Bacterial enzyme and enzyme synergic fixed biological active functional filler and application thereof in degradation of organic pollutants

Abstract

The application discloses a kind of bacteria enzyme synergic fixed biological activity functional filler and application in degrading organic pollutants, the bacteria enzyme synergic fixed biological activity functional filler is with bamboo fiber-bamboo biochar composite material as carrier, load composite microbial agent and immobilized enzyme made;The bamboo fiber-bamboo biochar composite material of the present application is made of bamboo fiber and bamboo charcoal composite with multiple pore size as carrier, and 2% of PAE is added to increase wet strength, adsorb small molecule metabolites, which can effectively solve the problem of secondary pollution, make the mixed waste gas purification process more green and safe, solve the defects of traditional waste gas biological purification.The bacteria enzyme synergic biological activity functional filler prepared by the method of the present application not only solves the process defects of traditional waste gas biological purification, but also has no secondary pollution problem, so that the mixed waste gas purification process is more green and safe.

Description

(I) TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental pollution treatment, in particular to a kind of bacteria enzyme synergistic fixed biological activity function filler and application in degrading organic pollutants. (II) BACKGROUND

[0002] Biological purification is a green low-carbon pollution treatment method, which has the characteristics of low cost and environmental friendliness compared with other physical and chemical methods. At present, the composite microbial agent for waste gas treatment has been industrialized. It can promote the formation of biofilm in the reactor, but it still cannot solve the problem of low treatment load. Immobilized enzyme technology can effectively improve the degradation efficiency of typical pollutants. It was first discovered as early as 1916 and has developed rapidly since the 1960s. The chemical and physical properties of immobilized microorganisms or immobilized enzymes are greatly influenced by the fixed carrier and the fixing method. Microorganisms and biological enzymes are only targeted at specific pollutants for degradation, so the "bacteria-enzyme" synergistic fixation of high efficiency plays the potential of their respective degradation, which is of research prospect.

[0003] Existing researches are mostly focused on single bacteria or single enzyme degradation of pollutants, and there is no research report on "bacteria-enzyme" synergistic degradation or co-fixation. Especially for pollutants containing heteroatoms (such as chlorine atoms), although microorganisms with degradation ability have been found, the slow degradation rate limits the application of biological method. In addition, bamboo materials have the advantages of renewable, degradable, biocompatibility, strong hydrophilicity, mechanical properties and heat resistance. However, in the field of environmental biological treatment, bamboo materials are rarely used as biological carriers. Traditional biological immobilization technology has blindness and randomness, and cannot be efficiently planted on the surface of the filler, so the performance of bacteria and enzymes cannot be maximized, resulting in low biological density in the unit volume device, which leads to poor degradation effect and large equipment size. Therefore, the research on new biological loading method and filler can effectively solve the defects of traditional biological purification technology. (III) SUMMARY

[0004] The present application aims to provide a kind of bacteria enzyme synergistic biological activity function filler and application in degrading organic pollutants, which increases the biological density on the unit carrier by precise fixation technology, and realizes the rapid and complete degradation of pollutants containing heteroatoms by "bacteria-enzyme" synergy, so as to realize the rapid degradation of refractory pollutants. It overcomes the shortcomings of single bacteria and enzyme degradation of complex waste gas in traditional biological purification technology, and the waste gas produced by degradation is easy to cause secondary pollution. In addition, the problems of blindness and randomness of immobilization technology are solved.

[0005] The technical solution adopted by the present application is:

[0006] The application provides a kind of bacteria enzyme synergic fixed biological activity functional filler, the bacteria enzyme synergic fixed biological activity functional filler is with bamboo fiber-bamboo biochar composite material as carrier, load composite bacterial agent and immobilized enzyme are made;The composite bacterial agent includes the wet bacteria body of strain for degrading straight-chain alkane, naphthenic hydrocarbon, ester or alcohol by expanding culture;The immobilized enzyme is prepared by immobilizing intracellular enzyme liquid from trichloromethane or propyl mercaptan degrading bacteria in metal organic framework (MOF);The bamboo fiber-bamboo biochar composite material is made by beating bamboo powder and bamboo fiber.

[0007] Preferably, the composite bacterial agent is added in an amount of 10-40% of the carrier mass, preferably 20% by weight of dry bacteria, and the immobilized enzyme is used in an amount of 1-5% of the carrier mass, preferably 3%.

[0008] Preferably, in the composite bacterial agent, the strain for degrading straight-chain alkane is Mycobacterium orleansense (M2021651) CCTCC, the strain for degrading naphthenic hydrocarbon is Acinetobacter venetianus (M2022719) CCTCC, and the strain for degrading ester or alcohol is Flavobacterium sp. (M2021801) CCTCC. Mycolicibacterium neworleansense )CCTCC NO:M 2022719, and the strain for degrading ester or alcohol is Flavobacterium sp. Acinetobacter venetianus )CCTCC NO:M 2022719, and the strain for degrading ester or alcohol is Flavobacterium sp. Microbacterium maritypicum )CCTCC NO:M 2022719, and the strain for degrading ester or alcohol is Flavobacterium sp.

[0009] Preferably, the composite bacterial agent is obtained by mixing the wet bacteria bodies of Mycobacterium orleansense (M2021651) CCTCC, Acinetobacter venetianus (M2022719) CCTCC, and Flavobacterium sp. (M2021801) CCTCC in a mass ratio of 1:1.2-1.8:1.5-2.5, preferably 1:1.5:2.

