Cutting fluid waste liquid degradation complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention discloses a cutting fluid waste liquid degradation complex microbial inoculant and a preparation method and application thereof, and belongs to the technical field of microbiological wastewater treatment, the microbial inoculant comprises immobilized thalli and immobilized enzyme, the immobilized thalli comprise a dry fermentation product and flora propagated on the fermentation product in situ, and the immobilized enzyme is immobilized enzyme. The flora comprises alcaligenes, bacteroides, bacillus subtilis and sporosarcina, and the immobilized enzyme comprises a biochar carrier, and laccase and lipase loaded on the biochar carrier. According to the invention, appropriate strains are mixed to construct the high-concentration organic matter waste liquid degradation composite microbial agent, the community rigidity is enhanced, the limitation of low degradation efficiency and poor stability of a single strain is made up, and the composite microbial agent can stably survive and rapidly proliferate in the cutting fluid waste liquid; complex components in the cutting fluid waste liquid can be efficiently degraded, the problems that a physical method and a chemical method are high in cost and prone to causing secondary pollution are solved, and treatment of the cutting fluid waste liquid is more environmentally friendly and efficient.

Description

Technical Field

[0001] The invention relates to a composite bacterial agent for degrading waste cutting fluid and a preparation method and application thereof, belonging to the technical field of wastewater treatment by microorganisms. Background Art

[0002] Cutting fluid plays the role of lubrication, cooling and rust prevention in the machining process. After 1 to 6 months of use, it loses its effectiveness due to loss of effective ingredients and deterioration and becomes waste fluid. The composition of cutting fluid waste is complex and difficult to treat. At present, three types of methods have been formed for the treatment of cutting fluid waste: physical, chemical and biological. The commonly used physical and chemical methods are not only incomplete, but also easy to cause secondary pollution, and the treatment cost is high. Biological methods have become a hot topic in the field of wastewater treatment due to their high efficiency, low cost and environmental friendliness. Studies have shown that it is feasible to isolate specific strains from cutting fluid waste to degrade high-concentration organic matter, but the proliferation and activity of strains are easily affected by environmental factors such as organic toxic substances, and the composition of cutting fluid waste is complex, resulting in unstable treatment efficiency of some strains for cutting fluid waste. Summary of the invention

[0003] In order to overcome the shortcomings of the prior art, the present invention provides a composite bacterial agent for degrading cutting fluid waste and a preparation method thereof, which has high treatment efficiency and strong impact resistance for degrading cutting fluid waste, and also provides a method for using the composite bacterial agent for degrading cutting fluid waste.

[0004] The technical solution adopted by the present invention to solve its technical problem is: In the first aspect, the present application provides a composite bacterial agent for degrading cutting fluid waste, including immobilized bacteria and immobilized enzymes, the immobilized bacteria including a dried fermentation product and a bacterial community that grows in situ on the fermentation product, the bacterial community including Alcaligenes, Bacteroides, Oceanic Bacillus and Sporosarcina, the immobilized enzyme including a biochar carrier and laccase and lipase loaded on the biochar carrier.

[0005] The composite bacterial agent for degrading cutting fluid waste provided in the present application can tolerate high-concentration organic cutting fluid waste, the bacterial community can grow and reproduce in high-concentration organic cutting fluid waste, and has high-efficiency degradation ability for organic matter in the waste fluid, thus making up for the shortcomings of low degradation efficiency and poor stability of single strains.

[0006] Furthermore, the mass ratio of the laccase to the lipase is 3:2.

[0007] Laccase mainly degrades refractory substances such as phenols and aromatic compounds, while lipase targets oils and esters. This ratio ensures that the composite enzyme plays a good catalytic degradation role in cutting fluid waste, enhances its adaptability to a variety of cutting fluid waste, and is conducive to improving treatment efficiency.

[0008] Furthermore, the alcaligenes is Alcaligenes, the Bacteroides is Bacteroides and Muribaculaceae, the oceanic Bacillus is Oceanobacillus, and the Sporosarcina is Sporosarcina.

