CN112871993B - Phase-change water production treatment process for perishable garbage - Google Patents
Phase-change water production treatment process for perishable garbage Download PDFInfo
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- CN112871993B CN112871993B CN202110187687.4A CN202110187687A CN112871993B CN 112871993 B CN112871993 B CN 112871993B CN 202110187687 A CN202110187687 A CN 202110187687A CN 112871993 B CN112871993 B CN 112871993B
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- perishable garbage
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- immobilized enzyme
- chitosan oligosaccharide
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Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B5/00—Operations not covered by a single other subclass or by a single other group in this subclass
Landscapes
- Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Processing Of Solid Wastes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention relates to the technical field of perishable garbage treatment, in particular to a perishable garbage phase-change water preparation treatment process, wherein perishable garbage is subjected to sorting to remove non-degradable substances, then crushed to pass through a 10-mesh sieve, placed in a degradation bin of phase-change water preparation treatment equipment, stirred by a stirring rod in the degradation bin and extruded with the side wall of the degradation bin, filtered to obtain filtrate, heated to 50-60 ℃, kept stand for layering, led out upper-layer crude grease, and other components are led into the equipment; a perishable garbage degrading microbial agent and an immobilized enzyme preparation are added into equipment, and a phase-change water production treatment process is operated for 36-48 hours under the conditions that the temperature is 15-40 ℃ and the relative humidity is 80-90%. The phase change water production treatment process can be remarkably promoted by the optimization device and method, normal-temperature rapid degradation is realized, macromolecular organic matters are degraded into carbon dioxide and water, and the reduction rate of perishable garbage is not less than 99.3%.
Description
Technical Field
The invention relates to the technical field of perishable garbage treatment, in particular to a perishable garbage phase-change water production treatment process.
Background
Along with the increasing living standard of people, the amount of kitchen garbage which is easy to decompose is increased while the material resources are greatly met, so that the decomposition work of the kitchen garbage has a rapid development trend at home and abroad. Particularly, the development level of the perishable waste treatment field in western countries with relatively developed technology level reaches a high level, and the perishable waste can be effectively treated and processed aiming at a large amount of perishable waste generated in daily life of the society, so that the perishable waste can be recycled. For example, a device developed in japan and capable of converting food waste such as banana peel, fish entrails, coffee grounds, etc. into fertilizer is gaining popularity after entering the market. However, large-scale perishable garbage treatment equipment is relatively large in floor area and design specification, at present, large-scale perishable garbage treatment work mainly depends on some production modes of assembly lines, the garbage treatment work is realized through a series of processes, and useful resources are extracted in the treatment process and are used as raw materials for production and utilization in other industries.
The perishable garbage treatment starts late in China, but the perishable garbage treatment is developed rapidly, particularly the domestic economy development and the garbage management system improvement, and the perishable garbage treatment market demand is huge. At present, the method for treating perishable garbage at home and abroad mainly comprises the following steps: simple stacking method, high-temperature fertilizer preparation method, incineration method, sanitary landfill method and comprehensive utilization method for producing organic compound fertilizer by garbage biological treatment. In addition, the method of feeding earthworms by using garbage, making bricks, making sugar, making fibers and the like is also available, but the method has small treatment scale and is not frequently adopted.
At present, the perishable garbage is treated at home and abroad basically by adopting a high-temperature anaerobic fertilizer preparation process and an anaerobic methane preparation process. The high-temperature composting process has the main disadvantages that firstly, the energy consumption is high, the temperature of equipment at least needs to be raised to more than 60 ℃, secondly, the utilization of regenerated fertilizer is hindered due to high salinity of perishable garbage, and thirdly, gases with odor, such as hydrogen sulfide, ammonia gas and the like, generated by high-temperature drying are concentrated, and the environment is poor. The main disadvantages of the anaerobic biogas preparation process are as follows: firstly, the process equipment is complex; secondly, the anaerobic fermentation process has higher requirement on material homogenization and unstable gas production; biogas slurry generated by anaerobic treatment needs to be matched with a huge sewage treatment system, and the concentration of sewage pollutants is extremely high; and fourthly, the equipment has high automation degree and higher requirement on management personnel. Fifthly, the initial volume of the treatment scale is high, the investment of the treatment cost is extremely high, and the occupied area is large.
The defects of the current production situation of perishable garbage treatment methods and equipment at home and abroad are combined, and the perishable garbage treatment mainly shows the following development trend in the future: 1) the construction scale is intensified and the occupied area is reduced, the traditional treatment technology occupies huge land, and with the shortage of land resources, the intensification and the occupied area reduction of a perishable garbage treatment system are a final target of the development of the industry; 2) the treatment method and equipment are efficient and intelligent, along with the improvement of labor cost and the rapid development of artificial intelligence, the method and equipment for treating perishable garbage are imperative, the labor cost input is effectively reduced, and the treatment efficiency is greatly improved; 3) the realization of the maximum working efficiency with lower cost investment is a great trend of the perishable garbage treatment industry due to low cost and diversification, and the research and development of a new treatment technology outside the traditional treatment mode also reflects the technical progress and the improvement of the treatment efficiency. Therefore, with the increasing situation of the refuse surrounding city, the development of a perishable refuse treatment process with low energy consumption, high reduction rate and high treatment efficiency is urgent.
The above background disclosure is only for the purpose of assisting understanding of the inventive concept and technical solutions of the present invention, and does not necessarily belong to the prior art of the present patent application, and should not be used for evaluating the novelty and inventive step of the present application in the case that there is no clear evidence that the above content is disclosed at the filing date of the present patent application.
Disclosure of Invention
Technical problem to be solved
In order to solve at least one of the technical problems mentioned in the background art, the present application provides an immobilized enzyme carrier that can immobilize a plurality of enzymes without preparing the carrier separately, and the immobilized enzyme preparation has an excellent recovery rate of enzyme activity.
The invention also provides a phase-change water-making treatment method for perishable garbage, which can remarkably promote the phase-change water-making treatment process through an optimization device and method, realize normal-temperature rapid degradation, degrade macromolecular organic matters into carbon dioxide and water, and realize the reduction rate of perishable garbage of not less than 99.3%.
(II) technical scheme
In order to solve the above technical problems or to achieve the above technical object, the present invention provides the following technical solutions.
[1] Application of chitosan oligosaccharide derivatives in preparation of immobilized enzyme carriers.
The chitosan oligosaccharide derivative is a product obtained by modifying chitosan oligosaccharide with gamma-aminobutyric acid.
The application specifically comprises the step of crosslinking the chitosan oligosaccharide derivative and graphene oxide by glutaraldehyde to prepare an immobilized enzyme carrier.
The method for preparing the chitosan oligosaccharide derivative comprises the following steps: dissolving gamma-aminobutyric acid in a large amount of MES buffer solution, sequentially adding EDC & HCl and NHS, stirring for at least 5h after all the materials are dissolved, adding chitosan oligosaccharide with the molecular weight of 1200-2000 Da, stirring at a high speed for 30-36 h, dialyzing by a 200-400 Da dialysis bag, and freeze-drying to obtain the chitosan oligosaccharide derivative.
Wherein the weight ratio of the gamma-aminobutyric acid to EDC & HCl, NHS and chitosan oligosaccharide is 1: 5.8-6.5: 3.4-3.8: 1.5-1.8; and/or
MES buffer solution is 0.1mol/L MES buffer solution with pH of 5.5; and/or
The stirring speed is 150-600 r/min; and/or
The high-speed stirring speed is 2400-4800 r/min; and/or
The reaction temperature is room temperature, specifically 25 +/-2 ℃; and/or
The dialysis time is 5-7 days.
The method comprises the steps of introducing a long-chain active group into chitosan oligosaccharide by using aminobutyric acid, grafting a new amide group on the chitosan oligosaccharide through an amidation reaction, prolonging the distance between an amino group and a main structure, then crosslinking the chitosan oligosaccharide and carboxyl and carbonyl on the surface of activated graphene oxide, replacing common chitosan and graphene oxide with the chitosan oligosaccharide to realize crosslinking through glutaraldehyde, obtaining an immobilized enzyme carrier with more excellent performance, improving the mechanical strength of a crosslinked product, enriching functional groups of the carrier, and enabling the obtained immobilized enzyme carrier to have a relatively broad-spectrum immobilization property and to be capable of efficiently solidifying lipase, neutral protease, cellulase, amylase and the like.
[2] A preparation method of an immobilized enzyme carrier comprises the following steps: preparing a chitosan oligosaccharide derivative aqueous solution, activating graphene oxide, mixing the chitosan oligosaccharide derivative and graphene oxide solution according to the weight ratio of 1: 1.5-2.0, reacting at room temperature for 1.5-3 h, centrifuging to obtain a precipitate, adding a glutaraldehyde solution for crosslinking for 1-2.5 h, removing uncrosslinked substances, collecting the precipitate, and freeze-drying to obtain the chitosan oligosaccharide derivative/graphene oxide composite material.