[0010] Preferably, the wet bacteria bodies of the composite bacterial agent are prepared as follows: inoculate Mycobacterium orleansense (M2021651) CCTCC, Acinetobacter venetianus (M2022719) CCTCC, and Flavobacterium sp. (M2021801) CCTCC into LB slant culture medium respectively, and culture at 30°C for 24 h to obtain slant bacteria bodies; inoculate the slant bacteria bodies into LB liquid culture medium respectively, and culture at a rotation speed of 160 rpm and a temperature of 30°C for 24 h; centrifuge the culture liquid, and collect the wet bacteria bodies.

[0011] The composition of the LB solid culture medium is: 5 g / L yeast extract, 10 g / L NaCl, 10 g / L peptone, 15-20 g / L agar, pH natural, and deionized water as the solvent;

[0012] The composition of the LB liquid culture medium is: 5 g / L yeast extract, 10 g / L NaCl, 10 g / L peptone, pH natural, and deionized water as the solvent.

[0013] Preferably, the intracellular enzyme solution derived from trichloromethane and propanethiol degrading bacteria is mixed with the intracellular enzyme solution of each of the Stenotrophomonas maltophilia (CCTCC NO: M 20191025) and Acetobacter (CCTCC NO: M 20221016) at a volume ratio of 1:0.5-0.7, preferably 1:0.6. The protein concentration of the intracellular enzyme solution of each of the Stenotrophomonas maltophilia (CCTCC NO: M 20191025) and Acetobacter (CCTCC NO: M 20221016) is 0.5-1.5 mg / mL, preferably 0.9 mg / mL. Stenotrophomonas maltophilia )CCTCC NO: M 20191025 and Acetobacter (CCTCC NO: M 20221016) is mixed at a volume ratio of 1:0.5-0.7, preferably 1:0.6. The protein concentration of the intracellular enzyme solution of each of the Stenotrophomonas maltophilia (CCTCC NO: M 20191025) and Acetobacter (CCTCC NO: M 20221016) is 0.5-1.5 mg / mL, preferably 0.9 mg / mL. Acetobacter sp. )CCTCC NO: M 20191025 and Acetobacter (CCTCC NO: M 20221016) is mixed at a volume ratio of 1:0.5-0.7, preferably 1:0.6. The protein concentration of the intracellular enzyme solution of each of the Stenotrophomonas maltophilia (CCTCC NO: M 20191025) and Acetobacter (CCTCC NO: M 20221016) is 0.5-1.5 mg / mL, preferably 0.9 mg / mL. Stenotrophomonas maltophilia )CCTCC NO: M 20191025 and Acetobacter (CCTCC NO: M 20221016) is mixed at a volume ratio of 1:0.5-0.7, preferably 1:0.6. The protein concentration of the intracellular enzyme solution of each of the Stenotrophomonas maltophilia (CCTCC NO: M 20191025) and Acetobacter (CCTCC NO: M 20221016) is 0.5-1.5 mg / mL, preferably 0.9 mg / mL. Acetobacter sp. )CCTCC NO: M 20191025 and Acetobacter (CCTCC NO: M 20221016) is mixed at a volume ratio of 1:0.5-0.7, preferably 1:0.6. The protein concentration of the intracellular enzyme solution of each of the Stenotrophomonas maltophilia (CCTCC NO: M 20191025) and Acetobacter (CCTCC NO: M 20221016) is 0.5-1.5 mg / mL, preferably 0.9 mg / mL.

[0014] Preferably, the intracellular enzyme solution is prepared as follows: the Stenotrophomonas maltophilia CCTCC NO: M 20191025 or Acetobacter CCTCC NO: M 20221016 is inoculated into LB slant medium, cultured at 30°C for 24 h to obtain slant bacteria; the slant bacteria are inoculated into LB liquid medium, cultured at 160 rpm and 30°C for 24 h, and then centrifuged at low temperature and high speed for 30 min (4°C, 8000 rpm), the bacterial precipitate is washed with 0.05 M phosphate buffer solution with pH 7, and the washing and centrifugation are repeated three times, the washed bacterial solution is broken by an ultrasonic cell disruptor under the condition of ice water bath (ultrasonic for 3 s, intermittent for 2 s, power 400 W, and cyclic work 99 times), and then centrifuged at low temperature and high speed for 30 min (4°C, 8000 rpm), and the supernatant is taken to obtain the intracellular enzyme solution of the Stenotrophomonas maltophilia CCTCC NO: M 20191025 and the intracellular enzyme solution of the Acetobacter CCTCC NO: M 20221016, respectively.

[0015] Preferably, the immobilized enzyme is prepared by the following steps: 2-methyl imidazole, 2-amino benzimidazole and phosphate buffer solution (pH 7, 0.05 M) are mixed and dissolved, then intracellular enzyme solution is added, glutaraldehyde is added, cross-linking is carried out at 30°C water bath under the condition of 250 rpm stirring for 10-20 min, then 100 mol / L zinc nitrate hexahydrate aqueous solution is added, stirring is continued for 10-20 min, after the reaction is completed, the solution is centrifuged at 4°C and 10000 rpm for 10 min, after the supernatant is separated, the precipitate is washed with phosphate buffer solution (pH 7, 0.05 M) and centrifuged, and the above steps are repeated three times; the obtained precipitate is vacuum dried at 0.05 MPa for 12 h to obtain the immobilized enzyme. The volume of 2-methyl imidazole added is 10% of the buffer volume; the volume of 2-amino benzimidazole added is 1% of the buffer volume; the intracellular enzyme solution is a mixture of Stenotrophomonas maltophilia CCTCC NO: M 20191025 intracellular enzyme solution and Acetobacter CCTCC NO: M 20221016 intracellular enzyme solution in a volume ratio of 1:0.5-0.7, and the volume of the intracellular enzyme solution is 100% of the buffer volume; the volume of glutaraldehyde is 2% of the buffer volume; and the volume of the zinc nitrate hexahydrate aqueous solution is 10% of the buffer volume.