[0009] Alcaligenes is a demulsifier that can produce biosurfactants with demulsification ability under certain culture conditions and has the ability to degrade crude oil and polycyclic aromatic hydrocarbons. Bacteroides can produce abundant carbohydrate-active enzymes that can degrade macromolecular peptides and polysaccharides. Oceanobacillus is isolated from petroleum-enriched bacteria and is an organic pollutant-degrading bacterium.

[0010] Furthermore, by mass, the bacterial community includes 25% to 45% of the Alcaligenes, 10% to 35% of the Muribaculaceae, 15% to 30% of the Bacteroides, 5% to 20% of the Oceanobacillus, and 5% to 20% of the Sporosarcina.

[0011] Reasonable strain ratio distribution is conducive to enhancing community rigidity, and the optimized ratio is conducive to dealing with a variety of different cutting fluid wastes while maintaining the activity and high degradation ability of the composite bacterial agent.

[0012] In a second aspect, the present application provides a method for preparing a composite bacterial agent for degrading waste cutting fluid, comprising the following steps: Activate and culture alcaligenes, Bacteroides, oceanic Bacillus and sporosarcina respectively; perform seed culture on each activated strain to obtain seed liquid of each strain; mix the seed liquid of each strain according to the proportion required for the designed immobilized bacterial body, and perform fermentation culture, freeze-drying the fermentation product including liquid and solid to obtain the immobilized bacterial body; The laccase solution and the lipase solution are mixed in a desired ratio for designing the immobilized enzyme, and a biochar carrier is added to obtain a mixed system. The mixed system is freeze-dried in a vacuum to obtain the immobilized enzyme.

[0013] The method ensures the activity and quantity of each strain, and the prepared composite bacterial agent has good stability, is easy to transport and store for a long time, and has higher degradation efficiency in the process of treating cutting fluid waste.

[0014] Furthermore, in the seed culture, the seed culture medium contains, by mass, 3 to 6 parts of NH4Cl, 2 to 5 parts of K2HPO4, 4 to 10 parts of KH2PO4, 20,000 to 45,000 parts of liquid paraffin, 0.8 to 1.2 parts of trace elements, and 0.8 to 1.2 parts of vitamins; the seed culture conditions are shaking culture at 25°C to 38°C for 24h-48h.

[0015] The efficiency of the strain in producing active substances to degrade cutting fluid waste is related to the type of carbon source in the culture medium. Both insoluble hydrocarbon matrix and soluble matrix can be used to cultivate microorganisms to produce demulsifiers. Petroleum hydrocarbon carbon source paraffin can induce demulsifiers to synthesize biosurfactants. Cutting fluid waste contains a large amount of hydrocarbon substances. This type of carbon source is one of the important reasons for the production of effective ingredients of demulsifiers. Adding liquid paraffin to the culture medium with various nutrients to acclimate the seed liquid is beneficial to improve the survival ability and degradation effect of the bacterial community in cutting fluid waste.

[0016] Furthermore, the step of mixing the seed liquid of each strain according to the proportion required for the designed immobilized bacterial body for fermentation and culturing comprises: The seed liquid of each strain is mixed according to the proportion required for the designed immobilized bacteria, added to the fermentation medium, and then bran equivalent to 50% to 60% of the mass of the fermentation medium is added, and fermented at pH 7.0 and temperature 35°C for 3d-5d; The fermentation medium comprises, by mass, 20 to 40 parts of flour, 8 to 10 parts of citric acid, 20 to 40 parts of beef extract, 1.5 to 2.5 parts of trace elements, 1.5 to 2.5 parts of vitamins, 20 to 40 parts of glycerol and 45 to 70 parts of amino acids; The amino acid is selected from at least one of lysine, histidine, glycine and alanine.

[0017] The fermentation medium formula provides the nutrients required for strain reproduction, especially flour, citric acid and beef extract, which promote the rapid proliferation of the flora. Bran is not only a good solid-state fermentation matrix, but also helps to immobilize the flora during subsequent freeze-drying.