The mass fraction of the chitosan oligosaccharide derivative in the aqueous solution of the chitosan oligosaccharide derivative is not higher than 2.0%.
Specifically, the activated graphene oxide comprises the steps of uniformly dispersing 1 part by weight of graphene oxide into a phosphate buffer solution with the pH value of 7.0, then adding 25-30 parts by weight of 0.5mmol/L EDC and 25-30 parts by weight of 0.5mmol/L NHS, and shaking and activating for at least 2 hours at 150-240 r/min.
The mass fraction of the glutaraldehyde solution is 0.2-0.5%, and the addition amount of the glutaraldehyde solution is 25-50 times of the wet weight of the precipitate.
And washing with deionized water to remove uncrosslinked substances.
The invention aims to provide a phase-change water treatment process for perishable garbage, and the inventor discovers that an immobilized enzyme carrier with more excellent performance can be obtained by using a chitosan oligosaccharide derivative of the application to replace common chitosan to be crosslinked with graphene oxide, an amide group is grafted on the chitosan oligosaccharide through amidation reaction, and then the chitosan oligosaccharide is crosslinked with carboxyl and carbonyl on the surface of the activated graphene oxide, so that the mechanical strength of a crosslinked substance is improved, the functional groups of the carrier are enriched, the obtained immobilized enzyme carrier has a broad-spectrum immobilization property, lipase, neutral protease, cellulase, amylase and the like can be efficiently immobilized, the carriers do not need to be prepared for the enzymes respectively, the cost of the preparation is obviously reduced, and the phase-change immobilized enzyme water treatment process for perishable garbage is further facilitated.
[3] An immobilized enzyme carrier obtained by the process described in the aforementioned item [2 ].
[4] An immobilized enzyme preparation based on the immobilized enzyme carrier described in the aforementioned item [3 ].
In some preferred embodiments, the immobilized enzyme preparation comprises:
lipases such as candida rugosa enzyme; or
Neutral proteases such as serrapeptases; or
Cellulases such as exo- β -glucanases, endo- β -glucanases or β -glucosidases; or
Amylases, such as alpha-amylase, beta-amylase or gamma-amylase.
In some preferred embodiments, the immobilized enzyme preparations are prepared by the following methods respectively:
preparing the immobilized enzyme carrier of the item [3] into a carrier solution with the concentration of 40-50 g/L by using a pH7.0PBS buffer solution, preparing a lipase solution with the concentration of 5g/L by using the pH7.0PBS buffer solution, mixing the solution in a shaking table with the volume ratio of 1: 5-8 at 37 ℃, reacting for 1-1.5 h under the condition of 150-240 r/min, collecting immobilized enzyme, washing for 2-3 times by using a PBS buffer solution, centrifuging, drying, and storing at low temperature to obtain an immobilized lipase preparation; or
Adding the immobilized enzyme carrier of the item [3] with the weight of 10-15% of the solution into a protease solution with the concentration of 5g/L, oscillating and adsorbing for 0.5-1.5 h at the temperature of 25 +/-2 ℃ and under the condition of 150-240 r/min, then adding a glutaraldehyde diluted solution until the volume fraction of glutaraldehyde is not higher than 0.5%, continuing to react for 0.5-2.0 h, washing for 2-3 times, centrifuging, drying and storing at low temperature to obtain an immobilized protease preparation; or
Preparing the immobilized enzyme carrier of the item [3] into a carrier solution with the concentration of 40-50 g/L by using a PBS (phosphate buffer solution) with the pH value of 6.0, preparing a cellulase solution with the concentration of 5g/L by using an acetic acid-sodium acetate buffer solution with the pH value of 4.8, mixing the carrier solution and the cellulase solution in a shaking table with the volume ratio of 1: 3-5 at 4 ℃, oscillating the carrier solution and the cellulase solution for 30-45 min at the interval of 30-45 min under the condition of 150-240 r/min, circulating for 2-3 times, standing overnight at the temperature of 4 ℃, washing for 2-3 times, and freeze-drying to obtain an immobilized cellulase preparation; or
Preparing the immobilized enzyme carrier of the item [3] into a carrier solution with the concentration of 40-50 g/L by using a PBS buffer solution with the pH value of 5.0, preparing an amylase solution with the concentration of 5g/L by using a PBS buffer solution with the pH value of 6.0, mixing the solution in a shaking table at the room temperature of 25 +/-2 ℃ according to the volume ratio of 3-5, oscillating and adsorbing the solution for 2-4 h under the condition of 150-240 r/min, standing the solution overnight at the temperature of 4 ℃, washing the solution for 2-3 times after centrifugal separation, and freeze-drying and storing the solution to obtain the immobilized amylase preparation.
Through different curing methods, the immobilized enzyme carrier obtained according to the application can be used for respectively immobilizing lipase, neutral protease, cellulase and amylase, different carriers do not need to be prepared for different enzymes respectively, the time cost and the economic cost of immobilized enzyme are greatly reduced, the immobilized enzyme carrier has a broad-spectrum immobilization property, the immobilized enzyme carrier can be used for efficiently curing lipase, neutral protease, cellulase, amylase and the like, the recovery rate of enzyme activity of an enzyme preparation cured by the four enzymes is not lower than 85%, particularly the recovery rate of enzyme activity of the lipase is not lower than 90%, the enzyme activity of the immobilized enzyme preparation is remarkably improved, the carriers do not need to be prepared for the enzymes respectively, the cost of the immobilized enzyme preparation is remarkably reduced, and the phase-change water treatment of perishable garbage is facilitated.
[5] A phase-change water production treatment method for perishable garbage comprises the following steps:
s1, picking out non-degradable substances from perishable garbage, crushing the materials to be sieved by a 10-mesh sieve, placing the materials in a degradation bin of phase change water treatment equipment, stirring the materials by a stirring rod in the degradation bin and extruding the materials with the side wall of the degradation bin, filtering filtrate, heating the filtered filtrate to 50-60 ℃, standing and layering the filtered filtrate, leading out upper-layer crude oil, and leading other components into the equipment;
s2, adding a perishable garbage degrading microbial agent and the immobilized enzyme preparation in item [3] into equipment, and running a phase-change water preparation treatment process for 36-48 h under the conditions that the temperature is 15-40 ℃ and the relative humidity is 80-90%;
and the perishable garbage reduction rate is not less than 99.3% when the phase change water production treatment process is finished.
In some preferred embodiments, the non-degradable substance in step S1 includes metal, fabric, plastic, glass, rubber, stone, and the like which are not degradable by microorganisms and/or enzymes.
In some preferred embodiments, the phase-change water production processing apparatus in step S1 includes:
the degradation bin is used for containing and degrading perishable garbage, a bulge is arranged on the side wall of the degradation bin, and a stirring rod is arranged inside the degradation bin;
the feeding port is arranged at the top of the degradation bin and used for feeding perishable garbage;
the stirring motor is arranged outside the degradation bin, is connected with the stirring rod and can drive the stirring rod to stir and finish stirring and extruding the perishable garbage in the degradation bin;
the filter screen is arranged at the bottom of the degradation bin and can be opened and closed;
and the sedimentation tank is arranged at the lower part of the degradation bin and can be heated for accommodating and settling the filtrate filtered out from the degradation bin.
In some more preferred embodiments, the protrusions on the side wall of the degradation bin are arc-shaped, point-shaped or strip-shaped. The side wall of the degradation bin is provided with the bulge part, when the stirring rod stirs perishable garbage in the degradation bin pipe, the distances between the perishable garbage and different areas of the side wall are different, so that the squeezing effect is realized, firstly, the squeezing of the perishable garbage can enable grease and sewage in the perishable garbage to be separated out as soon as possible and filtered into a sedimentation tank through a filter screen, and the grease is led out from the sedimentation tank to further refine the grease; and secondly, the extrusion of the perishable garbage can enable cellulose and the like to displace and even peel off, further accelerate the degradation of the perishable garbage by a microbial inoculum in a perishable garbage degrading microbial inoculum and an enzyme in an immobilized enzyme preparation, and remarkably improve the phase change water preparation treatment process.
In some more preferred embodiments, the sidewall of the settling tank of step S1 is provided with a viewing window, and the sidewall of the settling tank is provided with a switchable outlet; the oil-water separation condition in the sedimentation tank can be detected through the observation window, the water layer can be led out through the leading-out hole and injected into equipment after the oil and the water are obviously layered, and then the crude oil layer is led out for further recovery treatment.