[0016] Preferably, the bamboo fiber-bamboo biochar composite material is prepared by the following steps: (1) natural bamboo powder passing through a 100-mesh sieve is weighed, 0.8 M NaOH aqueous solution is added, stirring and impregnation is carried out at room temperature for 12 h, then the sample is dried at 60°C for 2 h, poured into a porcelain boat and placed in a tube furnace, nitrogen is introduced for oxygen deficiency, heated to 250°C at a rate of 5°C / min, kept for 1.5 h, then heated to 500°C, kept for 2.5 h, cooled, washed with deionized water, dried at 60°C to constant weight, ground through a 200-mesh sieve, and absolute dry bamboo charcoal powder is obtained; the volume of the NaOH aqueous solution is 1-5 mL / g of the mass of the natural bamboo powder, preferably 3 mL / g;

[0017] (2) at 25°C, the bamboo fiber is immersed in water for 12 h, then filtered to remove water, water is added, beating is carried out at room temperature until the beating degree is 50SR°, then the beating is stopped (preferably, 560 g of bamboo fiber is immersed in water for 12 h, then filtered to remove water, water is added to a constant volume of 23 L, then beating is carried out at room temperature for 1 h, 2.5 kg of a weighted counterweight is added and the beating is continued for 1 h, 5 kg of a weighted counterweight is added and the beating is continued until the beating degree is 50SR°), filtration is carried out, the filter cake is dried at 60°C to constant weight, and absolute dry bamboo fiber is obtained;

[0018] (3) taking absolute dry bamboo charcoal powder and absolute dry bamboo fiber, adding polyamide epoxy chloropropane resin (PAE) and deionized water, high-speed defibrillation for 15 min in a defibrator (model TD15-A) at a rotation speed of 10000 rpm to obtain a uniformly dispersed black viscous liquid; rapidly placing the black viscous liquid in a paper sheet former, filtering off the water, and vacuum pressing at 95 DEG C for 10 min to obtain a bamboo fiber-bamboo biochar composite material; the mass ratio of the absolute dry bamboo charcoal powder to the absolute dry bamboo fiber is 1:1-5, preferably 1:3; the mass ratio of the total mass of the absolute dry bamboo charcoal powder and the absolute dry bamboo fiber to the polyamide epoxy chloropropane resin is 1:0.01-0.04, preferably 1:0.02; the volume of the deionized water is 50-150 mL / g, preferably 100 mL / g, based on the total mass of the absolute dry bamboo charcoal powder and the absolute dry bamboo fiber.

[0019] The bacteria-enzyme synergistically fixed bioactive functional filler is prepared by the following method: adding the bamboo fiber-bamboo biochar composite material into an inorganic salt solution containing immobilized enzymes, soaking at 25 DEG C, turning over every 3 h, repeating for 4 times, taking out, drying at 30 DEG C to constant weight, then placing into an inorganic salt solution containing a composite bacterial agent, soaking at 25 DEG C, turning over every 3 h, repeating for 4 times, taking out, drying at 30 DEG C to constant weight, to obtain the bacteria-enzyme synergistically fixed bioactive functional filler; the mass ratio of the bamboo fiber-bamboo biochar composite material to the immobilized enzymes is 1:0.01-0.1, preferably 1:0.03; the dry mass ratio of the bamboo fiber-bamboo biochar composite material to the composite bacterial agent is 1:0.1-1, preferably 1:0.2.

[0020] The inorganic salt solution comprises K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl2·6H2O 0.2033 g / L, CaCl2·2H2O 0.0111 g / L, FeCl3 0.0162 g / L, trace elements 5 mL / L, a solvent is deionized water, and pH is 5-10; the trace elements comprise ZnCl2 0.088 g / L, MnCl2·4H2O 0.060 g / L, KI 0.01 g / L, Na2MoO4·2H2O 0.1 g / L, and H3BO3 0.05 g / L, a solvent is deionized water.

[0021] The application further provides a use of the bacteria-enzyme synergistically fixed bioactive functional filler in degrading organic pollutants, wherein the organic pollutants include n-hexane, cyclohexane, butyl acetate, trichloromethane or propanethiol.

[0022] Preferably, the application uses a biological trickling tower device to degrade organic pollutants: the bacteria enzyme synergistic fixed biological active functional filler is loaded into the filler layer of the biological trickling tower; after 24 hours of fresh air, the simulated exhaust gas generated by air bubbling of the organic pollutants is introduced into the biological trickling tower, and the inorganic salt solution is sprayed into the filler layer at the same time, and the treated tail gas is introduced into the fume hood; the inorganic salt solution is replaced every 3 days to achieve the degradation of organic pollutants.