[0018] Furthermore, the step of mixing the laccase solution and the lipase solution in a desired ratio for designing the immobilized enzyme and adding the biochar carrier to obtain the mixed system comprises: The laccase solution with a concentration of 5 g / L to 10 g / L is prepared, and the lipase solution with the same concentration as the laccase solution is prepared, and the laccase solution and the lipase solution are mixed in a volume ratio of 3:2 to obtain a mixed enzyme solution, and the biochar carrier is added in an amount of 20% of the mass of the mixed enzyme solution, and the mixture is placed in a shaker at 30° C. to 35° C. and shaken at a speed of 120 rpm to 180 rpm for 5 hours to obtain the mixed system.

[0019] The enzyme solution is prepared with appropriate concentration and fixed with biochar carrier to ensure the activity and loading effect of the enzyme, and enhance the treatment ability of the composite bacterial agent on oily and difficult-to-degrade organic matter.

[0020] In a third aspect, the present application provides an application of the composite bacterial agent for degrading cutting fluid waste as described in the first aspect, and the operation includes: adding the composite bacterial agent for degrading cutting fluid waste into cutting fluid waste.

[0021] Furthermore, the step of adding the composite bacterial agent for degrading the waste cutting fluid into the waste cutting fluid comprises: The immobilized bacterial bodies are activated and cultured, and then seed culture is performed to obtain bacterial colony seed liquid, and the bacterial colony seed liquid and the immobilized enzyme are added to the cutting fluid waste liquid at a mass ratio of 4:1.

[0022] Immobilized bacteria and immobilized enzymes have two degradation effects, and the two degradation effects overlap slightly. The specific bacteria in the immobilized bacteria have a high efficiency in degrading high-concentration organic matter in the cutting fluid waste, and the enzyme adsorbed and fixed by the biochar carrier is more stable during the degradation process. The above ratio of bacterial seed liquid and immobilized enzyme ensures the stability and degradation effect of the composite bacterial agent. The bacteria can provide the main biodegradation effect, and the enzyme fixed on the biochar carrier further enhances the degradation ability of organic pollutants while maintaining enzyme activity.

[0023] The beneficial effects of the present invention are as follows: the present invention mixes suitable strains to construct a composite bacterial agent for degrading high-concentration organic waste liquid, enhances community rigidity, and makes up for the limitations of low degradation efficiency and poor stability of a single strain; the composite bacterial agent can achieve stable survival and rapid proliferation in cutting fluid waste liquid, and through the synergistic effect of multiple strains and multiple enzymes, can efficiently degrade the complex components in the cutting fluid waste liquid, avoiding the high cost of physical and chemical methods and the problem of easy secondary pollution, making the treatment of cutting fluid waste liquid more environmentally friendly and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a comparison of the degradation effects of experimental groups 1 to 4 and the control group on high-concentration cutting waste fluid. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] It should be understood that, under the premise of no conflict, any and all embodiments of the present invention can be combined with the technical features in any other embodiment or multiple other embodiments to obtain another embodiment. The present invention includes such a combination to obtain another embodiment.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the standard meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0028] The embodiment of the present application provides a composite bacterial agent for degrading waste cutting fluid, including immobilized bacteria and immobilized enzymes. The immobilized enzymes include a biochar carrier and laccase and lipase loaded on the biochar carrier, laccase accounting for 60% of the mixed enzyme, and lipase accounting for 40% of the mixed enzyme. The immobilized bacteria include dried fermentation products and bacterial communities that grow in situ on the fermentation products, and the bacterial communities include Alcaligenes accounting for 25% to 45%, Muribaculaceae accounting for 10% to 35%, Bacteroides accounting for 15% to 30%, Oceanobacillus accounting for 5% to 20%, and Sporosarcina accounting for 5% to 20%.