In some preferred embodiments, the crude oil obtained in step S1 is passed through a 0.22 μm filter membrane, then heated to 100 ℃ to remove moisture, cooled, added with a mixture of peroxyacetic acid and barium peroxide in an amount of 0.10-0.15% by weight of the oil and in a weight ratio of 1: 3.5-5.0, mixed uniformly, placed in a closed kneader, added with dry ice in an amount of 0.01-0.05% by weight of the oil and crosslinked and reformed at 25-35 ℃ and 1.0-1.4 MPa to obtain the lubricating oil. Crude grease filtered in the degradation bin can be simply filtered and heated to filter impurities and moisture without adding a dispersing agent and a flocculating agent, the micro-explosion of carbon dioxide enables grease micromolecules to be activated, specific peroxide is used as a catalyst, molecular connection can still occur even under the condition of lower pressure, crosslinking reforming is carried out to macromolecular grease, and the high-temperature-resistant degradable grease has excellent viscosity-temperature characteristics and wear resistance, the recycling of perishable garbage grease can be realized at lower cost, and the industrial added value of perishable garbage is improved.
In some preferred embodiments, the adding amount of the perishable garbage degrading bacteria in the step S2 is 0.5-1.0 kg/t/12 h.
In some preferred embodiments, the perishable waste degrading microbial inoculum of step S2 is obtained by mixing cross-linked slurry containing sodium alginate and high-substitution sulfobutyl ether-beta-cyclodextrin with degrading microbial inoculum, adding gypsum powder, and granulating.
In some more preferred embodiments, the perishable waste degrading microbial inoculum of step S2 comprises a base strain consisting of an effective amount of bacillus subtilis subspecies, bacillus alcalophagus, bacillus amyloliquefaciens, bacillus cereus, pseudomonas herbaceous, pseudomonas buchneri, pseudomonas moldy, pseudomonas azotoformans, acetobacter aceti, bacillus thiogenes, and bacillus terreus, and a synergistic strain consisting of an effective amount of pseudomonas flavum, pseudomonas buchneri, and pseudomonas winkle. The microorganisms in the developed perishable garbage degrading microbial inoculum are detected safely, including an acute oral toxicity test, an algae growth inhibition test, an acute transdermal toxicity test, an acute inhalation toxicity test and a fish acute toxicity test, and are safe, reliable and environment-friendly according to the detection result.
In some preferred embodiments, the phase-change water production process of step S2 is performed by ventilation for 8-10 times/h.
In some preferred embodiments, the immobilized enzyme preparation described in item [3] of step S2 is added in an amount of 0.1 to 0.2kg/t/12 h.
As is known to all, the perishable garbage is easy to rot and deteriorate and pollute the environment, has high water content and high organic matter content proportion, is rotten and smells, breeds pathogenic bacteria and pathogenic microorganisms, and can cause the transmission and infection of the pathogenic bacteria if the perishable garbage is directly utilized without proper treatment; the perishable garbage phase-change water production treatment process is completely a novel perishable garbage treatment process, firstly, oil separation is carried out on perishable garbage, crude oil obtained by separation is subjected to cross-linking reforming under low pressure under the catalysis of specific peroxide, and lubricating oil with excellent high-temperature stability and viscosity-temperature characteristic is obtained, so that resource utilization of biological oil is realized, and the industrial added value of perishable garbage is improved; then, an optimized degradation bin is added, and a perishable garbage degradation microbial inoculum and an immobilized enzyme preparation are added, so that the perishable garbage is subjected to phase change water production treatment under a mild aerobic condition at normal temperature, normal-temperature rapid degradation is realized, macromolecules in the perishable garbage, such as protein, lipids, starch, sugar, cellulose and the like, are finally degraded into carbon dioxide and water, the carbon dioxide and the water are colorless and tasteless substances, the environment is greatly improved, the water is purified to reach more than five types of water quality on the earth surface, the water can be recycled, and the reduction rate of the perishable garbage is not lower than 99.3%.
The phase-change water production treatment process for perishable garbage provided by the application is completely a novel process for perishable garbage treatment, is simple in process, realizes harmless treatment on organic pollutants, does not need external heat energy supply in the operation process, is low in energy consumption, does not need squeezing dehydration in the early stage, and does not need high-temperature drying and pollutant concentration during treatment; the drainage reaches the B-level standard of the Water quality Standard for Sewage drainage into cities and towns GBT31962-2015, and is discharged into municipal pipe network, and the secondary standard of the waste gas in the discharge Standard for malodorous pollutants (GB14554-93) completely meets the environmental protection requirements specified by the state. The method can be widely used for the local decrement treatment of residential garbage, the local decrement treatment of perishable garbage in large commercial areas, the local decrement treatment of perishable garbage in institutions and canteens, the local decrement treatment of perishable garbage in schools and the perishable garbage treatment of large garbage transfer stations.
The perishable garbage phase change water production treatment process provided by the application fills the domestic gap of perishable garbage in-situ chemical treatment, and has obvious technical competitiveness: the process is simple, and the organic pollutants are treated in a harmless way. The energy consumption is low, and external heat energy supply is not needed in the operation process; the operation flexibility is large, the method can adapt to the characteristic of large change of the yield of the perishable garbage components, and the application prospect is good. The method not only can realize the treatment of 'reduction, resource and harmlessness' proposed by the state, but also can realize the treatment on the spot and the information management, achieves the aim of 'five-purpose', and completely accords with the current national environmental policy. The overall arrangement of the whole garbage disposal industry is changed, and the comprehensive treatment capability of the domestic garbage disposal environment is greatly improved.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
(III) advantageous effects
The technical scheme of the invention has the following advantages:
1) the chitosan oligosaccharide derivative can be used for replacing common chitosan and graphene oxide for crosslinking to obtain an immobilized enzyme carrier with more excellent performance, the mechanical strength of a crosslinked product is improved, the functional groups of the carrier are enriched, the obtained immobilized enzyme carrier has a relatively broad-spectrum immobilization property, lipase, neutral protease, cellulase, amylase and the like can be efficiently immobilized, and the carriers do not need to be prepared for the enzymes respectively;
2) the recovery rate of the enzyme activity of the immobilized enzyme carrier to the enzyme preparation solidified by the lipase, the neutral protease, the cellulase and the amylase is not lower than 85 percent, particularly the recovery rate of the enzyme activity of the lipase is not lower than 90 percent, and the enzyme activity of the immobilized enzyme preparation is remarkably improved;
3) by optimizing the side wall of the degradation bin, the perishable garbage can be extruded, and grease and sewage in the perishable garbage can be separated out into a sedimentation tank as soon as possible, so that the grease is further refined; the extrusion can also cause cellulose and the like to displace and even peel off, accelerate the degradation of the perishable garbage by microbial inoculum in the perishable garbage degrading microbial inoculum and enzyme in the immobilized enzyme preparation, and remarkably promote the phase change water preparation treatment process;
4) when the crude oil is refined, impurities and moisture can be filtered and removed through filtration and heating without adding a dispersing agent and a flocculating agent, the crude oil can be crosslinked and reformed into macromolecular oil at a lower pressure by using a specific peroxide as a catalyst, and the crude oil has excellent viscosity-temperature characteristics and wear resistance, can be recycled at a lower cost, and improves the industrial added value of perishable garbage;
5) the safety detection of the microorganisms in the perishable garbage degrading microbial agent is carried out, wherein the detection comprises an acute oral toxicity test, an algae growth inhibition test, an acute transdermal toxicity test, an acute inhalation toxicity test and a fish acute toxicity test, and the detection result shows that the microorganisms are safe and reliable and are environment-friendly;
6) through adding the optimized degradation bin and adding perishable garbage degradation microbial inoculum and immobilized enzyme preparation, the perishable garbage is subjected to phase change water production treatment under the relatively mild aerobic condition at normal temperature, the normal-temperature rapid degradation is realized, macromolecules in the perishable garbage, such as protein, lipids, starch, sugar, cellulose, and the like, are finally degraded into carbon dioxide and water, the carbon dioxide and the water are both colorless and tasteless substances, the environment is greatly improved, the water is purified to reach more than five types of water quality on the earth surface, the perishable garbage can be recycled, and the reduction rate of the perishable garbage is not lower than 99.3%.
The invention adopts the technical scheme for achieving the purpose, makes up the defects of the prior art, and has reasonable design and convenient operation.
Drawings
The foregoing and/or other objects, features, advantages and embodiments of the invention will be more readily understood from the following description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of a process for preparing a chitosan oligosaccharide derivative of the present invention;
FIG. 2 is a schematic view of a phase change water production treatment apparatus of the present invention;
FIG. 3 is a first longitudinal schematic view of a degradation bin of the phase-change water production treatment device of the present invention;
FIG. 4 is a second longitudinal schematic view of a degradation bin of the phase-change water production treatment device of the present invention;
FIG. 5 is a third longitudinal schematic view of a degradation bin of the phase-change water production treatment device of the present invention;
fig. 6 is a statistical representation of the perishable waste weight reduction rate of the perishable waste phase change water treatment protocol of the present invention.
Description of reference numerals: 1. a degradation bin; 2. a stirring rod; 3. a feeding port; 4. a stirring motor; 5. filtering with a screen; 6. a sedimentation tank; 7. a boss portion; 8. an observation window; 9. an outlet hole.