[0023] Preferably, the simulated exhaust gas contains n-hexane 400 mg / L, cyclohexane 300 mg / L, butyl acetate 800 mg / L, chloroform 15 mg / L and propyl mercaptan 600 mg / L.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] (1) The bamboo fiber-bamboo biochar composite material prepared by compounding bamboo fiber and bamboo charcoal with multiple pore sizes is used as a carrier, and 2% of PAE by mass fraction is added to increase the wet strength, adsorb small molecule metabolites, effectively solve the problem of secondary pollution, make the mixed exhaust gas purification process more green and safe, and solve the defects of traditional exhaust gas biological purification.

[0026] (2) The high-density distribution of microorganisms and biological enzymes on the bamboo fiber-bamboo biochar composite material can shorten the start-up period of the biological reactor, increase the biomass in the unit filler, improve the efficiency of the reactor operation, shorten the residence time, establish a new type of efficient complex typical pollutant removal system, and the microorganisms and biological enzymes can play an advantage in mineralization and removal of heteroatoms, and can realize rapid degradation of complex typical pollutants.

[0027] (3) The bacteria enzyme synergistic biological active functional filler prepared by the method of the application not only solves the process defects of traditional exhaust gas biological purification, but also has no secondary pollution problem, so that the mixed exhaust gas purification process is more green and safe.

[0028] (4) The bacteria enzyme synergistic biological active functional filler prepared by the method of the application is used for degrading organic pollutants in a biological trickling tower, and it is found that the abundance of characteristic degradation bacteria in the unit filler is high, the mixed exhaust gas purification effect is good, no additional carbon source is needed, only the nutrient solution needs to be replaced regularly, the operation cost is lower, the total organic matter content in the circulating liquid is low, no secondary pollution is produced, efficient degradation of multi-component pollutants is realized, no secondary pollution is produced, and green and safe degradation is realized.

[0029] (5) The bacterial-enzyme synergistic bioactive functional packing prepared by the method of the present invention has a degradation efficiency of 90%, 85%, 95%, and 95% for pollutants such as straight-chain alkanes, cyclic alkanes, esters, and alcohols, respectively. It has a good purification effect on mixed waste gas, especially for the degradation effect (including mineralization and dechlorination) of chloroform, which is a typical difficult-to-degrade substance. The mineralization efficiency and dechlorination efficiency of chloroform are higher than those of single bacterial or enzyme treatment technologies. (iv) Description of the attached drawings

[0030] Figure 1 This is a schematic diagram of the structure of a microbial enzyme synergistic bioactive functional filler; where: 1 represents the carrier material, 2 represents the composite microbial community, and 3 represents the immobilized enzyme.

[0031] Figure 2 This is a schematic diagram of a biological trickling filter treatment system; where: 4 represents a gas generator, 5 represents a mixing tank, 7 represents a biological trickling filter, and 8 represents a circulating liquid.

[0032] Figure 3 This is a diagram illustrating the effect of bacterial enzyme synergistic bioactive functional filler in degrading multi-component pollutants.

[0033] Figure 4 This is a comparison chart showing the efficiency of synergistic bioactive functional fillers (bacteria and enzymes) in the degradation, mineralization, and dechlorination of chloroform compared to those of single bacteria or enzymes.

[0034] Figure 5 It is a plate graph showing the changes in microbial abundance during operation.

[0035] Figure 6 This is a graph showing the relative abundance of microorganisms during the stable operation period.

[0036] Figure 7 This is a graph showing the total organic matter content of the circulating fluid. (V) Detailed Implementation Methods

[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0038] The Mycobacterium nov. used in the embodiments of this invention ( Mycolicibacterium neworleansense WCJ accession number is CCTCC NO: M2021651; Flavobacterium ( Microbacterium maritypicum HYY-2 accession number CCTCC NO: M 2021801; Acinetobacter veneriense ( Acinetobacter venetianus PFZR-1 accession number CCTCC NO:M 2022719. The bacteria used in the above examples are disclosed in patent applications CN113801821A, CN113699074A, and CN115786183A, respectively.

[0039] Stenotrophomonas maltophilia (Stenotrophomonas maltophilia GYH CCTCC NO: M20191025; Acetobacter xylinum (ATCC 53524) Acetobacter sp. WJJ CCTCC NO: M 20221016. The bacteria have been disclosed in CN11254091 A patent application.

[0040] The composition of the inorganic salt solution is: K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl2·6H2O 0.2033 g / L, CaCl2·2H2O 0.0111 g / L, FeCl3 0.0162 g / L, trace elements 5 mL / L, solvent is deionized water, pH = 5-10; the composition of the trace elements is: ZnCl2 0.088 g / L, MnCl2·4H2O, 0.060 g / L, KI 0.01 g / L, Na2MoO4·2H2O 0.1 g / L, H3BO3 0.05 g / L, solvent is deionized water.

[0041] Example 1: Preparation of a functional filler with biological activity by synergistic immobilization of bacteria and enzymes

[0042] 1. Compound microbial agent: respectively inoculate WCJ, Mycolicibacterium neworleansense PFZR-1 into LB slant medium, cultivate at 30 ℃ for 24 h, and obtain the slant bacteria; inoculate the slant bacteria into LB liquid medium, cultivate at 160 rpm and 30 ℃ for 24 h, centrifuge the culture solution, and collect the wet bacteria. Microbacterium maritypicum HYY-2, Acinetobacter venetianus PFZR-1 into LB slant medium, cultivate at 30 ℃ for 24 h, and obtain the slant bacteria; inoculate the slant bacteria into LB liquid medium, cultivate at 160 rpm and 30 ℃ for 24 h, centrifuge the culture solution, and collect the wet bacteria.