[0029] Alcaligenes is a demulsifying bacterium that can produce biosurfactants with demulsification ability under certain culture conditions and has the ability to degrade crude oil and polycyclic aromatic hydrocarbons.

[0030] Bacteroides contains abundant carbohydrate-active enzymes that can degrade large molecular peptides and polysaccharides.

[0031] Oceanobacillus is isolated from petroleum-enriched bacteria and is an organic pollutant-degrading bacterium.

[0032] Immobilized enzymes are easy to store, have high activity, are highly stable and can be reused, and have a wide range of applications.

[0033] Lipase, also known as triacylglycerol hydrolase, can catalyze lipid hydrolysis, exchange and synthesis at the water-oil interface. Lipase plays a key role in the hydrolysis process.

[0034] Laccase is a polyphenol oxidase that can efficiently catalyze the degradation of aromatic amines, phenols, and other heterocyclic carbon compounds without generating more toxic by-products.

[0035] The preparation method of the composite bacterial agent comprises the following steps: Alcaligenes, Muribaculaceae, Bacteroides, Oceanobacillus, and Sporosarcina were activated (here, activation of raw materials). The activation medium contained 10g of tryptone, 5g of yeast extract, and 10g of sodium chloride, which was diluted to 1L with sterile water, and then 15g of agar powder was added. The culture conditions were inverted culture at 25℃~38℃ for 12h-24h.

[0036] The activated Alcaligenes, Muribaculaceae, Bacteroides, Oceanobacillus, and Sporosarcina were cultured separately (here, the raw material was cultured). The seed culture medium contained 3g~6gNH4Cl, 2g~5g K2HPO4, 4g~10g KH2PO4, 25L~50L liquid paraffin, 1mL trace elements, and 1mL vitamins, and the volume was adjusted to 1L with sterile water. The culture conditions were shaking culture at 25℃~38℃ for 24h-48h.

[0037] The prepared seed liquid is mixed in proportion and fermented. The fermentation medium contains 20g~40g flour, 8g citric acid, 20g~40g beef extract, 2mL trace elements, 2mL vitamins, 20g~40g glycerol and 45g~70g amino acids (including lysine, histidine, glycine, alanine), and is fixed to 5L with sterile water to obtain a fermentation medium, and then bran equivalent to 50%-60% of the mass of the fermentation medium is added, and fermented at pH 7.0 and 35°C for 3d-5d.

[0038] The mixture of the fermentation liquid and the bran is taken out and freeze-dried in a vacuum to obtain the immobilized bacteria.

[0039] Prepare 5g / L~10g / L laccase solution with PBS solution, and prepare 5g / L~10g / L lipase solution with PBS solution. Weigh 10g of washed and dried biochar carrier, add 30mL laccase solution and 20mL lipase solution, mix well, place in a shaker, set the temperature to 30℃~35℃, and shake at 120rpm~180rpm for 5h. Take it out and wash it with PBS buffer, freeze-dry it in a vacuum to obtain the immobilized enzyme.

[0040] The steps of applying the above composite bacterial agent to the degradation of high-concentration organic cutting fluid waste are as follows: Activate the immobilized bacteria (here is the activation of the finished product), weigh 0.2g of immobilized bacteria and inoculate it into 100ml LB liquid culture medium, and culture it at 25℃~38℃ for 24h~48h.

[0041] The activated bacterial community is inoculated into the seed culture medium at 1%~5% (here is the seed culture for the finished product). The culture medium contains 3g~6g NH4Cl, 2g~5g K2HPO4, 4g~10g KH2PO4, 25L~50L liquid paraffin, 1mL trace elements, and 1mL vitamins. The volume is made up to 1L with sterile water. The culture conditions are 25℃~38℃ for 24h~48h to obtain the bacterial community seed solution.

[0042] The bacterial seed liquid is inoculated into high-concentration organic cutting fluid waste liquid at an inoculation ratio of 5% to 20%, and an immobilized enzyme equivalent to one-fourth of the mass of the bacterial seed liquid is added.