Detailed Description
In a preferred embodiment of the application, the perishable waste degrading bacteria agent is prepared by the following method:
1) and preparing high-substitution sulfobutyl ether-beta-cyclodextrin: dissolving the recrystallized beta-cyclodextrin in a large amount of 10-20% sodium hydroxide solution, dispersing until the solution is colorless and transparent, slowly dripping 1, 4-sulfobutyl lactone with the molar weight being 10-20 times of that of the beta-cyclodextrin into the solution, stirring and reacting for 6-12 hours at 50-70 ℃, maintaining the pH of a reaction solution to 11-12, adjusting the pH of the reaction solution to be neutral when the reaction solution is yellow and clear, concentrating to 1/3-1/2 volume, slowly dripping the reaction solution into an ethanol solution to generate a precipitate, filtering, drying the precipitate, precipitating again with absolute ethanol, and performing vacuum drying to obtain the beta-cyclodextrin solid phase-change material;
2) compounding crosslinking slurry: preparing an aqueous solution with the total concentration of sodium alginate and high-substitution-degree sulfobutyl ether-beta-cyclodextrin being not higher than 2.0% at 40-80 ℃, and then adding 5-50 mmol/L of calcium, potassium, zinc, copper and iron elements to prepare a cross-linking slurry;
3) and compounding degrading bacteria liquid: compounding a basic bacterium liquid containing effective amounts of bacillus subtilis subspecies, bacillus alcalophilus, bacillus amyloliquefaciens, bacillus cereus, pseudomonas palustris, pseudomonas buchneri, pseudomonas moldy, pseudomonas azotoformis, acetobacter aceti, bacillus thiogenes and bacillus terreus with a synergistic bacterium liquid containing effective amounts of pseudomonas yellowish, pseudomonas buchneri and pseudomonas winkle to obtain a degrading bacterium liquid;
4) preparing a perishable garbage degrading microbial inoculum: and cooling the cross-linked slurry to 20-40 ℃, uniformly mixing the cross-linked slurry with the degrading agent bacterial liquid, adding gypsum powder, uniformly mixing to obtain a carrier matrix containing the degrading agent bacterial agent, and granulating and drying to obtain the perishable garbage degrading bacterial agent.
The method for preparing the perishable garbage degrading microbial inoculum further comprises the following limiting factors:
a) in the step 1), the speed of slowly dropping 1, 4-sulfobutyl lactone is 1.0-5.0 mL/1 min; and/or
b) In the step 1), the stirring speed is 150-900 r/min; and/or
c) In the step 1), 0.5-2.0 mol/L sodium hydroxide solution and 0.2-1.0 mol/L hydrochloric acid are used for maintaining or regulating the pH of the reaction solution; and/or
d) In the step 1), the volume content of the ethanol solution is not less than 85 vol%; and/or
e) In the step 1), the precipitation drying means drying at 45-60 ℃ for at least 6 hours; and/or
f) In the step 1), vacuum drying means drying in a vacuum drying oven at 45-70 ℃ to constant weight; and/or
g) In the step 1), the substitution degree of the high-substitution-degree sulfobutyl ether-beta-cyclodextrin is not less than 5.0, preferably not less than 6.0, more preferably 6.0-10.0, and most preferably 6.5-8.0; and/or
h) In the step 2), the viscosity of the sodium alginate is 100-500 cps; and/or
i) In the step 2), the weight ratio of sodium alginate to high-substitution-degree sulfobutyl ether-beta-cyclodextrin is 1-5: 1; and/or
j) In the step 2), the calcium, potassium, zinc, copper and iron elements can be respectively provided by calcium chloride, monopotassium phosphate, zinc chloride, copper sulfate and ferric chloride without limitation; and/or
k) In the step 3), the basic bacteria and the synergistic bacteria are subjected to amplification culture, the concentration content difference of each strain in each basic bacteria or each strain in the synergistic bacteria of the degrading bacteria liquid is not more than 10 times, and the content of the viable bacteria of the basic bacteria is 5 multiplied by 1010～1×1011cfu/mL, the viable bacteria content of the synergistic bacteria is 1 multiplied by 109～5×109cfu/mL; and/or
l) in the step 4), the weight ratio of the cross-linking slurry to the degrading agent bacterial liquid to the gypsum powder is 3-8: 1: 3-10; and/or
m) in the step 4), granulating, namely specifically, preparing a carrier matrix containing a degrading agent microbial inoculum into cylindrical particles with the diameter of 5-15 mm and the length of 10-20 mm; and/or
n) in the step 4), drying means placing the perishable garbage degrading microbial inoculum particles in a ventilated place for natural air drying for at least 24 hours.
The preparation method specifically comprises the steps of mixing a bacterial solution containing the basic bacteria and the synergistic bacteria with a cross-linking slurry containing high-substitution sulfobutyl ether-beta-cyclodextrin and sodium alginate in a specific ratio, adding gypsum powder for granulation to obtain the perishable garbage degrading microbial agent, cross-linking and concentrating the bacteria by using the cross-linking slurry and limiting the bacteria in a certain spatial range, wherein the microbial agent particles have certain mechanical strength, the obtained immobilized microorganisms are live bacteria, activation is not needed during use, and during application, the microorganisms bound in pores of a carrier overflow to degrade perishable garbage and finally bind an immobilized enzyme preparation, the protein, lipid, starch, sugar, cellulose and other macromolecules in the perishable garbage are finally degraded into carbon dioxide and water, the water is purified to reach the five water qualities on the earth surface, the water can be recycled, and the reduction rate of the perishable garbage is not less than 99.3%.
The recovery rate of the enzyme activity of the immobilized lipase preparation of the present application was determined according to the method described in the prior art "https:// wenku. baidu. com/view/03010b240242a 8956aeceeef 40f. html".
The method for measuring the recovery rate of the enzyme activity of the immobilized protease preparation comprises the following steps: dissolving the immobilized protease preparation in PBS buffer solution with the pH value of 7.0 to prepare a solution with the mass fraction of 1%, measuring the enzyme activity by an ultraviolet absorption method, comparing the enzyme activity of the protease when preparing the immobilized protease preparation, and calculating the recovery rate of the enzyme activity.
The recovery rate of enzyme activity of the immobilized cellulase preparation of the application is determined according to the method for determining enzyme activity of immobilized cellulase in section 2.3.3.4 of the prior art of immobilization of cellulase and application thereof (Master academic thesis of Jiangsu university, Li Yue, 2015).
The recovery rate of the enzyme activity of the amylase in the application is determined according to the research on the cross-linked chitosan immobilized alpha-amylase (No. 4 of 27 of the university of Xihua, Nature science) in the prior art, and specifically, the activity of the immobilized enzyme is determined firstly, and then the recovery rate of the enzyme activity is determined according to the ratio of the activity of the immobilized enzyme to the activity of the original enzyme solution.
The starting materials described herein are commercially available and include, but are not limited to:
bacillus subtilis subspecies ATCC 6633, Bacillus alcalophilus ATCCBA-125, Bacillus cereus CMCC (B)63301, Pseudomonas graminis CGMCC 1.1797, Pseudomonas brucei ATCC 49642;
bacillus amyloliquefaciens, Pseudomonas moldavica, Pseudomonas azotoformans, Acetobacter aceti, Bacillus thiogenes, Geobacillus, Pseudomonas flaccida, Pseudomonas brucei and Pseudomonas winkle were all purchased from the institute of Biotechnology, Beijing, Ministry of Industrial science and technology.
The present invention is described in detail below.
Example 1:
the embodiment provides an immobilized enzyme carrier, which is prepared through the following steps: as shown in the flow chart of figure 1, 1g of gamma-aminobutyric acid is dissolved in 150mL0.1mol/L MES buffer solution with pH value of 5.5, 6.0g of EDC & HCl and 3.5g of NHS are sequentially added, after being completely dissolved, 300r/min is stirred for 6h, 1.8g of chitosan oligosaccharide with the relative molecular weight of 1500Da is added, 3000r/min is stirred for 36h at high speed, then 400Da dialysis bags are used for dialysis, and the chitosan oligosaccharide derivatives are obtained by freeze drying, and 1% chitosan oligosaccharide derivative aqueous solution is prepared; uniformly dispersing 1g of graphene oxide into 200g of phosphate buffer solution with pH7.0, then adding 28g of 0.5mmol/L EDC and 28g of 0.5mmol/L NHS, and oscillating and activating for 4h at 180 r/min; mixing the two solutions according to the weight ratio of the chitosan oligosaccharide derivative to the graphene oxide of 1:1.6, reacting for 2 hours at room temperature, centrifuging to obtain a precipitate, adding 0.3% glutaraldehyde solution with the weight 40 times of the wet weight of the precipitate for crosslinking for 1.5 hours, washing with deionized water to remove uncrosslinked substances, collecting the precipitate, and freeze-drying to obtain the chitosan oligosaccharide/graphene oxide composite material.
