate of the VFAs is low in the fermentation process, and the thalli are collected by solid-liquid separation to obtain 2.0g/L of dry thalli;
5) treating dry bacteria with high concentration VFAs solution at 80 deg.C for 2 hr to lyse cells, and extracting with n-hexane to obtain oil with oil content of 0.9g/L and oil content of 45% (w/w).
Comparative example 2
1) Preparing corn stalk hydrolysate: weighing 100g of corn straw which is sieved by a 20-mesh sieve, adding the corn straw into 1L of 2% (w/w) dilute sulfuric acid, and pretreating at 120 ℃ for 60min, wherein the solid-to-liquid ratio is 10% (w/w); adjusting the solid-to-liquid ratio to 10% (w/w), adding cellulase 15FPU/g, beta-glucosidase 15CBU/g and xylanase 100U/g, adjusting pH to 4.8, and hydrolyzing in a water bath shaker at 50 ℃ and 200rpm for 48h to obtain hydrolysate; boiling the hydrolysate for 5min, vacuum filtering to obtain supernatant, and adjusting the concentrations of glucose and xylose to 22.1g/L and 15.5g/L, adjusting pH to 6.0, and sterilizing at 121 deg.C for 15 min;
2) inoculating oleaginous yeast C.oleginosum ATCC 20509 (purchased from American type culture Collection) into a seed culture medium (10 g/L of glucose, 5g/L of yeast powder and 5g/L of peptone), and performing shaking culture at 30 ℃ and 200rpm for 24 hours to obtain a seed solution;
3) inoculating the oil-producing microorganism seed solution prepared in the step (2) into the culture medium obtained in the step 1), wherein the inoculation amount is 10% (v/v), and performing aeration culture at 30 ℃ for 240 h;
4) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquid is 0g/L, and performing solid-liquid separation to collect thalli to obtain 12.8g/L dry thalli;
5) treating dry bacteria with 5% VFAs solution at 80 deg.C for 2 hr to lyse cells, and extracting with n-hexane to obtain oil with oil content of 5.5g/L and oil content of 43% (w/w).
Example 1:
1) preparing high-concentration VFAs and regenerated corn straws produced by anaerobic fermentation of waste peels: placing the waste pericarp in a 6L fermentation tank with a jacket, adjusting pH value of the fermentation tank to 5.5, reacting at 35 deg.C, performing anaerobic fermentation for 36h, and performing solid-liquid separation to obtain high-concentration VFAs solution containing VFAs 65 g/L; weighing 100g of corn straws sieved by a 20-mesh sieve, adding the corn straws into a VFAs solution according to a solid-liquid ratio of 20% (w/w), pretreating at 120 ℃ for 60min, and then adding water until the solid-liquid mass ratio is 10%, so as to obtain acidic regenerated straws; weighing 100g of corn straws which are sieved by a 20-mesh sieve, adding the corn straws into a calcium hydroxide solution with the concentration of 2% (w/w) according to the solid-to-liquid ratio of 10% (w/w), pretreating at 100 ℃ for 60min, and then adding water until the solid-to-liquid mass ratio is 5%, so as to obtain alkalescent regenerated straws; directly neutralizing the two regenerated straws, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 15min for later use;
2) lipomyces hircus C.oleginosus ATCC 20509 (purchased from American type culture Collection) is subjected to shaking culture at 30 ℃ and 200rpm for 24 hours in a liquid culture medium (10 g/L of glucose, 5g/L of yeast powder and 5g/L of peptone) to obtain a seed solution;
3) inoculating the oleaginous microorganism seed solution prepared in the step 2) into the oleaginous microorganism culture medium obtained in the step 1), wherein the inoculation amount is 10% (v/v), and performing aerobic culture at 30 ℃ for 120h, wherein the concentration of residual VFAs in the fermentation liquid is 0 g/L;
4) adding 20FPU/g of cellulase, 15CBU/g of beta-glucosidase and 100U/g of xylanase into the fermentation liquor obtained in the step 3), adjusting the pH to 4.8, ventilating and culturing at 30 ℃, and performing fermentation culture for 120 hours while hydrolyzing;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 0.7g/L, performing solid-liquid separation to collect thalli to obtain 20.7g/L dry thalli, treating the dry thalli with a high-concentration VFAs solution at the high temperature of 80 ℃ for 2h to crack cells, and then extracting oil with normal hexane, wherein the oil content is 11.4g/L and the oil content is 55.1% (w/w); obtaining a solution containing VFAs in the liquid phase, adjusting the concentration, and recycling the solution for pretreatment of lignocellulose and oil extraction by cell lysis.