[0043] Mix the wet bacteria of WCJ, Mycolicibacterium neworleansense HYY-2, Microbacterium maritypicum PFZR-1 at a wet weight mass ratio of 1:1.5:2, that is, the mixed microbial agent. Acinetobacter venetianus The composition of LB solid culture medium is: 5 g / L yeast extract, 10 g / L NaCl, 10 g / L peptone, 15-20 g / L agar, pH natural, solvent is deionized water.

[0044] The composition of LB liquid culture medium is: 5 g / L yeast extract, 10 g / L NaCl, 10 g / L peptone, pH natural, solvent is deionized water.

[0045] 2. Immobilized enzyme

[0046]

[0047] ​(1) Bioenzyme extracted from Stenotrophomonas maltophilia CCTCC NO: M 20191025

[0048] Stenotrophomonas maltophilia CCTCC NO: M 20191025 was inoculated into LB slant medium and cultured at 30 ℃ for 24 h to obtain slant bacteria; the slant bacteria were inoculated into LB liquid medium, respectively, and cultured at 30 ℃ and a rotation speed of 160 rpm for 24 h; the culture solution was centrifuged at low temperature and high speed for 30 min (4 ℃, 8000 rpm), the bacterial precipitate was washed with 0.05 M phosphate buffer solution with a pH of 7, and the washing and centrifugation were repeated three times; the washed bacterial solution was broken in an ice water bath using an ultrasonic disrupter (ultrasonic for 3 s, intermittent for 2 s, power 400 W, and cyclic work 99 times), and then centrifuged at low temperature and high speed for 30 min (4 ℃, 8000 rpm); the supernatant was the intracellular enzyme solution of Stenotrophomonas maltophilia, which was diluted to a protein concentration of 0.9 mg / mL using 0.05 M phosphate buffer solution after the protein concentration was measured by the Bradford method, and was stored in a 4 ℃ refrigerator for use.

[0049] Protein concentration determination: the bovine serum standard protein solution was diluted by different multiples, 1 mL of each was added to 5 mL of coomassie brilliant blue staining agent, and the absorbance was measured at 595 nm after standing for five minutes, and the standard protein concentration curve was drawn. 1 mL of the sample to be tested was added to a 10 mL centrifuge tube, and 5 mL of the prepared coomassie brilliant blue staining agent was added, and the absorbance was measured after standing for 5 min and was brought into the standard protein curve to calculate the protein concentration.

[0050] (2) Bioenzyme extracted from Acetobacter CCTCC NO: M 20221016

[0051] In step (1), Stenotrophomonas maltophilia CCTCC NO: M 20191025 was replaced by Acetobacter CCTCC NO: M 20221016, and the other operations were the same, to prepare the intracellular enzyme solution of Acetobacter, which was diluted to a protein concentration of 0.9 mg / mL by 0.05 M phosphate buffer solution.

[0052] (3) Immobilized enzyme

[0053] Into a 50 mL beaker, 1.5 mL of 2-methylimidazole, 0.15 mL of 2-aminobenzimidazole and 15 mL of phosphate buffer solution (pH 7, 0.05 M) were sequentially added, and after dissolution, 9 mL of the intracellular enzyme solution of S. maltophilia prepared in step (1) and 6 mL of the intracellular enzyme solution of Acetobacter prepared in step (2) were added. Then, 0.3 mL of glutaraldehyde was added, and the crosslinking was performed at 30°C in a water bath with stirring at a speed of 250 rpm for 10-20 min. Then, 1.5 mL of 100 mol / L zinc nitrate hexahydrate aqueous solution was added, and the stirring was continued for 10-20 min. After the reaction was completed, the solution was centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was separated. The precipitate was washed and centrifuged with phosphate buffer solution (pH 7, 0.05 M) for three times. The obtained precipitate was dried under vacuum at 0.05 MPa for 12 h to obtain 2.05 g of the immobilized enzyme.

[0054] The immobilized enzyme 0.1 g was inoculated into 50 mL of an inorganic salt solution containing 8 mg / L of chloroform and 200 mg / L of propanethiol, and was cultured at 30°C and 160 rpm for 60 h. The concentrations of chloroform and propanethiol in the culture solution were detected by sampling. When the degradation efficiencies of chloroform and propanethiol both reached 100%, the culture solution was centrifuged at 8,000 rpm for 15 min, and the precipitate was dried at 30°C to constant weight to obtain 0.5 g of activated immobilized enzyme, which was used for the next step.

[0055] 3. Bamboo fiber-bamboo biochar composite material:

[0056] 10 g of natural bamboo powder sieved through a 100-mesh sieve was weighed and added into 30 mL of 0.8 M NaOH aqueous solution. After stirring and soaking at room temperature for 12 h, the mixture was filtered, and the filter cake was dried at 60°C for 2 h. The dried sample was poured into a porcelain boat and placed in a tube furnace. Nitrogen was introduced to create an oxygen-deficient environment. The temperature was raised to 250°C at a rate of 5°C / min, and then the temperature was maintained for 1.5 h. Then, the temperature was continuously raised to 500°C, and the temperature was maintained for 2.5 h. After cooling, the sample was washed with deionized water, and then dried at 60°C to constant weight. The dried sample was ground and sieved through a 200-mesh sieve to obtain 5 g of absolutely dry bamboo charcoal powder.