The embodiment also provides various immobilized enzyme preparations based on the carrier, which specifically comprise:
immobilized lipase preparation: the carrier obtained in the embodiment is prepared into a carrier solution with the concentration of 45g/L by using PBS buffer solution with the pH value of 7.0, is prepared into a candida rugosa lipase solution with the concentration of 5g/L by using PBS buffer solution with the pH value of 7.0, is mixed in a shaking table with the volume ratio of 1:6 at the temperature of 37 ℃ to react for 1.5h under the condition of 240r/min, is washed for 3 times by using PBS buffer solution after being collected and immobilized, and is obtained after centrifugal drying and low-temperature storage;
immobilized protease preparation: adding the carrier obtained in the embodiment with the solution weight of 12% into a serrapeptase solution with the concentration of 5g/L, oscillating and adsorbing for 1.5h at the temperature of 25 +/-2 ℃ and 240r/min, then adding a glutaraldehyde diluted solution until the volume fraction of glutaraldehyde is 0.4%, continuing to react for 1.5h, washing for 3 times, centrifuging, drying and storing at low temperature to obtain the serrapeptase;
immobilized cellulase preparation: the carrier obtained in the embodiment is prepared into a carrier solution with the concentration of 45g/L by PBS buffer solution with the pH value of 5.0, and is prepared into a beta-glucosidase solution with the concentration of 5g/L by acetic acid-sodium acetate buffer solution with the pH value of 4.8, the beta-glucosidase solution is mixed in a shaking table with the volume ratio of 1:4 at the temperature of 4 ℃, the shaking table is shaken for 45min at the interval of 45min at the speed of 180r/min, the mixture is circulated for 3 times and then is kept stand overnight at the temperature of 4 ℃, and the carrier is obtained by centrifugal water washing for 3 times and then freeze-drying;
immobilized amylase preparation: the carrier obtained in the embodiment is prepared into a carrier solution with the concentration of 45g/L by using PBS buffer solution with the pH value of 6.0, is prepared into an alpha-amylase solution with the concentration of 5g/L by using PBS buffer solution with the pH value of 6.0, is mixed in a shaking table with the temperature of 25 +/-2 ℃ according to the volume ratio of 1:4, is vibrated and adsorbed for 2.5h under the condition of 240r/min, is kept stand overnight at the temperature of 4 ℃, is washed for 3 times after centrifugal separation, and is freeze-dried and stored to obtain the carrier.
Example 2:
the embodiment provides an immobilized enzyme carrier, which is prepared through the following steps: dissolving 1g of gamma-aminobutyric acid in 150mL0.1mol/L MES buffer solution with pH value of 5.5, sequentially adding 6.4g of EDC & HCl and 3.8g of NHS, stirring for 5h at 450r/min after all the gamma-aminobutyric acid is dissolved, adding 1.6g of chitosan oligosaccharide with the relative molecular weight of 1800Da, stirring for 30h at high speed at 3600r/min, dialyzing by a 400Da dialysis bag, and freeze-drying to obtain a chitosan oligosaccharide derivative, and preparing 1% chitosan oligosaccharide derivative aqueous solution; uniformly dispersing 1g of graphene oxide into 200g of phosphate buffer solution with pH7.0, then adding 25g of 0.5mmol/L EDC and 25g of 0.5mmol/L NHS, and oscillating and activating for 2h at 240 r/min; mixing the two solutions according to the weight ratio of the chitosan oligosaccharide derivative to the graphene oxide of 1:1.8, reacting for 1.5h at room temperature, centrifuging to obtain a precipitate, adding 0.5% glutaraldehyde solution with the wet weight of 30 times of that of the precipitate for crosslinking for 1.5h, washing with deionized water to remove uncrosslinked substances, collecting the precipitate, and freeze-drying to obtain the chitosan oligosaccharide/graphene oxide composite material.
The embodiment also provides various immobilized enzyme preparations based on the carrier, which specifically comprise:
immobilized lipase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized protease preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized cellulase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized amylase preparation: the preparation method is the same as the corresponding method of example 1.
Example 3:
this example provides an immobilized enzyme carrier, which was prepared in the same manner as in example 1 except that the relative molecular weight of chitosan oligosaccharide in this example was 1000Da, and the immobilized enzyme carrier was prepared in the same manner as in example 1.
The embodiment also provides various immobilized enzyme preparations based on the carrier, which specifically comprise:
immobilized lipase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized protease preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized cellulase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized amylase preparation: the preparation method is the same as the corresponding method of example 1.
Example 4:
this example provides an immobilized enzyme carrier, which was prepared in the same manner as in example 1 except that the relative molecular weight of chitosan oligosaccharide in this example was 3200Da, and the immobilized enzyme carrier was prepared in the same manner as in example 1.
The embodiment also provides various immobilized enzyme preparations based on the carrier, which specifically comprise:
immobilized lipase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized protease preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized cellulase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized amylase preparation: the preparation method is the same as the corresponding method of example 1.
Example 5:
the embodiment provides an immobilized enzyme carrier, which is prepared through the following steps: preparing 1% aqueous solution of chitosan oligosaccharide with relative molecular weight of 2800 Da; uniformly dispersing 1g of graphene oxide into 200g of phosphate buffer solution with pH7.0, then adding 25g of 0.5mmol/L EDC and 25g of 0.5mmol/L NHS, and oscillating and activating for 2h at 240 r/min; mixing the two solutions according to the weight ratio of 1:1.8 of chitosan oligosaccharide to graphene oxide, reacting for 1.5h at room temperature, centrifuging to obtain precipitate, adding 0.5% glutaraldehyde solution with the wet weight of 30 times of that of the precipitate for crosslinking for 1.5h, washing with deionized water to remove uncrosslinked substances, collecting the precipitate, and freeze-drying to obtain the chitosan oligosaccharide/graphene oxide composite material.
The embodiment also provides various immobilized enzyme preparations based on the carrier, which specifically comprise:
immobilized lipase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized protease preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized cellulase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized amylase preparation: the preparation method is the same as the corresponding method of example 1.
Example 6:
the embodiment provides an immobilized enzyme carrier, which is prepared through the following steps: activating chitosan with relative molecular weight of 10000Da and deacetylation degree of 95% with 120 weight times of 2.0% acetic acid solution; then, uniformly dispersing 1g of graphene oxide into 200g of phosphate buffer solution with pH7.0, then adding 25g of 0.5mmol/L EDC and 25g of 0.5mmol/L NHS, and oscillating and activating for 2h at 240 r/min; mixing the two solutions according to the weight ratio of 1:1.8 of chitosan to graphene oxide, reacting for 1.5h at room temperature, centrifuging to obtain a precipitate, adding a 1.0% glutaraldehyde solution with the wet weight of 50 times of that of the precipitate for crosslinking for 4h, washing with deionized water to remove uncrosslinked substances, collecting the precipitate, and freeze-drying to obtain the graphene oxide.
The embodiment also provides various immobilized enzyme preparations based on the carrier, which specifically comprise:
immobilized lipase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized protease preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized cellulase preparation: the preparation method is the same as that of the corresponding method of the example 1;
immobilized amylase preparation: the preparation method is the same as the corresponding method of example 1.
Experimental example 1:
and (3) sequentially measuring the recovery rate of the enzyme activity of each immobilized enzyme preparation obtained in the embodiments 1-6, respectively, and measuring the recovery rate of the enzyme activity of each immobilized enzyme preparation again after the immobilized enzyme preparation is subjected to heat preservation at 50 ℃ for 180min, wherein the thermal stability of the immobilized enzyme preparation is represented by the high and low recovery rates of the enzyme activity. The enzyme activity recovery rate and the thermal stability statistical result of each immobilized enzyme preparation are shown in table 1.
TABLE 1 recovery of enzyme activity and thermal stability of immobilized enzyme preparations
As can be seen from table 1, the immobilized lipase preparations, the immobilized protease preparations, the immobilized cellulase preparations and the immobilized amylase preparations prepared from the vectors obtained in examples 1 and 2 of the preferred embodiment of the present application have excellent enzyme activity recovery rates, particularly, the enzyme activity recovery rate of lipase is not less than 90%, which significantly improves the enzyme activity of the immobilized enzyme preparations, and indicates that the vectors obtained in the scheme of the present application can be used for efficiently curing lipase, neutral protease, cellulase, amylase and the like, have a relatively broad-spectrum immobilization property, and do not need to prepare carriers for the above enzymes respectively, thereby reducing process complexity and cost, and the immobilized enzyme preparations obtained by the method also have relatively excellent heat stability, and the enzyme activity recovery rates are not significantly reduced after 180min at 50 ℃. It can also be seen from table 1 that too high or too low average molecular weight of chitosan oligosaccharide during preparation of the carrier is not beneficial to loading enzyme by the carrier, and the chitosan oligosaccharide is not modified and the excellent broad-spectrum immobilization property and heat stability of the carrier obtained by the scheme of the present application cannot be obtained by directly preparing the carrier with chitosan.