Example 2
1) Preparation of high-concentration VFAs and regenerated straws produced by anaerobic fermentation of industrial wastewater: putting industrial wastewater into a 6L fermentation tank containing a jacket, adjusting the pH value of the fermentation tank to 5.5, adjusting the reaction temperature to 37 ℃, performing anaerobic fermentation for 30h, and performing solid-liquid separation to obtain a high-concentration VFAs solution containing 70g/L VFAs; weighing 100g of straw which is sieved by a 20-mesh sieve, adding the straw into VFAs solution according to the solid-liquid ratio of 10% (w/w), pretreating at 140 ℃ for 60min, and then adding water until the solid-liquid mass ratio is 5%, so as to obtain acidic regenerated straw; weighing 100g of straw which is sieved by a 20-mesh sieve, adding the straw into a calcium hydroxide solution with the concentration of 2% (w/w) according to the solid-to-liquid ratio of 10% (w/w), pretreating at 100 ℃ for 120min, and then adding water until the solid-to-liquid mass ratio is 5%, so as to obtain the alkaline regenerated straw; directly neutralizing two kinds of regenerated straws, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min;
2) sporobolomyces roseus IAM 13481 (purchased from JCM culture collection of Japan) is inoculated in a seed culture medium (30 g/L of glucose, 5g/L of ammonium sulfate, 0.5g/L of yeast powder, 1g/L of potassium dihydrogen phosphate and 0.5g/L of magnesium sulfate heptahydrate) and is subjected to shaking culture at 30 ℃ and 200rpm for 24 hours to obtain a microorganism seed solution;
3) inoculating 15% (v/v) of the oleaginous microorganism seed solution prepared in the step 2) into the oleaginous microorganism culture medium obtained in the step 1), and performing aerobic culture at 30 ℃ for 120h, wherein VFAs are not detected in the fermentation liquid;
4) adding 25FPU/g of cellulase into the fermentation liquor obtained in the step 3), adjusting the pH value to 4.8 by using 25CBU/g of beta-glucosidase, and performing aeration culture at the temperature of 30 ℃ while performing fermentation culture for 160 h;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 1g/L, performing solid-liquid separation to collect thalli to obtain 28.6g/L dry thalli, treating the dry thalli with a high-concentration VFAs solution at the high temperature of 80 ℃ for 2h to crack cells, and then extracting oil with n-hexane, wherein the oil content is 17.4g/L and the oil content is 60.8% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
Example 3
1) Preparing high-concentration VFAs and regenerated rice hulls produced by anaerobic fermentation of kitchen waste: placing kitchen garbage in a 6L fermentation tank with a jacket, adjusting the pH value of the fermentation tank to 5.5, adjusting the reaction temperature to 37 ℃, performing anaerobic fermentation for 40h, and performing solid-liquid separation to obtain a high-concentration VFAs solution containing 65g/L VFAs; weighing 100g of rice hulls, adding the rice hulls into a VFAs solution according to a solid-liquid ratio of 10% (w/w), pretreating at 120 ℃ for 120min, and then adding water until the solid-liquid mass ratio is 5% to obtain acidic regenerated rice hulls; weighing 100g of rice hulls, adding the rice hulls into a calcium hydroxide solution with the concentration of 5% (w/w) according to the solid-liquid ratio of 20% (w/w), pretreating at 100 ℃ for 60min, and then adding water until the solid-liquid mass ratio is 10%, so as to obtain alkaline regenerated rice hulls; directly neutralizing the two regenerated rice hulls, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 15min for later use;
2) geotrichum robustum CICC 1256 (purchased from China center for culture Collection of industrial microorganisms) is inoculated in YEPD seed culture medium (20 g/L glucose, 10g/L yeast powder and 20g/L peptone), and is subjected to shaking culture at 25 ℃ and 200rpm for 28h to obtain seed liquid;
3) inoculating 5% (v/v) of the oleaginous microorganism seed solution prepared in the step 2) into the oleaginous microorganism culture medium obtained in the step 1), and performing aerobic culture at 25 ℃ for 180 hours, wherein the concentration of residual VFAs in the fermentation liquid is 1.7 g/L;