[0057] At room temperature, 560 g of bamboo fiber was soaked in 5 L of water for 12 h, and then the water was filtered off. The bamboo fiber was added with water to a final volume of 23 L, and then the mixture was poured into a Valli beater. The bamboo fiber was beaten at room temperature for 1 h, and then 2.5 kg of a weight was added to continue beating for another 1 h. After adding a weight of 5 kg, the beating was continued until the beating degree reached 50 SR°, and then the beating was stopped. The bamboo fiber was filtered, and the filter cake was dried at 60°C to constant weight to obtain 560 g of absolutely dry bamboo fiber.

[0058] Take 5 g of absolutely dry bamboo charcoal powder and 15 g of absolutely dry bamboo fiber, add 0.4 g of polyamide epoxy chloropropane resin (PAE), and dilute to 2 L with deionized water. Disperse at a speed of 10000 rpm for 15 min in a defibrator (model TD15-A) to obtain a black, uniformly dispersed thick liquid. Place the black viscous liquid in a paper sheet former, filter out the water, and press at 95°C under vacuum for 10 min to obtain 20.8 g of bamboo fiber-bamboo biochar composite material with a size of 15 mm 10 mm 10 mm.

[0059] 4、Bacteria-enzyme synergistically immobilized bioactive functional filler

[0060] Take 3.00 g of the bamboo fiber-bamboo biochar composite material prepared in step 3, add 50 mL of an inorganic salt solution containing 100 mg of the immobilized enzyme activated in step 2, and soak at 25°C, turning over every 3 h. Repeat 4 times, then take out and dry at 30°C. Then place in 50 mL of an inorganic salt solution containing 600 mg of the composite microbial agent prepared in step 1, soak at room temperature, turning over every 3 h. Repeat 4 times, then take out and dry at 30°C to obtain 3.5 g of bacteria-enzyme synergistically immobilized bioactive functional filler. The schematic diagram is shown in Figure 1 .

[0061] Comparative Example 1: Single bacteria immobilized biofiller

[0062] Take 3.00 g of the bamboo fiber-bamboo biochar composite material prepared in step 3 of Example 1, add 50 mL of an inorganic salt solution containing 600 mg of the composite microbial agent prepared in step 1, and soak at room temperature, turning over every 3 h. Repeat 4 times, then take out and dry at 30°C to obtain a single bacteria immobilized biofiller.

[0063] Comparative Example 2: Single enzyme immobilized biofiller

[0064] Take 3.00 g of the bamboo fiber-bamboo biochar composite material prepared in step 3 of Example 1, add 50 mL of an inorganic salt solution containing 100 mg of the immobilized enzyme activated in step 2, and soak at 25°C, turning over every 3 h. Repeat 4 times, then take out and dry at 30°C to obtain a single enzyme immobilized biofiller.

[0065] Example 2: Bacteria-enzyme synergistically immobilized bioactive functional filler for treating multi-component mixed waste gas

[0066] 1. Mixed waste gas removal effect

[0067] Use Figure 2The bio-trickling filter device shown in the figure was filled with the functional filler with bacteria-enzyme synergistically immobilized biological activity prepared by the method of Example 1, and the filling amount was 3 L. After the bio-trickling filter was connected with fresh air for 24 h, the simulated waste gas was generated by bubbling the waste gas (containing n-hexane 400 mg / L, cyclohexane 300 mg / L, butyl acetate 800 mg / L, chloroform 15 mg / L and propyl mercaptan 600 mg / L) into the mixing tank by the air pump, and the simulated waste gas was connected with the bio-trickling filter. At the same time, the inorganic salt solution (module 8) was sprayed on the filler layer of the bio-trickling filter, and the tail gas treated by the filler layer of the bio-trickling filter was connected with the fume hood. The inorganic salt solution was replaced every 3 days.

[0068] The samples were taken from the exhaust port on the 3rd, 7th, 10th, 30th, 60th and 90th day respectively, and the concentrations of various pollutants were detected by Agilent 6890 gas chromatograph (Agilent, USA). The degradation effect is shown in Figure 3 .

[0069] The concentrations of various pollutants were detected by Agilent 6890 gas chromatograph (Agilent, USA), and HP-Innowax type capillary column (30 m) x 0.32 mm x 0.5 μm) was configured. Nitrogen was used as carrier gas; gas flow rate 1 mL / min, split ratio 15:1; gas sample amount 0.8 mL.

[0070] The results show that the removal effect of the bio-trickling filter device filled with the functional filler with bacteria-enzyme synergistically immobilized biological activity on n-hexane, cyclohexane, butyl acetate and propyl mercaptan is about 90%, and the removal effect on chloroform is about 80%, which shows that the functional filler with bacteria-enzyme synergistically immobilized biological activity can efficiently and stably remove multi-component mixed waste gas, especially the typical refractory chloroform.

[0071] 2, Microbial population quantity in the biofilm formation and start-up period

[0072] At 1 d, 3 d, 7 d and 30 d, the microbial population on the filler layer was collected by sampling rod through the sampling port on the bio-trickling filter, and then diluted 10 5 times with sterile water after stirring, and then coated on LB solid culture medium, and then placed in a 30 ℃ incubator for 24 h. The microbial population quantity was calculated by cross streak method. The microbial population quantity of the maximum dilution was compared by sampling and coating once a day according to the above method, and the results are shown in Figure 5The results show that the microbial abundance increases rapidly within 7 days, indicating that the biofilm formation is very rapid. After 7 days, the microbial abundance on the biofilm is consistent with the number of microorganisms in the stable operation period, indicating that 7 days is the start-up period of microbial biofilm formation, and the reactor reaches a stable state after about 7 days. The biofilm formation period of the bacteria-enzyme synergistic bioactive functional filler is greatly shortened.