[0043] Experimental comparison The COD (chemical oxygen demand) of the high-concentration organic cutting fluid waste to be treated was measured to be 130,000 mg / L.

[0044] Among the raw materials used for the immobilized enzymes, the laccase was Solebold L8540 and the lipase was Solebold TYPEⅡ L8070.

[0045] Example 1 Alcaligenes, Muribaculaceae, Bacteroides, Oceanobacillus, and Sporosarcina were activated (here, activation of raw materials). The activation medium contained 10g of tryptone, 5g of yeast extract, and 10g of sodium chloride, which was diluted to 1L with sterile water, and then 15g of agar powder was added. The culture conditions were inverted culture at 25℃~38℃ for 12h.

[0046] The activated Alcaligenes, Muribaculaceae, Bacteroides, Oceanobacillus, and Sporosarcina were cultured as seeds (here, the raw materials were cultured as seeds). The seed culture medium contained 6g NH4Cl, 5g K2HPO4, 8g KH2PO4, 50L liquid paraffin, 1mL trace elements, and 1mL vitamins, and was diluted to 1L with sterile water. The culture conditions were shaking culture at 25℃~38℃ for 24h.

[0047] The seed liquid of Alcaligenes accounts for 35%, the seed liquid of Muribaculaceae accounts for 20%, the seed liquid of Bacteroides accounts for 25%, the seed liquid of Oceanobacillus accounts for 10%, and the seed liquid of Sporosarcina accounts for 10%. The prepared seed liquids are mixed according to the proportions and fermented, and the inoculation ratio is 5% of the mass of the fermentation medium. The fermentation medium contains 20g flour, 8g citric acid, 30g beef extract, 2mL trace elements, 2mL vitamins, 20g glycerol and 45g amino acids, and is fixed to 5L with sterile water to obtain a fermentation medium, and then bran equivalent to 50% of the mass of the fermentation medium is added, and fermented at pH 7.0 and temperature 35℃ for 3d.

[0048] The mixture of the fermentation liquid and the bran is taken out and freeze-dried in a vacuum to obtain the immobilized bacteria.

[0049] Prepare 5g / L laccase solution with PBS solution, and prepare 5g / L lipase solution with PBS solution. Weigh 10g of washed and dried biochar carrier, add 30mL laccase solution and 20mL lipase solution, mix well, place in a shaker, set the temperature to 30℃~35℃, shake at 120rpm for 5h. Take out and wash with PBS buffer, freeze-dry in vacuum to obtain immobilized enzyme.

[0050] Comparative Example 1 The difference from Example 1 is that: (1) only Alcaligenes and Muribaculaceae are activated and seed cultured, and during fermentation culture, only Alcaligenes seed solution and Muribaculaceae seed solution are inoculated, and the mixing ratio of the two is 30:20. (2) There is no need to prepare immobilized enzyme. The remaining steps are the same as in Example 1, including that during fermentation culture, the inoculation ratio is 5% of the mass of the fermentation medium.

[0051] Comparative Example 2 The difference from Example 1 is that: (1) only Alcaligenes, Muribaculaceae and Bacteroides were activated and cultured, and during fermentation culture, only the seed solution of Alcaligenes, the seed solution of Muribaculaceae and the seed solution of Bacteroides were inoculated, and the mixing ratio of the three was 30:20:25. (2) There was no need to prepare immobilized enzyme. The remaining steps were the same as in Example 1, including that during fermentation culture, the inoculation ratio was 5% of the mass of the fermentation medium.

[0052] Comparative Example 3 The difference from Example 1 is that: (1) only Alcaligenes, Muribaculaceae, Bacteroides, and Oceanobacillus are activated and cultured, and during fermentation culture, only the seed solution of Alcaligenes, the seed solution of Muribaculaceae, the seed solution of Bacteroides, and the seed solution of Oceanobacillus are inoculated, and the mixing ratio of the four is 30:20:25:10. (2) There is no need to prepare immobilized enzyme. The remaining steps are the same as in Example 1, including that during fermentation culture, the inoculation ratio is 5% of the mass of the fermentation medium.

