CN109400410B - Method for producing biological organic matter by using threonine fermentation waste liquid - Google Patents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05C—NITROGENOUS FERTILISERS
- C05C11/00—Other nitrogenous fertilisers
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
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- C05C9/00—Fertilisers containing urea or urea compounds
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/02—Other organic fertilisers from peat, brown coal, and similar vegetable deposits
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- C05—FERTILISERS; MANUFACTURE THEREOF
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- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/90—Mixtures of one or more fertilisers with additives not having a specially fertilising activity for affecting the nitrification of ammonium compounds or urea in the soil
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G5/00—Fertilisers characterised by their form
- C05G5/20—Liquid fertilisers
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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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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Abstract
The invention belongs to the technical field of biology, and discloses a method for producing biological organic matters by using threonine fermentation waste liquid, which comprises the following steps: step 1) preparing threonine fermentation liquor, step 2) preparing protein powder, step 3) preparing threonine, step 4) collecting threonine fermentation waste liquor, and step 5) preparing biological organic matters. The method prepares the biological organic matter while preparing the threonine, can be used as a liquid fertilizer, changes waste into valuable, and achieves two purposes at one stroke.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing biological organic matters by using threonine fermentation waste liquid.
Background
Threonine is an essential amino acid, is the second limiting amino acid of pig feed and the third limiting amino acid of poultry feed, and gradually becomes a main limiting factor influencing the growth of livestock and poultry along with the wide application of lysine and methionine synthetic products in compound feed. Threonine is mainly used as a feed additive, the production steps of the threonine are concentration, crystallization and mycoprotein extraction, and mother liquor generated in the process is difficult to treat and high in cost, and byproducts with high cost performance are difficult to obtain.
At present, a small part of waste water of the amino acid fermentation industry is used for developing liquid fertilizers, and a small part of waste water is used for a spraying granulation process of solid organic fertilizers. However, the amount of liquid required for spray granulation is small, and a large amount of industrial waste liquid of amino acid cannot be consumed. Meanwhile, due to the basic characteristics of the liquid, negative factors such as strong acidity, strong corrosivity, heavy odor, difficulty in optimizing the components of the amino acid waste liquid, poor field effect, even harm to certain crops and the like exist, so that the utilization of the amino acid waste liquid in the aspect of organic fertilizer is always limited, and the produced fertilizer, namely solid waste or liquid fertilizer, is difficult to popularize. These all inhibit the recycling of the industrial wastewater of amino acids. With the development of the amino acid industry, the amount of the produced amino acid wastewater is more and more. The water resource is more and more scarce, the environmental protection pressure is more and more, and the treatment of the waste water in the amino acid industry is more and more a difficult problem which troubles the development of the amino acid industry.
The amino acid fermentation waste liquid still contains a large amount of amino acid residues and organic matter residues. On one hand, the COD of the amino acid wastewater is high, and the amino acid wastewater is difficult to treat, and on the other hand, the amino acid wastewater has the potential of utilization in the aspects of planting and breeding industries and the like. At present, the microbial biotechnology utilizes the metabolic function of microorganisms to realize characteristic transformation of organic components in materials, form characteristic secondary metabolites and change the physicochemical and biochemical properties of the materials. Meanwhile, certain microorganisms can promote the growth of crops, play a role in improving the energy absorption of soil, bring the effects of soil improvement and plant fertilizers and are the main force of microbial fertilizers. The prior patent technology of the applicant improves the threonine fermentation waste liquid, wherein the 'fertilizer prepared by using threonine fermentation waste' can prepare solid fertilizer from the fermentation waste while preparing threonine, thereby obtaining better economic benefit; the compound fertilizer prepared by fermenting the waste by using threonine is prepared by using the fermented waste.
Disclosure of Invention
The invention aims to provide a method for producing biological organic matters by using threonine fermentation waste liquid.
In order to realize the purpose, the invention adopts the following technical scheme:
the method for producing the biological organic matter by using the threonine fermentation waste liquid comprises the following steps: step 1) preparing threonine fermentation liquor, step 2) preparing protein powder, step 3) preparing threonine, step 4) collecting threonine fermentation waste liquor, and step 5) preparing biological organic matters.