[0073] 3. Microbial community structure on the bacteria-enzyme synergistic immobilized bioactive functional filler in the stable operation period

[0074] The surface microorganisms of the filler layer in the stable operation period were sampled, and after total DNA extraction, PCR amplification steps, the PCR products were entrusted to Miseq 341F-806R platform of Mingke Biological Co., Ltd. for high-throughput sequencing analysis work. The sequencing results are shown in Figure 6 , and the non-labeled proteomics identification of the immobilized crude enzyme on the surface of the filler layer in the stable operation period is shown in Table 1. The results show that the added complex microbial agent becomes the dominant microorganism in the stable operation period, especially for the removal of high-load butyl acetate, the relative abundance of its characteristic degrading bacteria reaches more than 30%, and the relative abundance of the characteristic bacteria of the remaining typical pollutants is more than 20%, and several microorganisms such as Pseudomonas are produced in the reactor. The protein identification overview shows that there are many active proteins, and under the impact of high-load pollutants, the number of proteins can still maintain a high level. The enzymes involved in dehalogenation reaction mainly include oxidase, dehydrogenase and dehalogenase.

[0075] Table 1: Protein identification overview

[0076]

[0077] 4. Total organic matter content determination of the circulating liquid samples during the operation process

[0078] The circulating liquid samples were sampled from the circulating liquid at 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day, and the total organic matter content determination of the circulating liquid samples during the operation process was carried out by using Sievers 860 total organic carbon TOC analyzer. The results are shown in Figure 7 . The results show that the total organic matter content in the circulating liquid is at a low level, and the total organic carbon content in the effluent is < 20 mg / L, which is far lower than the wastewater comprehensive discharge standard in GB 8978-1996, indicating that the bacteria-enzyme synergistic immobilized bioactive functional filler has good adsorption and degradation ability for metabolic small molecule intermediates, and there is no secondary pollution after degradation.

[0079] Example 3: Comparison and investigation of the mineralization and dechlorination efficiency of the typical pollutant trichloromethane

[0080] The single bacteria immobilized biological filler prepared in Comparative Example 1, the single enzyme immobilized filler prepared in Comparative Example 2, and the bacteria-enzyme synergistically immobilized biological active filler prepared in Example 1 were used to replace the bacteria-enzyme synergistically immobilized biological active filler prepared in Example 2, the mixture of trichloromethane and air was used to replace the combined exhaust gas, the concentration of trichloromethane was 15 mg / L, and other operations were the same.

[0081] At 12, 24, 36, 48, 60, 72, and 84 h, samples were taken from the circulating liquid, diluted 100 times with deionized water, purified through an ion chromatography special pretreatment column, and then detected for the concentration of chloride ions by ion chromatography, and samples were taken from the outlet tail gas and detected for the concentration of CO2 by gas chromatography, and the results are shown in Table 1. Figure 4

[0082] The ion chromatography detection conditions were as follows: the model was Dionex ICS2000, the detection column was IonPac AS18, the sample injection amount was 1 mL, and the detector temperature was set to 30 °C.

[0083] The gas chromatography detection conditions were as follows: the model was Agilent 6890, the detector was a TCD detector, the gas injection amount was 0.8 mL, the carrier gas was nitrogen, the detector temperature was set to 180 °C, the injector temperature was set to 90 °C, and the column oven temperature was set to 40 °C.

[0084] Figure 4 It is shown that the single bacteria has a better mineralization effect on pollutants than the single enzyme, but has a poorer heteroatom removal effect, the single enzyme is on the contrary, the bacteria-enzyme synergistically immobilized biological active filler has both dechlorination and mineralization effects, and the bacteria and the enzyme promote each other, the dechlorination and mineralization effects are maintained at more than 90%, which is much greater than the treatment effect of the single bacteria or the enzyme. The bacteria-enzyme synergistically immobilized biological active filler can effectively improve the removal of complex pollutants containing heteroatoms.​

Claims

1. A bacteria-enzyme synergistically immobilized bioactive functional filler, characterized in that, The bioactive functional filler with synergistic bacterial and enzyme immobilization is made by loading a composite bacterial agent and immobilized enzyme onto a bamboo fiber-bamboo biochar composite material. The composite bacterial agent includes wet cells of strains that degrade straight-chain alkanes, cycloalkanes, esters, or alcohols through expanded culture. The immobilized enzyme is prepared by immobilizing intracellular enzyme solutions derived from chloroform or propane-thiol-degrading bacteria within a metal-organic framework. The bamboo fiber-bamboo biochar composite material is made by pulping bamboo powder and bamboo fiber. The composite bacterial agent is derived from *Mycobacterium nov. Orleans* (…). Mycolicibacterium neworleansense CCTCC NO: M 2021651, Acinetobacter venetianis ( Acinetobacter venetianus CCTCC NO:M 2022719, Flavobacterium ( Microbacterium maritypicum CCTCC NO:M 2021801 The wet bacterial cells were obtained by mixing them at a mass ratio of 1:1.2-1.8:1.5-2.

5.

2. The bacterial enzyme synergistic immobilization bioactive functional filler as described in claim 1, characterized in that, The complex microbial agent is added in an amount of 10-40% of the weight of the dry bacteria to the mass of the carrier, and the immobilized enzyme is used in an amount of 1-5% of the mass of the carrier.