[0053] Test Example 1 The purchased Alcaligenes were activated, and 0.2 g of immobilized bacteria were weighed and inoculated into 100 ml of LB liquid culture medium. The culture conditions were 25° C. to 38° C. for 24 h.

[0054] The activated bacterial flora is inoculated into the seed culture medium at 1%~5% (here is the seed culture for the finished product). The culture medium contains 5g NH4Cl, 3g K2HPO4, 8g KH2PO4, 25L liquid paraffin, 1mL trace elements, 1mL vitamins, and is diluted to 1L with sterile water. The culture conditions are 25℃~38℃ for 24h to obtain the seed solution.

[0055] The seed liquid was inoculated into high-concentration organic cutting fluid waste liquid at an inoculation ratio of 15%. The mixture was left standing for 36 days, and the COD values ​​before inoculation and 30 days after inoculation were compared.

[0056] Test Example 2 The difference from Test Example 1 is that purchased Muribaculaceae was used, and the remaining steps are the same as Test Example 1.

[0057] Test Example 3 The difference from Test Example 1 is that purchased Bacteroides is used, and the remaining steps are the same as Test Example 1.

[0058] Test Example 4 The difference from Test Example 1 is that purchased Oceanobacillus was used, and the remaining steps are the same as those of Test Example 1.

[0059] Test Example 5 The difference from Test Example 1 is that purchased Sporosarcina is used, and the remaining steps are the same as those of Test Example 1.

[0060] Test Example 6 The difference from Test Example 1 is that the bacterial cells prepared in Comparative Example 1 are used, and the remaining steps are the same as those in Test Example 1.

[0061] Test Example 7 The difference from Test Example 1 is that the bacterial cells prepared in Comparative Example 2 are used, and the remaining steps are the same as those in Test Example 1.

[0062] Test Case 8 The difference from Test Example 1 is that the bacterial cells prepared in Comparative Example 3 are used, and the remaining steps are the same as those in Test Example 1.

[0063] Test Example 9 The difference from Test Example 1 is that the bacteria prepared in Example 1 are used, and the remaining steps are the same as those in Test Example 1.

[0064] The test results of test examples 1 to 9 are shown in Table 1 below.

[0065] Table 1 strain COD removal rate (%) Test Example 1 Alcaligenes 42.08 Test Example 2 Muribaculaceae 41.28 Test Example 3 Bacteroides 36.01 Test Example 4 Oceanobacillus 30.46 Test Example 5 Sporosarcina 31.55 Test Example 6 Alcaligenes + Muribaculaceae 58.32 Test Example 7 Alcaligenes+Muribaculaceae+Bacteroides 67.47 Test Case 8 Alcaligenes+Muribaculaceae+Bacteroides+Oceanobacillus 72.66 Test Example 9 Alcaligenes+Muribaculaceae+Bacteroides+Oceanobacillus +Sporosarcina 76.89 The results showed that there was a synergistic effect between the mixed bacteria, and the mixed bacteria were more efficient in degrading the cutting fluid waste than a single bacteria species, and the bacteria prepared in Example 1 had a stronger degradation effect.

[0066] Test Example 10 The difference from Test Example 9 is that the inoculation rate of the bacterial seed liquid is 12% during inoculation, and an immobilized enzyme equivalent to one quarter of the mass of the bacterial seed liquid is added at the same time. The remaining steps are the same as Test Example 9.

[0067] Test Example 11 The difference from Test Example 9 is that the inoculation rate of the bacterial seed liquid is 7.5% during inoculation, and an immobilized enzyme with the same mass as the bacterial seed liquid is added at the same time. The remaining steps are the same as Test Example 9.

[0068] Test Example 12 The difference from Test Example 9 is that the inoculation rate of the bacterial seed liquid is 3% during inoculation, and four times the mass of the bacterial seed liquid is added as immobilized enzyme. The remaining steps are the same as Test Example 9.