Further, the method comprises the steps of:
step 1) preparing threonine fermentation liquor: inoculating threonine-producing escherichia coli engineering bacteria into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 8% for fermentation for 36h, then inoculating chlamydomonas reinhardtii according to the inoculation amount of 10%, continuing to ferment for 36h, stopping fermentation, and collecting threonine fermentation liquor;
step 2) protein powder preparation: the threonine fermentation liquor is firstly centrifuged for 5min at 4000rpm by a disc centrifuge, upper-layer liquid and mycoprotein sediment are collected, and the mycoprotein sediment is dried and crushed to prepare protein powder;
step 3) preparation of threonine: filtering the upper layer liquid with ceramic membrane, collecting filtrate and retentate, separating the filtrate with horizontal screw centrifuge at centrifugal speed of 5000rpm for 3min, and collecting supernatant and precipitate; then filtering through an ultrafiltration membrane, and collecting filtrate; concentrating and crystallizing the filtrate, centrifuging to collect crystals and mother liquor, drying the crystals, compressing the crystals into sheets, and putting the sheets into a granulation tower to be in a boiling state under the action of hot air flow; drying by a fluidized bed, and crushing and granulating to obtain threonine;
step 4), collecting threonine fermentation waste liquid: mixing the retentate, the precipitate and the mother liquor obtained in the step 3) to obtain threonine fermentation waste liquor;
step 5), preparing biological organic matters: heating the threonine fermentation waste liquid to 100 ℃, treating for 10min under the condition of heat preservation, cooling to room temperature, adding ammonia water to adjust the pH value to 6-7, then sequentially inoculating bacillus subtilis and trichoderma viride for fermentation treatment, wherein the inoculation amount is 3-5%, the fermentation temperature is 30-35 ℃, the fermentation time is 3 days, ending the fermentation, adding potassium dihydrogen phosphate, humic acid and urea into the fermentation liquid, stirring and mixing uniformly, filtering, and collecting filtrate to obtain the biological organic matter.
Further, the components of the fermentation medium are as follows: 20g/L of glucose, 15g/L of glycerol, 15g/L of corn steep liquor, 2g/L of ammonium sulfate, 0.2g/L of monopotassium phosphate, 0.2g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and the pH value of 6.5.
Further, the fermentation conditions are: the temperature was 30 ℃, the pot pressure was 0.04MPa, the aeration rate was 0.5vvm, and the rotation speed was 100 rpm.
Further, the molecular weight cut-off of the ceramic membrane was 1 ten thousand Da.
Further, the ultrafiltration membrane has a molecular weight cut-off of 300 Da.
Further, in the step 5), the ratio of the fermentation liquor to the monopotassium phosphate to the humic acid to the urea is 1L:5g:3g:2 g.
The starting point and the beneficial effects of the research of the invention mainly comprise but are not limited to the following aspects:
according to the invention, glucose and glycerol are selected as fermentation carbon sources, the thallus density is low in the early stage of fermentation, the oxygen supply is sufficient, and Escherichia coli preferentially utilizes glucose as a carbon source, so that thallus proliferation and threonine production can be promoted; in the middle and later period of fermentation, glucose is exhausted, and at the moment, the Escherichia coli utilizes glycerol as a carbon source, and the carbon flow entering glycolysis is reduced due to the low glycerol absorption rate of cells, so that the accumulation amount of acetic acid is reduced, and the yield of threonine is increased;
the chlamydomonas reinhardtii is inoculated in the fermentation, so that acetic acid in the fermentation liquor can be used as a carbon source to perform non-light action, and glycerol is difficult to be used as the carbon source, so that the inhibition effect on threonine production of escherichia coli is relieved, and trace photosynthesis can be performed to release oxygen for threonine production by escherichia coli fermentation. By adding chlamydomonas reinhardtii, the threonine yield can be improved, and the mycoprotein yield is correspondingly improved;
threonine, mycoprotein and biological organic matter are simultaneously prepared in the fermentation process, an intermediate pretreatment step is omitted, the direct biological utilization of sewage is realized, the discharge is reduced while the waste is changed into the valuable, and the economic benefit is remarkable;
in the process of preparing the biological organic matter, the fermentation waste liquid is treated by the compound bacterial liquid, COD and ammonia nitrogen are degraded, active substances are generated, the growth of plants is facilitated, and a certain prevention and control effect on plant diseases and insect pests is achieved; humic acid contains functional groups such as carboxyl, phenolic hydroxyl and the like, has strong ion exchange capacity and adsorption capacity, has obvious synergistic effect on urea, can generate a complex with urea, and can slow down urea decomposition, reduce volatilization and prolong fertilizer efficiency.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