4) adding 25FPU/g of cellulase into the fermentation liquor obtained in the step 3), adjusting the pH value to 4.8 by using 25CBU/g of beta-glucosidase, and performing aeration culture at the temperature of 30 ℃ while performing fermentation culture for 240 hours while performing hydrolysis;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 1.8g/L, performing solid-liquid separation to collect thalli to obtain 19.9g/L dry thalli, treating the dry thalli with a high-concentration VFAs solution at a high temperature of 78 ℃ for 1.5h to crack cells, and then extracting oil with normal hexane, wherein the oil content is 10.5g/L and the oil content is 52.8% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
Example 4
1) Preparation of high-concentration VFAs and regenerated wheat straw produced by anaerobic fermentation of industrial wastewater: VFAs are prepared in the step 1) of the example 2, 80g of wheat straw with the diameter less than 1mm is weighed, added into a VFAs solution according to the solid-liquid ratio of 10% (w/w), pretreated for 80min at 130 ℃, and then added with water until the solid-liquid mass ratio is 5%, so that the acidic regenerated wheat straw is obtained; weighing 80g of wheat straw with the diameter less than 1mm, adding the wheat straw into a calcium oxide solution with the concentration of 5% (w/w) according to the solid-liquid ratio of 10% (w/w), pretreating at 120 ℃ for 70min, and adding water until the solid-liquid mass ratio is 5%, thereby obtaining alkaline regenerated wheat straw; directly neutralizing two kinds of regenerated wheat straw, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min;
2) trichosporon cutaneum AS 2.571 (purchased from China general microbiological culture Collection center) is inoculated in a liquid seed culture medium (20 g/L glucose, 5g/L yeast powder and 5g/L peptone), and is subjected to shaking culture at 32 ℃ and 200rpm for 20 hours to obtain a seed solution;
3) inoculating the oil-producing microorganism seed solution prepared in the step 2) into the oil-producing microorganism culture medium obtained in the step 1), wherein the inoculation amount is 20% (v/v), and performing aeration culture at 32 ℃ for 192h, wherein the concentration of residual VFAs in the fermentation liquid is 0 g/L;
4) adding 20FPU/g cellulase and 200U/g xylanase into the fermentation liquor obtained in the step 3), adjusting the pH to 4.8, ventilating and culturing at 30 ℃, and performing fermentation culture for 160h while hydrolyzing;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 0g/L, performing solid-liquid separation to collect thalli to obtain 25.9g/L dry thalli, treating the dry thalli with a high-concentration VFAs solution at the high temperature of 80 ℃ for 2h to crack cells, and then extracting oil with normal hexane, wherein the oil content is 15.2g/L and the oil content is 58.7% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
Example 5
1) Preparing high-concentration VFAs and regenerated corncobs produced by anaerobic fermentation of waste pericarps: preparation of VFAs as same as step 1) of example 1); weighing 100g of corncobs sieved by a 20-mesh sieve, adding the corncobs into VFAs solution according to a solid-to-liquid ratio of 20% (w/w), pretreating at 120 ℃ for 60min, and then adding water until the solid-to-liquid mass ratio is 15%, so as to obtain acidic regenerated corncobs; weighing 100g of corncobs sieved by a 20-mesh sieve, adding the corncobs into 0.5L of 5% (w/w) calcium hydroxide solution with the solid-liquid ratio of 20% (w/w), pretreating at 100 ℃ for 60min, and then adding water until the solid-liquid mass ratio is 15%, so as to obtain alkaline regenerated corncobs; directly neutralizing the regenerated corncob, adjusting the pH to 7.0, and sterilizing at 121 ℃ for 15min for later use;
2) cryptococcus albidus ATCC 56298 (purchased from American type culture Collection) is inoculated in YEPD seed culture medium (20 g/L glucose, 10g/L yeast powder and 20g/L peptone), and is subjected to shaking culture at 20 ℃ and 200rpm for 28h to obtain seed liquid;