Example 7:
the embodiment provides a perishable garbage degrading microbial inoculum, which is prepared by the following steps:
1) and preparing high-substitution sulfobutyl ether-beta-cyclodextrin: dissolving 0.01mol of recrystallized beta-cyclodextrin in 500g of 12% sodium hydroxide solution, dispersing until the solution is colorless and transparent, slowly dripping 0.15mol of 1, 4-sulfobutyl lactone into the solution at 5.0mL/min, stirring at 55 ℃ for 12h, maintaining the pH of a reaction solution to 11 (maintaining by 0.5mol/L sodium hydroxide solution and 0.5mol/L hydrochloric acid, the same applies below), adjusting the pH to be neutral when the reaction solution is yellow and clear, concentrating to 1/3 volume, slowly dripping into 90% vol ethanol solution within 1h to generate precipitate, filtering, drying the precipitate at 50 ℃ for 12h, precipitating again by absolute ethanol, drying in a vacuum drying oven at 50 ℃ until the weight is constant, and determining that the average substitution degree of the sulfobutyl ether-beta-cyclodextrin is 6.4;
2) compounding crosslinking slurry: preparing sodium alginate (200 +/-20 cps) and sulfobutyl ether-beta-cyclodextrin obtained in step 1) into a water solution with the total concentration of 1.5% according to the weight ratio of 2:1 at 60 ℃, and then adding 20mmol/L calcium chloride, 30mmol/L potassium dihydrogen phosphate, 5mmol/L zinc chloride, 5mmol/L copper sulfate and 20mmol/L ferric chloride to prepare cross-linked slurry;
3) and compounding degrading bacteria liquid: culturing the strains in an enlarged way, and then compounding the strains with 8 multiplied by 109cfu/mL Bacillus subtilis subspecies, 8 × 109cfu/mL Bacillus alcalophagus, 8X 109cfu/mL Bacillus amyloliquefaciens, 8X 109cfu/mL Bacillus cereus, 8X 109cfu/mL Pseudomonas herbicola, 8X 109cfu/mL Pseudomonas brunetti, 8X 109cfu/mL Pseudomonas fragi, 8X 109cfu/mL Pseudomonas azotoformans, 8X 109cfu/mL Acetobacter aceti, 8X 109cfu/mL Sulfur-producing Bacillus, 8X 109cfu/mL Geobacillus, 5X 108cfu/mL Pseudomonas flacci, 5X 108cfu/mL Pseudomonas buchneri and 8X 108cfu/mL pseudomonas winkle degrading bacteria liquid;
4) preparing a perishable garbage degrading microbial inoculum: and cooling the cross-linked slurry to 25 ℃, uniformly mixing the cross-linked slurry with the degrading agent bacterial liquid, adding gypsum powder, uniformly mixing to obtain a carrier matrix containing the degrading agent bacterial agent, extruding the cross-linked slurry, the degrading agent bacterial liquid and the gypsum powder according to the weight ratio of 5:1:6 to prepare cylindrical particles with the diameter of 10mm and the length of 20mm, and naturally airing the cylindrical particles in a ventilated place for 48 hours to obtain the perishable garbage degrading bacterial agent.
Example 8:
the embodiment provides a perishable garbage degrading microbial inoculum, which is prepared by the following steps:
1) and preparing high-substitution sulfobutyl ether-beta-cyclodextrin: dissolving 0.01mol of recrystallized beta-cyclodextrin in 500g of 10% sodium hydroxide solution, dispersing until the solution is colorless and transparent, slowly dripping 0.18mol of 1, 4-sulfobutyl lactone into the solution at 5.0mL/min, stirring at 60 ℃ for reaction at 450r/min for 12h, maintaining the pH of the reaction solution to 11.5, adjusting the pH of the reaction solution to be neutral when the reaction solution is yellow and bright, concentrating to 1/2 volume, slowly generating precipitate in ethanol solution with 85 vol% in 1h, drying the precipitate at 50 ℃ for 12h after filtering, precipitating again with absolute ethanol, drying in a vacuum drying oven at 55 ℃ until the weight is constant, and obtaining the sulfobutyl ether-beta-cyclodextrin with the average substitution degree of 6.7 after determination;
2) compounding crosslinking slurry: preparing sodium alginate (200 +/-20 cps) and sulfobutyl ether-beta-cyclodextrin obtained in step 1) into an aqueous solution with the total concentration of 2.0% according to the weight ratio of 3:1 at 55 ℃, and then adding 40mmol/L calcium chloride, 10mmol/L potassium dihydrogen phosphate, 20mmol/L zinc chloride, 20mmol/L copper sulfate and 40mmol/L ferric chloride to prepare cross-linked slurry;
3) and compounding degrading bacteria liquid: culturing the strains in an enlarged way, and then compounding the strains with 8 multiplied by 109cfu/mL Bacillus subtilis subspecies, 8 × 109cfu/mL Bacillus alcalophagus, 5X 109cfu/mL Bacillus amyloliquefaciens, 6X 109cfu/mL Bacillus cereus, 9X 109cfu/mL Pseudomonas herbicola, 9X 109cfu/mL Pseudomonas buchneri, 4X 109cfu/mL Pseudomonas fragi, 8X 109cfu/mL Pseudomonas azotoformans, 8X 109cfu/mL Acetobacter aceti, 9X 109cfu/mL Sulfur-producing Bacillus, 8X 109cfu/mL Geobacillus, 9X 108cfu/mL Pseudomonas flacci, 8X 108cfu/mL Pseudomonas buchneri and 9X 108Degrading bacterium of cfu/mL pseudomonas winkleLiquid;
4) preparing a perishable garbage degrading microbial inoculum: and cooling the cross-linked slurry to 25 ℃, uniformly mixing the cross-linked slurry with the degrading agent bacterial liquid, adding gypsum powder, uniformly mixing to obtain a carrier matrix containing the degrading agent bacterial agent, extruding the cross-linked slurry, the degrading agent bacterial liquid and the gypsum powder in a weight ratio of 4:1:7 to prepare cylindrical particles with the diameter of 10mm and the length of 10mm, and naturally airing the cylindrical particles in a ventilated place for 36 hours to obtain the perishable garbage degrading bacterial agent.
Example 9:
the embodiment provides a perishable garbage degrading microbial inoculum, which is prepared by the following steps:
1) and preparing high-substitution sulfobutyl ether-beta-cyclodextrin: dissolving 0.01mol of recrystallized beta-cyclodextrin in 500g of 12% sodium hydroxide solution, dispersing until the solution is colorless and transparent, slowly dripping 0.20mol of 1, 4-sulfobutyl lactone into the solution at 4.0mL/min, stirring at 60 ℃ for reaction at 600r/min for 9h, maintaining the pH of the reaction solution to 12, adjusting the pH to be neutral when the reaction solution is yellow and bright, concentrating to 1/3 volume, slowly dripping into an ethanol solution with the volume content of 90 vol% to generate a precipitate, filtering, drying the precipitate at 55 ℃ for 12h, precipitating again with absolute ethanol, drying in a vacuum drying oven at 60 ℃ until the weight is constant, and obtaining the sulfobutyl ether-beta-cyclodextrin with the average substitution degree of 7.3 by determination;
2) compounding crosslinking slurry: preparing sodium alginate (200 +/-20 cps) and sulfobutyl ether-beta-cyclodextrin obtained in step 1) into a water solution with the total concentration of 1.8% according to the weight ratio of 1:1 at 50 ℃, and then adding 50mmol/L calcium chloride, 50mmol/L potassium dihydrogen phosphate, 20mmol/L zinc chloride, 20mmol/L copper sulfate and 10mmol/L ferric chloride to prepare cross-linked slurry;
3) and compounding degrading bacteria liquid: culturing the strains in an enlarged way, and then compounding the strains containing 6 multiplied by 109cfu/mL Bacillus subtilis subspecies, 8 × 109cfu/mL Bacillus alcalophagus, 8X 109cfu/mL Bacillus amyloliquefaciens, 9X 109cfu/mL Bacillus cereus, 9X 109cfu/mL Pseudomonas herbicola, 8X 109cfu/mL Pseudomonas brunetti, 6X 109cfu/mL Pseudomonas fragi, 6X 109cfu/mL Pseudomonas azotoformans, 5X 109cfu/mL Acetobacter aceti, 6X 109cfu/mL Sulfur-producing Bacillus, 8X 109cfu/mL Geobacillus, 1X 109cfu/mL Pseudomonas flacci, 1.5X 109cfu/mL Pseudomonas buchneri and 1X 109cfu/mL pseudomonas winkle degrading bacteria liquid;
4) preparing a perishable garbage degrading microbial inoculum: and cooling the cross-linked slurry to 25 ℃, uniformly mixing the cross-linked slurry with the degrading agent bacterial liquid, adding gypsum powder, uniformly mixing to obtain a carrier matrix containing the degrading agent bacterial agent, extruding the cross-linked slurry, the degrading agent bacterial liquid and the gypsum powder according to the weight ratio of 5:1:8 to prepare cylindrical particles with the diameter of 15mm and the length of 20mm, and naturally airing the cylindrical particles in a ventilated place for 48 hours to obtain the perishable garbage degrading bacterial agent.