The method for producing the biological organic matter by using the threonine fermentation waste liquid comprises the following steps:
step 1) preparing seed solution of escherichia coli engineering bacteria K12 delta dapA (the concentration of the seed solution is 1 multiplied by 10)8cfu/mL) is inoculated into a fermentation tank containing a fermentation medium according to the inoculation amount of 8 percent for fermentation, the temperature is 30 ℃, the tank pressure is 0.04MPa, the ventilation volume is 0.5vvm, the rotating speed is 100rpm, the fermentation time is 36h, and then chlamydomonas reinhardtii (the concentration of chlamydomonas reinhardtii is 1 multiplied by 10) is inoculated according to the inoculation amount of 10 percent5cfu/mL), continuously fermenting and culturing for 36h, stopping fermentation, and collecting fermentation liquor;
the fermentation medium comprises the following components: 20g/L of glucose, 15g/L of glycerol, 15g/L of corn steep liquor, 2g/L of ammonium sulfate, 0.2g/L of monopotassium phosphate, 0.2g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and the pH value of 6.5;
step 2), firstly, centrifuging the fermentation liquor by a disc centrifuge at 4000rpm for 5min, collecting upper-layer liquid and mycoprotein precipitation, drying and crushing the mycoprotein precipitation to prepare protein powder;
step 3), filtering the upper layer liquid by a ceramic membrane (with molecular weight cutoff of 1 ten thousand Da), collecting filtrate and retentate, separating the filtrate by a horizontal screw centrifuge at the centrifugal speed of 5000rpm for 3min, and collecting supernatant (with protein content less than 0.5%) and precipitate; then filtering with ultrafiltration membrane, collecting filtrate, and intercepting with ultrafiltration membrane with molecular weight of 300 Da; crystallizing the filtrate with intermittent single-effect concentration crystallizing pan, centrifuging to collect crystal and mother liquor, drying at 120 deg.C until water content is 0.8%, compressing into tablet, and placing into granulation tower to be in boiling state under the action of hot air flow; drying with a 65 ℃ fluidized bed, and crushing and granulating to obtain threonine;
step 4) mixing the retentate, the precipitate and the mother liquor obtained in the step 3) to obtain threonine fermentation waste liquid;
step 5) heating the threonine fermentation waste liquid to 100 ℃, treating for 10min under the condition of heat preservation, then cooling to room temperature, adding ammonia water to adjust the pH value to 6.5, and then sequentially inoculating bacillus subtilis (the concentration is 1 multiplied by 10)8cfu/mL) and Trichoderma viride (at a concentration of 1X 10)8cfu/mL), the inoculation amount is 5%, the fermentation temperature is 32 ℃, the fermentation time is 3 days, the fermentation is finished, monopotassium phosphate, humic acid and urea are added into the fermentation liquor, the mixture is stirred and mixed evenly, then the filtration is carried out, and the filtrate is collected, so that the biological organic matter is obtained; the ratio of the fermentation liquor to the monopotassium phosphate to the humic acid to the urea is 1L to 5g to 3g to 2 g.
Example 2
The influence of different factors on the threonine yield and the acetic acid yield in the fermentation method of the invention is as follows:
setting a group:
experimental groups: example 1;
control group 1: the same procedure as in example 1 was repeated except that Chlamydomonas reinhardtii was not added;
control group 2: the glycerol was replaced with glucose of equal mass, as in example 1;
control group 3: the same procedure as in example 1 was repeated except that Chlamydomonas reinhardtii was not added and that glycerol was replaced with glucose of the same mass.
The threonine and acetic acid content of the final fermentation broths of each group is shown in table 1:
TABLE 1
Group of | Threonine g/L | Acetic acid g/L |
Experimental group | 127.9 | 0.7 |
Control group 1 | 103.5 | 12.6 |
Control group 2 | 115.7 | 4.9 |
Control group 3 | 97.1 | 14.8 |
And (4) conclusion: the experiment group can perform non-light action by using acetic acid in threonine fermentation liquor as a carbon source through performing auxiliary fermentation treatment on chlamydomonas reinhardtii, so that the inhibition effect on threonine production of escherichia coli is eliminated, and trace photosynthesis can be performed to release oxygen for the threonine production by escherichia coli fermentation; meanwhile, glycerol replaces part of glucose, and Escherichia coli utilizes the glycerol as a carbon source along with the consumption of the glucose, so that the accumulation amount of acetic acid is reduced and the yield of threonine is improved due to the low glycerol absorption rate of cells.