3) inoculating 12% (v/v) of the oleaginous microorganism seed solution prepared in the step 2) into the oleaginous microorganism culture medium obtained in the step 1), and performing aerobic culture at 20 ℃ for 130h, wherein the concentration of residual VFAs in the fermentation liquid is 0 g/L;
4) adding 15FPU/g of cellulase, 10CBU/g of beta-glucosidase and 200U/g of xylanase into the fermentation liquor obtained in the step 3), adjusting the pH to 4.8, ventilating and culturing at 30 ℃, and performing fermentation culture for 150 hours while hydrolyzing;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 1.5g/L, performing solid-liquid separation to collect thalli to obtain dry thalli 26.9g/L, treating the dry thalli with high-concentration VFAs solution at the high temperature of 75 ℃ for 1h to crack cells, and then extracting oil with normal hexane, wherein the oil content is 14.2g/L, and the oil content is 52.8% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
Example 6
1) Preparation of high-concentration VFAs and regenerated bagasse produced by anaerobic fermentation of activated sludge: putting the activated sludge into a 6L fermentation tank containing a jacket, adjusting the pH value of the fermentation tank to 5.0, adjusting the reaction temperature to 40 ℃, performing anaerobic fermentation for 38 hours, and performing solid-liquid separation to obtain a high-concentration VFAs solution containing 50g/L VFAs; weighing 100g of bagasse sieved by a 40-mesh sieve, adding the bagasse into a VFAs solution according to a solid-liquid ratio of 20% (w/w), pretreating at 160 ℃ for 60min, and then adding water until the solid-liquid mass ratio is 10% to obtain acidic regenerated bagasse; weighing 100g of bagasse sieved by a 40-mesh sieve, adding the bagasse into a calcium oxide solution with the concentration of 5% (w/w) according to the solid-liquid ratio of 20% (w/w), pretreating at 100 ℃ for 120min according to the solid-liquid ratio of 20% (w/w), and then adding water until the solid-liquid mass ratio is 10%, thereby obtaining the alkalescent regenerated bagasse; directly neutralizing two kinds of regenerated bagasse, adjusting pH to 7.0, and sterilizing at 121 deg.C for 30 min;
2) rhodosporidium toruloides AS 2.1389 (purchased from China general microbiological culture Collection center) is inoculated in a seed culture medium (20 g/L glucose, 5g/L corn steep liquor), and is subjected to shaking culture at 37 ℃ and 200rpm for 24h to obtain a seed solution;
3) inoculating 20% (v/v) of the oleaginous microorganism seed solution prepared in the step 2) into the oleaginous microorganism culture medium obtained in the step 1), and performing aerobic culture at 37 ℃ for 144h, wherein the concentration of residual VFAs in the fermentation liquid is 0 g/L;
4) adding 15FPU/g of cellulase, 10CBU/g of beta-glucosidase and 200U/g of xylanase into the fermentation liquor obtained in the step 3), adjusting the pH to 4.8, ventilating and culturing at 30 ℃, and performing fermentation culture while hydrolyzing for 144 h;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 0g/L, performing solid-liquid separation to collect thalli to obtain 26.4g/L dry thalli, treating the dry thalli with a high-concentration VFAs solution at a high temperature of 78 ℃ for 1h to crack cells, and then extracting oil with normal hexane, wherein the oil content is 15.1g/L and the oil content is 57.2% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
Example 7
1) Preparing high-concentration VFAs and regenerated cotton stalks produced by anaerobic fermentation of hydrogen production wastewater: putting the hydrogen production wastewater into a 6L fermentation tank containing a jacket, adjusting the pH value of the fermentation tank to 4.5, adjusting the reaction temperature to 30 ℃, performing anaerobic fermentation for 32 hours, and performing solid-liquid separation to obtain a high-concentration VFAs solution containing 62g/L VFAs; weighing 100g of cotton stalks which are sieved by a 40-mesh sieve, adding the cotton stalks into VFAs solution according to a solid-liquid ratio of 10% (w/w), pretreating for 80min at 150 ℃, and then adding water until the solid-liquid mass ratio is 8% to obtain subacid regenerated cotton stalks; weighing 100g of cotton stalks which are sieved by a 40-mesh sieve, adding the cotton stalks into a calcium hydroxide solution with the concentration of 3% (w/w) according to the solid-liquid ratio of 10% (w/w), pretreating at 100 ℃ for 60min, and then adding water until the solid-liquid mass ratio is 8%, so as to obtain alkaline regenerated cotton stalks; directly neutralizing two regenerated cotton stalks, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min;