[0069] The test results of Test Examples 9 to 12 are shown in Table 2 below.

[0070] Table 2

[0071] The results show that when used in combination with immobilized enzymes, the degradation effect on cutting fluid can be enhanced. The inoculation rate affects the degradation effect, and adding a small amount of immobilized enzymes at the same time as inoculation can improve the degradation efficiency. The bacterial community cultured in Example 1 has a good reproduction rate in high-concentration cutting fluid waste liquid. With the assistance of immobilized enzymes, it still has a good degradation effect even at a low inoculation rate.

[0072] Experimental Group 1 The immobilized bacteria of Example 1 were activated (here, the finished product was activated), and 0.2 g of the immobilized bacteria were weighed and inoculated into 100 ml of LB liquid culture medium. The culture conditions were 25° C. to 38° C. for 24 h.

[0073] The activated bacterial community was inoculated into the seed culture medium at 5% (here is the seed culture for the finished product). The culture medium contained 6g NH4Cl, 5g K2HPO4, 4g KH2PO4, 30L liquid paraffin, 1mL trace elements, and 1mL vitamins. The volume was made up to 1L with sterile water. The culture conditions were 25℃~38℃ for 24h to obtain the bacterial community seed solution.

[0074] The bacterial seed liquid was inoculated into high-concentration organic cutting fluid waste liquid at an inoculation ratio of 5%, and an immobilized enzyme equivalent to one-fourth of the mass of the bacterial seed liquid was added.

[0075] Experimental Group 2 The difference from experimental group 1 is that when the bacterial seed liquid was inoculated into the high-concentration organic cutting fluid waste liquid, the inoculation ratio was 10%, and the other steps were the same.

[0076] Experimental Group 3 The difference from experimental group 1 is that when the bacterial seed liquid was inoculated into the high-concentration organic cutting fluid waste liquid, the inoculation ratio was 15%, and the other steps were the same.

[0077] Experimental Group 4 The difference from experimental group 1 is that when the bacterial seed liquid was inoculated into the high-concentration organic cutting fluid waste liquid, the inoculation ratio was 20%, and the other steps were the same.

[0078] Blank Group Add tap water equivalent to 10% of the volume of the high-concentration organic cutting fluid waste to be treated.

[0079] The control group inoculated 10% of the commercially available composite bacterial agent into high-concentration organic cutting fluid wastewater according to the instructions.

[0080] Subsequently, samples were taken from the high-concentration organic cutting fluid wastewater every 5 days to test the COD value. The COD removal effects of the experimental group and the control group were as follows: Figure 1 As shown. Figure 1It can be seen that high-concentration cutting fluid waste will also degrade spontaneously in the air, but the degradation rate is slow. Adding bacterial agents can greatly increase the degradation rate. With the cooperation of immobilized enzymes, the bacterial seed liquid has a good degradation effect as long as it is inoculated at 5%; with the increase of the amount of composite bacterial agents, the degradation rate will also increase, and the effect of the bacterial seed liquid at an inoculation rate of 15% and 20% is not much different. When the amount of immobilized enzyme used is one-fourth of the bacterial seed liquid, the inoculation rate of the bacterial seed liquid is preferably 5%~15%.

[0081] The above is a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A composite bacterial agent for degrading waste cutting fluid, characterized in that: The invention comprises immobilized bacteria and immobilized enzymes. The immobilized bacteria comprises dried fermentation products and bacterial communities propagated in situ on the fermentation products. The bacterial communities comprise alcaligenes, bacteroides, oceanic bacillus and sporosarcina. The immobilized enzymes comprise biochar carriers and laccase and lipase loaded on the biochar carriers.

2. The composite bacterial agent for degrading waste cutting fluid according to claim 1, characterized in that: The mass ratio of the laccase to the lipase is 3:

2.