Example 3
The fertilizer efficiency test of the prepared biological organic matter comprises the following steps:
taking tomatoes as an example, the yield and quality of the tomatoes are detected.
Experimental groups: spraying the biological organic matter;
control group: spraying the same amount of clear water.
Selecting a greenhouse in an inner Mongolia area; the planting area of each cell is 10m multiplied by 10m, 3 times of repetition is set, 6 test cells are set in total, and random block arrangement is carried out. The tomato variety is Fengli No. 1, 100kg of liquid fertilizer is sprayed per mu, and other planting conditions are the same. The results are shown in table 2:
TABLE 2
Group of | Weight of single fruit g | Soluble solids% | Soluble sugar% | Titratable acid% |
Experimental group | 168.9 | 4.73 | 4.03 | 0.36 |
Control group | 162.4 | 4.55 | 3.89 | 0.41 |
And (4) conclusion: as shown in table 2, compared to the control group without organic matter, the organic matter of the experimental group can increase the single fruit weight, increase the specific gravity of the soluble solid and soluble sugar, reduce the specific gravity of titratable acid, and improve the tomato quality.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (5)
1. The method for producing the biological organic matter by using the threonine fermentation waste liquid is characterized by comprising the following steps of:
step 1) preparing threonine fermentation liquor: inoculating threonine-producing escherichia coli engineering bacteria into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 8% for fermentation for 36h, then inoculating chlamydomonas reinhardtii according to the inoculation amount of 10%, continuing to ferment for 36h, stopping fermentation, and collecting threonine fermentation liquor;
step 2) protein powder preparation: centrifuging threonine fermentation liquor by a disc centrifuge at 4000rpm for 5min, collecting upper layer liquid and mycoprotein precipitation, drying and crushing the mycoprotein precipitation to prepare protein powder;
step 3) preparation of threonine: filtering the supernatant obtained in the step 2) by a ceramic membrane, collecting filtrate and retentate, separating the filtrate by a horizontal decanter centrifuge, and collecting supernatant and precipitate; filtering the supernatant with ultrafiltration membrane, and collecting the ultrafiltrate; concentrating and crystallizing the ultrafiltrate, centrifugally collecting crystals and mother liquor, drying the crystals, compressing the crystals into sheets, and putting the sheets into a granulation tower to be in a boiling state under the action of hot air flow; drying by a fluidized bed, and crushing and granulating to obtain threonine;
step 4), collecting threonine fermentation waste liquid: mixing the retentate, the precipitate and the mother liquor obtained in the step 3) to obtain threonine fermentation waste liquor;
step 5), preparing biological organic matters: heating threonine fermentation waste liquid to 100 ℃, treating for 10min under the condition of heat preservation, then cooling to room temperature, adding ammonia water to adjust the pH value to 6-7, then sequentially inoculating bacillus subtilis and trichoderma viride for fermentation treatment, wherein the inoculation amount is 3-5%, the fermentation temperature is 30-35 ℃, the fermentation time is 3 days, ending the fermentation, adding potassium dihydrogen phosphate, humic acid and urea into the fermentation liquid, stirring and mixing uniformly, filtering, and collecting filtrate to obtain biological organic matters;
in the step 5), the ratio of the fermentation liquor to the monopotassium phosphate to the humic acid to the urea is 1L to 5g to 3g to 2 g.
2. The method of claim 1, wherein the fermentation medium comprises: 20g/L of glucose, 15g/L of glycerol, 15g/L of corn steep liquor, 2g/L of ammonium sulfate, 0.2g/L of monopotassium phosphate, 0.2g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and the pH value of 6.5.
3. The method according to claim 1, wherein the fermentation conditions in step 1) are: the temperature was 30 ℃, the pot pressure was 0.04MPa, the aeration rate was 0.5vvm, and the rotation speed was 100 rpm.
4. The method according to claim 1, wherein the ceramic membrane has a molecular weight cut-off of 1 ten thousand Da.
5. The method of claim 1, wherein the ultrafiltration membrane has a molecular weight cut-off of 300 Da.
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