2) inoculating Mortierella isabellina ATCC 42613 (purchased from American type culture Collection) into PDA seed culture medium, and performing shaking culture at 34 deg.C and 200rpm for 24 hr to obtain seed solution;
3) inoculating the oil-producing microorganism seed solution prepared in the step 2) into the oil-producing microorganism culture medium obtained in the step 1), wherein the inoculation amount is 20% (v/v), and performing aeration culture at 35 ℃ for 180h, wherein the concentration of residual VFAs in the fermentation liquid is 0 g/L;
4) adding 20FPU/g of cellulase, 8CBU/g of beta-glucosidase and 100U/g of xylanase into the fermentation liquor obtained in the step 3), adjusting the pH to 4.8, ventilating and culturing at 30 ℃, and performing fermentation culture for 140 hours while hydrolyzing;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 0g/L, performing solid-liquid separation to collect thalli to obtain 23.7g/L dry thalli, treating the dry thalli with a high-concentration VFAs solution at a high temperature of 78 ℃ for 1h to crack cells, and then extracting oil with n-hexane, wherein the oil content is 12.9g/L and the oil content is 54.4% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
Example 8
1) Preparing high-concentration VFAs and regenerated sawdust produced by anaerobic fermentation of kitchen waste: placing kitchen garbage in a 6L fermentation tank with a jacket, adjusting the pH value of the fermentation tank to 5.0, adjusting the reaction temperature to 40 ℃, performing anaerobic fermentation for 2 days, and performing solid-liquid separation to obtain a high-concentration VFAs solution containing 58g/L VFAs; weighing 80g of sawdust sieved by a 20-mesh sieve, adding the sawdust into a VFAs solution according to a solid-liquid ratio of 10% (w/w), pretreating at 200 ℃ for 30min, and then adding water until the solid-liquid mass ratio is 5% to obtain subacid regenerated sawdust; weighing 80g of sawdust which is sieved by a 20-mesh sieve, adding the sawdust into a calcium hydroxide solution with the concentration of 5% (w/w) according to the solid-liquid ratio of 10% (w/w), pretreating at 120 ℃ for 120min, and then adding water until the solid-liquid mass ratio is 5%, so as to obtain alkaline regenerated sawdust; directly neutralizing the two kinds of regenerated sawdust, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min;
2) yarrowia lipolytica Po1g (purchased from Yeaster Biotech Co., Ltd., Taiwan) was inoculated in PDA seed culture medium, and cultured at 25 deg.C and 200rpm for 28h under shaking to obtain seed solution;
3) inoculating the oleaginous microorganism seed solution prepared in the step 2) into the oleaginous microorganism culture medium obtained in the step 1), wherein the inoculation amount is 10% (v/v), and performing aerobic culture at 25 ℃ for 108h, wherein the concentration of residual VFAs in the fermentation liquid is 3.2 g/L;
4) adding 15FPU/g of cellulase, 10CBU/g of beta-glucosidase and 100U/g of xylanase into the fermentation liquor obtained in the step 3), adjusting the pH to 4.8, ventilating and culturing at 30 ℃, and performing fermentation culture for 120 hours while hydrolyzing;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 0g/L, performing solid-liquid separation to collect thalli to obtain 17.8g/L dry thalli, treating the dry thalli with a high-concentration VFAs solution at a high temperature of 78 ℃ for 1h to crack cells, and then extracting oil with normal hexane, wherein the oil content is 7.5g/L and the oil content is 42.1% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
Example 9