3. The composite bacterial agent for degrading waste cutting fluid according to claim 1, characterized in that: The alcaligenes is Alcaligenes, the Bacteroides is Bacteroides and Muribaculaceae, the oceanic Bacillus is Oceanobacillus, and the Sporosarcina is Sporosarcina.

4. The composite bacterial agent for degrading waste cutting fluid according to claim 3, characterized in that: By mass, the bacterial flora includes 25% to 45% of the Alcaligenes, 10% to 35% of the Muribaculaceae, 15% to 30% of the Bacteroides, 5% to 20% of the Oceanobacillus, and 5% to 20% of the Sporosarcina.

5. A method for preparing a composite bacterial agent for degrading waste cutting fluid, characterized in that: The following steps are involved: Activate and culture alcaligenes, Bacteroides, oceanic Bacillus and sporosarcina respectively; perform seed culture on each activated strain to obtain seed liquid of each strain; mix the seed liquid of each strain according to the proportion required for the designed immobilized bacterial body, and perform fermentation culture, freeze-drying the fermentation product including liquid and solid to obtain the immobilized bacterial body; The laccase solution and the lipase solution are mixed in a desired ratio for designing the immobilized enzyme, and a biochar carrier is added to obtain a mixed system. The mixed system is freeze-vacuum dried to obtain the immobilized enzyme.

6. The method for preparing a composite bacterial agent for degrading waste cutting fluid according to claim 5, characterized in that: In the seed culture, the seed culture medium contains, by mass, 3 to 6 parts of NH4Cl, 2 to 5 parts of K2HPO4, 4 to 10 parts of KH2PO4, 20,000 to 45,000 parts of liquid paraffin, 0.8 to 1.2 parts of trace elements, and 0.8 to 1.2 parts of vitamins; the seed culture conditions are shaking culture at 25°C to 38°C for 24h-48h.

7. The method for preparing a composite bacterial agent for degrading waste cutting fluid according to claim 5, characterized in that: The step of mixing the seed liquid of each strain according to the proportion required for the design of the immobilized bacterial body and fermenting and culturing comprises: The seed liquid of each strain is mixed according to the proportion required for the designed immobilized bacteria, added to the fermentation medium, and then bran equivalent to 50% to 60% of the mass of the fermentation medium is added, and fermented at pH 7.0 and temperature 35°C for 3d-5d; The fermentation medium comprises, by mass, 20 to 40 parts of flour, 8 to 10 parts of citric acid, 20 to 40 parts of beef extract, 1.5 to 2.5 parts of trace elements, 1.5 to 2.5 parts of vitamins, 20 to 40 parts of glycerol and 45 to 70 parts of amino acids; The amino acid is selected from at least one of lysine, histidine, glycine and alanine.

8. The method for preparing a composite bacterial agent for degrading waste cutting fluid according to claim 5, characterized in that: The step of mixing the laccase solution and the lipase solution in a desired ratio for designing the immobilized enzyme and adding the biochar carrier to obtain a mixed system comprises: The laccase solution with a concentration of 5 g / L to 10 g / L is prepared, and the lipase solution with the same concentration as the laccase solution is prepared, and the laccase solution and the lipase solution are mixed in a volume ratio of 3:2 to obtain a mixed enzyme solution, and the biochar carrier is added in an amount of 20% of the mass of the mixed enzyme solution, and the mixture is placed in a shaker at 30° C. to 35° C. and shaken at a speed of 120 rpm to 180 rpm for 5 hours to obtain the mixed system.

9. The use of the composite bacterial agent for degrading waste cutting fluid according to any one of claims 1 to 4, characterized in that: The composite bacterial agent for degrading waste cutting fluid is added into waste cutting fluid.

10. The use according to claim 9, characterized in that: The step of adding the composite bacterial agent for degrading the waste cutting fluid into the waste cutting fluid comprises: The immobilized bacterial bodies are activated and cultured, and then seed culture is performed to obtain bacterial colony seed liquid, and the bacterial colony seed liquid and the immobilized enzyme are added to the cutting fluid waste liquid at a mass ratio of 4:1.

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