Example 10:
the embodiment provides a perishable garbage degrading microbial inoculum, which is prepared by the following steps:
1) and preparing high-substitution sulfobutyl ether-beta-cyclodextrin: same as step 1) of example 9;
2) compounding crosslinking slurry: same as step 2) of example 9;
3) and compounding degrading bacteria liquid: culturing the strains in an enlarged way, and then compounding the strains containing 6 multiplied by 109cfu/mL Bacillus subtilis subspecies, 8 × 109cfu/mL Bacillus alcalophagus, 8X 109cfu/mL Bacillus amyloliquefaciens, 9X 109cfu/mL Bacillus cereus, 9X 109cfu/mL Pseudomonas herbicola, 8X 109cfu/mL Pseudomonas brunetti, 6X 109cfu/mL Pseudomonas fragi, 6X 109cfu/mL Pseudomonas azotoformans, 5X 109cfu/mL Acetobacter aceti, 6X 109cfu/mL Sulfur-producing Bacillus, 8X 109cfu/mL Geobacillus, 1X 109cfu/mL Pseudomonas flaccid and 1X 109cfu/mL pseudomonas winkle degrading bacteria liquid;
4) preparing a perishable garbage degrading microbial inoculum: same as step 4 of example 9).
Experimental example 2:
the experimental example provides a series of perishable garbage phase-change water-making treatment schemes for a certain batch of perishable garbage and verifies the reduction rate, selects the perishable garbage in life in a certain area of Hangzhou city in Zhejiang province uniformly, picks out the non-degradable substances such as metal, fabric, plastic, glass, rubber, stone, ceramic and the like, the perishable garbage mainly comprises melon peel, rice, flour products, meat, vegetables, fish, broken bones and the like, has the water content of 78.8 percent and the organic matter moisture content of 74.2 +/-2.5 percent, is crushed to 10 meshes of sieves, has the water content of 81.5 percent, is placed in the phase-change water-making treatment equipment shown in figure 2, and comprises a degradation bin 1 for containing and degrading the perishable garbage, a bulge part 7 is arranged on the side wall of the degradation bin 1, a stirring rod 2 is arranged inside the degradation bin 1, the perishable garbage can be put into the degradation bin 1 through a feeding port 3 arranged on the top of the degradation bin 1, stirring by a stirring rod 2 in the degradation bin 1 and extruding with the side wall of the degradation bin 1 for 1h, extruding by the stirring rod 2 and a convex part 7 on the side wall, filtering out a liquid phase in the degradation garbage into a sedimentation tank 6 through a filter screen 5 arranged at the bottom of the degradation bin 1, heating filtrate in the sedimentation tank 6 to 60 ℃, standing for layering, leading out upper-layer crude grease for additional treatment through a leading-out hole 9 after seeing that oil and water in the sedimentation tank 6 are separated through an observation window 8 on the side wall of the sedimentation tank 6, and leading other components into equipment again; in particular, the degradation bin 1 of the experimental example has the structure shown in fig. 3, that is, the protrusion 7 therein is arc-shaped, and it is verified that when the protrusion 7 is point-shaped and strip-shaped, the degradation bin 1 can achieve the same effects of extruding the perishable garbage, accelerating the degradation speed and improving the treatment process.
Adding 0.2kg/t/12h of the immobilized enzyme preparation (the same amount of immobilized lipase preparation, immobilized protease preparation, immobilized cellulase preparation and immobilized amylase preparation) obtained in the embodiments 1-6 and/or 0.8kg/t/12h of the perishable garbage degrading bacteria agent obtained in the embodiments 7-9 into equipment, and changing a water treatment process to operate for 48h under the conditions that the temperature is 35 ℃, the relative humidity is 85% and ventilation is 10 times/h; details of the protocol the perishable waste degraded by the protocol described in table 2 is from the same batch as shown in table 2.
TABLE 2 phase Change Water treatment protocol for perishable waste
Note: "/" indicates that the item is not added.
Sorting and screening undegraded perishable garbage degrading bacteria agent and immobilized enzyme preparation particles after degradation is finished, counting the dry weight of insoluble substances of the perishable garbage, and calculating the weight reduction rate of the insoluble substances relative to the initial perishable garbage. The weight reduction rates obtained in the statistical schemes 1 to 12 are shown in fig. 6. As can be seen from FIG. 6, the treatment methods in the preferred embodiments 1, 2, 7, and 8 of the present application all achieved a reduction rate of not less than 99.3%, particularly, the reduction rate of the scheme 7 is more than 99.5%, and correspondingly, the enzyme activity recovery rate of each immobilized enzyme preparation in the embodiments 3 to 6 does not achieve the excellent effect, so that the corresponding reduction rate of the schemes 3 to 6 is slightly reduced under the same treatment time, compared with analysis schemes 7-9, it can be seen that the loss of microorganisms can also cause degradation efficiency to decrease, and compared with schemes 10 and 11, it can be seen that the loss rate of perishable garbage can be significantly decreased without adding immobilized enzyme preparation or perishable garbage degrading bacteria agent, and it can also be seen from scheme 12 that a sufficient amount of enzyme can be used to replace the immobilized enzyme preparation to achieve approximate degradation efficiency, however, it can cause risk factors such as cost increase and uncontrollable storage of enzyme agent; therefore, the perishable waste degrading microbial inoculum is prepared by optimizing the microbial inoculum, the perishable waste is subjected to phase change water production treatment by combining an immobilized enzyme preparation, normal-temperature rapid degradation is realized, macromolecules in the perishable waste, such as proteins, lipids, starch, sugar, cellulose and the like are finally degraded into carbon dioxide and water, the carbon dioxide and the water are colorless and tasteless substances, the environment is greatly improved, the water is purified to reach the five types of water quality on the earth surface, the perishable waste can be recycled, the perishable waste is not lower than 99.3 percent of reduction rate, the process is simple, no external heat energy is required to be provided in the operation process, the energy consumption is low, the squeezing dehydration is not required in the earlier stage, high-temperature drying is not required during treatment, and pollutants are concentrated.
In addition, the inventor also verifies the corresponding scheme when no lipase and/or neutral protease and/or cellulase and/or amylase is added and when bacillus subtilis subspecies and/or bacillus alcaligenes and/or bacillus amyloliquefaciens and/or bacillus cereus and/or pseudomonas herbaceous and/or pseudomonas brucei and/or pseudomonas moldy and/or pseudomonas azogenes and/or acetobacter aceti and/or bacillus thiogenes and/or bacillus terreus and/or pseudomonas yellowish and/or pseudomonas brucei and/or pseudomonas winkle in a microbial agent are deleted, and finds that the perishable waste reduction rate obtained by the scheme has a variable reduction rate of 75-95% under the same perishable waste phase transition water production treatment method as the embodiment, far from the reduction rates of 99.3% that are achievable with the preferred embodiment described herein. The structure is arranged in a degradation bin, and the protruding parts on the side wall of the structure are strip-shaped as shown in figure 4 and point-shaped as shown in figure 5, which are beneficial to the precipitation of grease and sewage and the extrusion of cellulose and the like, and the phase change water preparation treatment process is promoted.
Example 11:
taking part of the crude oil obtained in the experimental example 2, filtering the crude oil with a 0.22-micron filter membrane, heating the crude oil to 100 ℃ to remove moisture, cooling the crude oil, adding a mixture of peroxyacetic acid and barium peroxide in a weight ratio of 1:4 of 0.12% of the weight of the oil, uniformly mixing the mixture, putting the mixture into a closed kneader, adding dry ice in a weight ratio of 0.04% of the weight of the oil, and performing crosslinking and reforming for 45min at 30 ℃ and 1.0-1.4 MPa to obtain the lubricating oil.
Example 12:
taking part of the crude oil obtained in the experimental example 2, filtering the crude oil with a 0.22-micron filter membrane, heating the crude oil to 100 ℃ to remove moisture, cooling the crude oil, adding a mixture of peracetic acid and barium peroxide in a weight ratio of 1:5.0 to 0.15% of the weight of the oil, uniformly mixing the mixture, putting the mixture into a closed kneading machine, adding dry ice in a weight ratio of 0.05 per thousand of the weight of the oil, and performing crosslinking and reforming for 45min at 32 ℃ and 1.0-1.4 MPa to obtain the lubricating oil.
Example 13:
and (2) filtering part of the crude oil obtained in the experimental example 2 by a 0.22-micron filter membrane, heating to 100 ℃ to remove moisture, cooling, adding peroxyacetic acid accounting for 0.15% of the weight of the oil, uniformly mixing, placing in a closed milling machine, adding dry ice accounting for 0.05% of the weight of the oil, and performing crosslinking reforming for 45min at the temperature of 32 ℃ and under the pressure of 1.0-1.4 MPa to obtain the lubricating oil.