1) Preparing high-concentration VFAs and regenerated beet pulp produced by anaerobic fermentation of activated sludge: putting the activated sludge into a 6L fermentation tank with a jacket, adjusting the pH value of the fermentation tank to 4.5, adjusting the reaction temperature to 38 ℃, performing anaerobic fermentation for 30h, and performing solid-liquid separation to obtain a high-concentration VFAs solution containing 47g/L VFAs; weighing 100g of 40-mesh-sieve beet pulp, adding the beet pulp into VFAs solution according to a solid-to-liquid ratio of 10% (w/w), pretreating at 130 ℃ for 60min, and then adding water until the solid-to-liquid mass ratio is 5%, thereby obtaining subacid regenerated beet pulp; weighing 100g of beet pulp sieved by a 40-mesh sieve, adding the beet pulp into a calcium hydroxide solution with the concentration of 5% (w/w) according to the solid-to-liquid ratio of 10% (w/w), pretreating at 100 ℃ for 120min, and then adding water until the solid-to-liquid mass ratio is 5%, thereby obtaining alkaline regenerated beet pulp; directly neutralizing the two regenerated beet pulp, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min;
2) rhodotorula glutinis Glutinis AS 2.703 (purchased from China general microbiological culture Collection center) is inoculated in YEPD seed culture medium (glucose 20g/L, yeast powder 10g/L, peptone 5g/L), and is subjected to shaking culture at 28 ℃ and 200rpm for 20h to obtain seed liquid;
3) inoculating the oleaginous microorganism seed solution prepared in the step 2) into the oleaginous microorganism culture medium obtained in the step 1), wherein the inoculation amount is 2% (v/v), and performing aerobic culture at 30 ℃ for 72 hours, wherein the concentration of residual VFAs in the fermentation liquid is 1.1 g/L;
4) adding 10FPU/g of cellulase, 20CBU/g of beta-glucosidase and 100U/g of xylanase into the fermentation liquor obtained in the step 3), adjusting the pH to 4.8, ventilating and culturing at 30 ℃, and performing fermentation culture for 120 hours while hydrolyzing;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 0.5g/L, performing solid-liquid separation to collect thalli to obtain dry thalli 16.1g/L, treating the dry thalli with high-concentration VFAs solution at the high temperature of 78 ℃ for 1h to crack cells, and then extracting oil with n-hexane, wherein the oil content is 8.6g/L and the oil content is 53.4% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
Example 10
1) Preparing high-concentration VFAs and regenerated water hyacinth which are produced by anaerobic fermentation of hydrogen production wastewater: putting the hydrogen production wastewater into a 6L fermentation tank containing a jacket, adjusting the pH value of the fermentation tank to 4.9, adjusting the reaction temperature to 37 ℃, performing anaerobic fermentation for 2 days, and performing solid-liquid separation to obtain a high-concentration VFAs solution containing 61g/L VFAs; weighing 100g of water hyacinth which is sieved by a 20-mesh sieve, adding the water hyacinth into VFAs solution according to the solid-liquid ratio of 10% (w/w), pretreating at 130 ℃ for 60min, and then adding water until the solid-liquid mass ratio is 5%, thereby obtaining the subacid regenerated water hyacinth; weighing 100g of water hyacinth which is sieved by a 20-mesh sieve, adding the water hyacinth into a calcium oxide solution with the concentration of 5% (w/w) according to the solid-liquid ratio of 10% (w/w), pretreating at 140 ℃ for 120min, and then adding water until the solid-liquid mass ratio is 5%, thereby obtaining the alkaline regenerated water hyacinth; directly neutralizing two kinds of regenerated water hyacinth, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min;
2) the Lipomyces starkeyi AS 2.1560 (purchased from China general microbiological culture Collection center) is inoculated in a seed culture medium (20 g/L glucose, 5g/L yeast powder and 3 g/L peptone), and is subjected to shaking culture at 28 ℃ and 200rpm for 48h to obtain seed liquid;
3) inoculating 5% (v/v) of the oleaginous microorganism seed solution prepared in the step 2) into the oleaginous microorganism culture medium obtained in the step 1), and performing aerobic culture at 28 ℃ for 180 hours, wherein the concentration of residual VFAs in the fermentation liquid is 1.5 g/L;
4) adding 20FPU/g of cellulase, 8CBU/g of beta-glucosidase and 100U/g of xylanase into the fermentation liquor obtained in the step 3), adjusting the pH to 4.8, ventilating and culturing at 30 ℃, and performing fermentation culture for 120 hours while hydrolyzing;
5) terminating fermentation, wherein the concentration of residual sugar in the fermentation liquor is 0g/L, performing solid-liquid separation to collect thalli to obtain 22.1g/L dry thalli, treating the dry thalli with a high-concentration VFAs solution at the high temperature of 80 ℃ for 1h to crack cells, and then extracting oil with n-hexane, wherein the oil content is 12.5g/L and the oil content is 56.6% (w/w); the solution containing VFAs is obtained in the liquid phase, adjusted for concentration, and recycled for pretreatment of the lignocellulose.
The results of comparative examples 1, 2 and examples 1 to 10 described above are shown in the following table:
by comparing the experimental results of comparative examples 1 and 2 and example 1, it was found that the biomass (i.e., dry biomass content) and the amount of oil and fat were significantly increased after the direct neutralization of the lignocellulosic feedstocks pretreated with VFAs and the lignocellulosic feedstocks pretreated with alkali, and that the VFAs and the major carbohydrates glucose and xylose in the lignocellulosic hydrolysate were substantially completely utilized, and the total oil and fat yield was significantly increased.
Furthermore, examples 2-10 show that the co-transformation of VFAs with lignocellulosic hydrolysates to microbial oils enables full utilization of VFAs and carbohydrates and leads primarily to oil synthesis. Therefore, the oil yield and the raw material utilization efficiency of the oleaginous microorganism cultured by the scheme of the invention are both improved, and the cost can be obviously reduced when the oleaginous microorganism is produced in large quantities.
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 process for the production of microbial greases using VFAs and lignocellulosic feedstocks, comprising the steps of:
step S1, preparing VFAs solution through anaerobic fermentation;
step S2, mixing the lignocellulose raw material with a certain amount of VFAs solution uniformly, carrying out high-temperature pretreatment, and then supplementing water to obtain an acidic regenerated lignocellulose raw material;
step S3, uniformly mixing the lignocellulose raw material with a certain amount of alkali solution, carrying out high-temperature pretreatment, and then supplementing water to obtain an alkaline regenerated lignocellulose raw material;
step S4, directly neutralizing the acidic and alkaline regenerated lignocellulose raw materials, adjusting the pH value to be neutral, and then performing sterilization treatment to obtain an oil-producing microorganism culture medium;
step S5, culturing oleaginous microorganisms in a seed culture medium to obtain oleaginous microorganism seed liquid;
step S6, inoculating the oil-producing microorganism seed liquid into the oil-producing microorganism culture medium for ventilation fermentation culture until the concentration of residual VFAs in the fermentation culture liquid is lower than 5 g/L;
step S7, adjusting the pH value, adding a proper amount of lignocellulose degrading enzyme, hydrolyzing the regenerated lignocellulose raw material into fermentable biomass sugar, and continuing fermentation until the concentration of residual total sugar in the fermentation liquor is lower than 5g/L, and then stopping fermentation;
and S8, performing solid-liquid separation to collect oil-producing microbial cells, treating the oil-producing microbial cells at high temperature by using a VFAs solution, cracking the cells, extracting oil by using an extracting agent, and returning to the step S2 to pretreat lignocellulose after the concentration of the VFAs is adjusted, wherein the VFAs is remained in the residual liquid phase after extraction.