Example 14:
and (2) filtering part of the crude oil obtained in the experimental example 2 by a 0.22-micron filter membrane, heating to 100 ℃ to remove moisture, cooling, adding barium peroxide accounting for 0.15% of the weight of the oil, uniformly mixing, placing in a closed milling machine, adding dry ice accounting for 0.05% of the weight of the oil, and performing crosslinking reforming for 45min at 32 ℃ under the condition of 1.0-1.4 MPa to obtain the lubricating oil.
Example 15:
taking part of the crude oil obtained in the experimental example 2, filtering the crude oil with a 0.22-micron filter membrane, heating the crude oil to 100 ℃ to remove moisture, cooling the crude oil, adding a mixture of peroxyacetic acid and barium peroxide in a weight ratio of 1:8 of 0.15% of the weight of the oil, uniformly mixing the mixture, putting the mixture into a closed kneader, adding dry ice in a weight ratio of 0.05 ‰ of the weight of the oil, and performing crosslinking and reforming for 45min at 32 ℃ and under the pressure of 1.0-1.4 MPa to obtain the lubricating oil.
Example 16:
taking part of the crude oil obtained in the experimental example 2, filtering the crude oil with a 0.22-micron filter membrane, heating the crude oil to 100 ℃ to remove moisture, cooling the crude oil, adding a mixture of peroxyacetic acid and barium peroxide in a weight ratio of 1:2 of 0.15% of the weight of the oil, uniformly mixing the mixture, putting the mixture into a closed kneader, adding dry ice in a weight ratio of 0.05 ‰ of the weight of the oil, and performing crosslinking and reforming for 45min at 32 ℃ and under the pressure of 1.0-1.4 MPa to obtain the lubricating oil.
Experimental example 3:
for comparison, a part of the crude oil obtained in experimental example 2 is taken, filtered through a 0.22-micron filter membrane, heated to 100 ℃ to remove moisture, cooled, added with ammonium persulfate accounting for 0.4% of the weight of the oil, uniformly mixed and placed in a closed kneader, added with dry ice accounting for 0.05 per mill of the weight of the oil, and cross-linked and reformed for 45min at the temperature below 32 ℃ and under the pressure of 1.5-2.0 MPa to obtain lubricating oil, which is marked as lubricating oil C.
40. Kinematic viscosity values of examples 11 to 16 and lubricating oil C were measured at 50 ℃ respectively, and wear resistance was measured in a four-ball testing machine with steel balls of 1200r/min, 25 + -2 ℃, 294N, 30min, and phi 12.7 according to the prior art, and statistical results are shown in Table 3.
TABLE 3 lubricating oil Performance measurement results
| Lubricating oil | Kinematic viscosity (40 ℃ C.) | Kinematic viscosity (50 ℃ C.) | Wear resistance (Spot grinding diameter mm) |
| Example 11 | 121 | 118 | 0.28 |
| Example 12 | 125 | 121 | 0.30 |
| Example 13 | 129 | 108 | 0.49 |
| Example 14 | 126 | 106 | 0.50 |
| Example 15 | 128 | 112 | 0.46 |
| Example 16 | 125 | 111 | 0.52 |
| Lubricating oil C | 124 | 98 | 0.58 |
As can be seen from table 3, the lubricating oils obtained in examples 11 and 12 according to the preferred embodiments of the present invention have excellent kinematic viscosity and viscosity-temperature characteristics, the viscosity decreases only to a low extent with an increase in temperature, and the lubricating oil has excellent viscosity-temperature characteristics, which are deteriorated only by peracetic acid or barium peroxide and a large or small weight ratio of the two, and further, the lubricating oil obtained according to the preferred embodiments of the present invention has very excellent wear resistance, so that the recycling of the perishable waste oil can be realized at a low cost, and the industrial added value of the perishable waste can be increased.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The invention is not the best known technology.
Claims (9)
1. The application of the chitosan oligosaccharide derivative in the preparation of immobilized enzyme carriers is characterized in that:
the chitosan oligosaccharide derivative is a product obtained by modifying chitosan oligosaccharide with gamma-aminobutyric acid;
the method for preparing the chitosan oligosaccharide derivative comprises the following steps: dissolving gamma-aminobutyric acid in a large amount of MES buffer solution, sequentially adding EDC & HCl and NHS, stirring for at least 5h after all the materials are dissolved, adding chitosan oligosaccharide with the molecular weight of 1200-2000 Da, stirring at a high speed for 30-36 h, dialyzing by a 200-400 Da dialysis bag, and freeze-drying to obtain the chitosan oligosaccharide derivative.
2. Use according to claim 1, characterized in that: the application specifically comprises the step of crosslinking the chitosan oligosaccharide derivative and graphene oxide by glutaraldehyde to prepare an immobilized enzyme carrier.
3. A preparation method of an immobilized enzyme carrier is characterized by comprising the following steps: preparing an aqueous solution of the chitosan oligosaccharide derivative in the application of claim 1 or 2, activating graphene oxide, mixing the two solutions according to the weight ratio of 1: 1.5-2.0 of the chitosan oligosaccharide derivative to the graphene oxide, reacting at room temperature for 1.5-3 h, centrifuging to obtain a precipitate, adding a glutaraldehyde solution for crosslinking for 1-2.5 h, removing uncrosslinked substances, collecting the precipitate, and freeze-drying to obtain the chitosan oligosaccharide derivative-graphene oxide.
4. An immobilized enzyme carrier obtained by the process of claim 3.
5. An immobilized enzyme preparation based on the immobilized enzyme carrier of claim 4, characterized in that:
the immobilized enzyme preparation comprises:
a lipase or a neutral protease or a cellulase or an amylase.
6. A phase change water production treatment method for perishable garbage is characterized by comprising the following steps:
s1, picking out non-degradable substances from perishable garbage, crushing the materials to be sieved by a 10-mesh sieve, placing the materials in a degradation bin of phase change water treatment equipment, stirring the materials by a stirring rod in the degradation bin and extruding the materials with the side wall of the degradation bin, filtering filtrate, heating the filtered filtrate to 50-60 ℃, standing and layering the filtered filtrate, leading out upper-layer crude oil, and leading other components into the equipment;
s2, adding a perishable garbage degrading microbial agent and the immobilized enzyme preparation of claim 5 into equipment, and carrying out phase change water preparation treatment process for 36-48 h under the conditions of temperature of 15-40 ℃ and relative humidity of 80-90%;
and the perishable garbage reduction rate is not less than 99.3% when the phase change water production treatment process is finished.
7. The method of claim 6, wherein: the phase change water production treatment device comprises:
the degradation bin is used for containing and degrading perishable garbage, a bulge is arranged on the side wall of the degradation bin, and a stirring rod is arranged inside the degradation bin;
the feeding port is arranged at the top of the degradation bin and used for feeding perishable garbage;
the stirring motor is arranged outside the degradation bin, is connected with the stirring rod and can drive the stirring rod to stir and finish stirring and extruding the perishable garbage in the degradation bin;
the filter screen is arranged at the bottom of the degradation bin and can be opened and closed;
and the sedimentation tank is arranged at the lower part of the degradation bin and can be heated for accommodating and settling the filtrate filtered out from the degradation bin.
8. The method of claim 7, wherein: the bulge of the side wall of the degradation bin is arc-shaped, point-shaped or strip-shaped.
9. The method according to any one of claims 6 to 8, wherein: the perishable garbage degrading microbial inoculum is prepared by mixing cross-linked slurry containing sodium alginate and high-substitution sulfobutyl ether-beta-cyclodextrin with degrading microbial inoculum, adding gypsum powder and granulating.
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| JPS6458400A (en) * | 1987-08-31 | 1989-03-06 | Nikko Service Kk | Method and apparatus for industrial waste treatment |
| CN1389502A (en) * | 2002-07-24 | 2003-01-08 | 山东美林亚生物科技有限公司 | Heavy metal chelant composite containing chitosan derivative |
| PL2774481T3 (en) * | 2013-03-08 | 2019-03-29 | Symrise Ag | Antimicrobial compositions |
| CN104383887B (en) * | 2014-11-12 | 2017-02-15 | 青岛海洋生物医药研究院股份有限公司 | Application of chitosan, chitosan oligosaccharide and additives thereof to preparation of adsorbing agent for adsorbing air pollutants |
| CN104828953B (en) * | 2015-04-14 | 2017-01-11 | 浙江海洋学院 | Water treatment agent and preparation method thereof |
| CN104922145B (en) * | 2015-05-25 | 2018-05-01 | 福州乾正药业有限公司 | Composition of γ-aminobutyric acid and chitosan oligosaccharide and its preparation method and application |
| US20170226535A1 (en) * | 2015-09-16 | 2017-08-10 | Sweetwater Energy, Inc. | Specialized Activated Carbon Derived From Pretreated Biomass |
| CN106542568B (en) * | 2016-10-27 | 2018-08-31 | 中国农业科学院油料作物研究所 | A kind of immobilised enzymes, fixed enzyme vector and preparation method thereof |
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