2. The method of claim 1, wherein the step S1 is embodied as follows: selecting organic wastes from different sources, placing the organic wastes in a fermentation tank, adjusting the pH value to be 4.5-6.5 and the reaction temperature to be 30-40 ℃, carrying out anaerobic fermentation, and carrying out solid-liquid separation to obtain a VFAs solution with high concentration, wherein the VFAs solution contains short-chain VFAs capable of being used by oleaginous microorganisms.
3. The process for the production of microbial oils and fats using VFAs and lignocellulosic feedstocks according to claim 2 wherein the VFAs solution is a mixture of short chain fatty acids at a total concentration of 10g/L to 70g/L, including acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and n-valeric acid, with the highest acetic acid content in the mixture, followed by propionic acid and butyric acid.
4. The method of claim 1, wherein in step S2, the lignocellulosic raw material is mixed with VFAs solution uniformly at a solid-liquid mass ratio of 10% to 30% and pretreated at high temperature, and then supplemented with water to a solid-liquid mass ratio of 5% to 20% to obtain an acidic regenerated lignocellulosic raw material; in the step S3, the lignocellulose raw material and an alkali solution with the mass concentration of 0.5-5% are uniformly mixed according to the solid-liquid mass ratio of 10-30%, the mixture is pretreated at high temperature, water is supplemented until the solid-liquid mass ratio is 5-20%, so that an alkaline regenerated lignocellulose raw material is obtained, and the alkali solution is prepared from calcium oxide or calcium hydroxide.
5. The method for producing microbial oils and fats using VFAs and lignocellulosic feedstocks as claimed in claim 4, wherein the high temperature pretreatment is carried out at 80 ℃ to 180 ℃ for 15min to 4h in steps S2, S3.
6. The method for producing microbial oils and fats using VFAs and lignocellulosic feedstocks according to claim 1, wherein in step S6, the seed solution of the oleaginous microorganism is inoculated in an amount of 2% to 20% by volume ratio and subjected to aeration culture at 20 ℃ to 37 ℃; in step S7, the pH is adjusted to 4-8.
7. The method of claim 1, wherein the VFAs solution is treated at a high temperature of 70-85 deg.C for lysing the somatic cells of the oleaginous microorganisms for 0.5-2 h.
8. The process of claim 7 for the production of microbial oils from VFAs and lignocellulosic feedstocks wherein the lignocellulose degrading enzymes are enzymes having the ability to reduce the degree of polymerization of carbohydrate polymers, including one or a combination of more of cellulase, β -glucosidase, xylanase, β -xylosidase, pectinase, and fermentable biomass sugars obtained from hydrolysis comprise glucose and xylose, and further comprise arabinose, galactose, mannose, cellobiose, and galacturonic acid.
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| LIU JUAN等: "《Microbial Lipid Production from High Concentration of Volatile Fatty Acids via Trichosporon cutaneum for Biodiesel Preparation》", 《APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY》, 22 March 2022 (2022-03-22), pages 1 - 12 * |
| 刘娟: "《皮状丝孢酵母转化木质纤维素糖和醋酸产油脂的研究》", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 * |
| 刘娟: "《皮状丝孢酵母转化木质纤维素糖和醋酸产油脂的研究》", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》, no. 1, 15 January 2022 (2022-01-15), pages 017 - 191 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115449409A (en) * | 2022-10-19 | 2022-12-09 | 中润油新能源股份有限公司 | Microbial fermentation methanol gasoline and preparation method thereof |
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