3. The bacterial enzyme synergistic immobilization bioactive functional filler as described in claim 1, characterized in that, The intracellular enzyme solution derived from chloroform and propane-thiol degrading bacteria was prepared by separately adding Stenotrophomonas maltophilia (… Stenotrophomonas maltophilia CCTCC NO: M 20191025 and Acetic Acid Bacillus ( Acetobacter sp. The intracellular enzyme solutions of each of the CCTCC NO: M20221016 were mixed at a volume ratio of 1:0.5-0.

7.

4. The bacterial enzyme synergistic immobilization bioactive functional filler as described in claim 3, characterized in that, The intracellular enzyme liquid is prepared by the following steps: inoculating the Stenotrophomonas maltophilia CCTCC NO: M 20191025 or the Acetobacter sp. CCTCC NO: M 20221016 into LB slant culture medium, respectively, and culturing at 30°C for 24 hours to obtain slant bacteria; inoculating the slant bacteria into LB liquid culture medium, respectively, and culturing at a rotation speed of 160 rpm and a temperature of 30°C for 24 hours; centrifuging the culture liquid at a low temperature and a high speed of 8000 rpm for 30 minutes at 4°C; rinsing the bacterial precipitate with a phosphate buffer solution with a pH of 7 and a concentration of 0.05 M; repeating the washing and centrifuging three times; crushing the cells in an ice water bath, an ultrasonic wave for 3 seconds, an interval of 2 seconds, a power of 400 W, and a cycle of 99 times; centrifuging the crushed cells at a low temperature and a high speed of 8000 rpm for 30 minutes at 4°C; and obtaining the intracellular enzyme liquid of the Stenotrophomonas maltophilia CCTCC NO: M 20191025 and the intracellular enzyme liquid of the Acetobacter sp. CCTCC NO: M 20221016 from the supernatant, respectively.

5. The bacterial enzyme synergistic immobilization bioactive functional filler as described in claim 1, characterized in that, The immobilized enzyme is prepared by the following steps: mixing and dissolving 2-methylimidazole, 2-aminobenzimidazole, and a phosphate buffer solution with a pH of 7 and a concentration of 0.05 M, adding the intracellular enzyme liquid, adding glutaraldehyde, stirring at a rotation speed of 250 rpm in a 30°C water bath for 10-20 minutes, adding 100 mol / L zinc nitrate hexahydrate aqueous solution, continuing to stir for 10-20 minutes, centrifuging the solution at a rotation speed of 10000 rpm for 10 minutes at 4°C after the reaction is completed, rinsing the precipitate with a phosphate buffer solution with a pH of 7 and a concentration of 0.05 M, and repeating the centrifuging three times; vacuum drying the obtained precipitate at 0.05 MPa for 12 hours to obtain the immobilized enzyme; the volume of 2-methylimidazole added is 10% of the volume of the buffer solution; the volume of 2-aminobenzimidazole added is 1% of the volume of the buffer solution; the volume of the intracellular enzyme liquid is 100% of the volume of the buffer solution; the volume of glutaraldehyde is 2% of the volume of the buffer solution; and the volume of the zinc nitrate hexahydrate aqueous solution is 10% of the volume of the buffer solution.

6. The bacteria-enzyme synergistically immobilized bioactive functional filler according to claim 1, wherein the bacteria-enzyme synergistically immobilized bioactive functional filler is a bacteria-enzyme synergistically immobilized bioactive functional filler for removing formaldehyde. The bamboo fiber-bamboo biochar composite material is prepared by the following steps: (1) a natural bamboo powder passing through a 100-mesh sieve is weighed, and then added into a 0.8M NaOH aqueous solution; the mixture is stirred at room temperature for 12 hours, and then dried at 60°C for 2 hours; the dried sample is poured into a porcelain boat and placed in a tube furnace; nitrogen is introduced to create an oxygen-deficient environment; the temperature is raised to 250°C at a rate of 5°C / min, and then kept at 250°C for 1.5 hours; then the temperature is continuously raised to 500°C, and kept at 500°C for 2.5 hours; after cooling, the sample is taken out, washed with deionized water, dried at 60°C to a constant weight, ground through a 200-mesh sieve, and then an absolutely dry bamboo charcoal powder is obtained; (2) the bamboo fiber is immersed in clean water at 25°C for 12 hours, then filtered and dried; clean water is added, and the bamboo fiber is beaten at room temperature until the beating degree reaches 50SR°, then the beating is stopped; the bamboo fiber is filtered and dried at 60°C to a constant weight, and then an absolutely dry bamboo fiber is obtained; (3) the absolutely dry bamboo charcoal powder and the absolutely dry bamboo fiber are taken, and then added into polyamide epoxy chloropropane resin and deionized water; the mixture is high-speed defibrated in a defibrator at a speed of 10000 rpm for 15 minutes, and then a uniformly dispersed black viscous liquid is obtained; the black viscous liquid is quickly placed in a paper sheet former, and then filtered and dried; the mixture is vacuum-pressed at 95°C for 10 minutes, and then a bamboo fiber-bamboo biochar composite material is prepared; the mass ratio of the absolutely dry bamboo charcoal powder to the absolutely dry bamboo fiber is 1:1-5; the mass ratio of the total mass of the absolutely dry bamboo charcoal powder and the absolutely dry bamboo fiber to the polyamide epoxy chloropropane resin is 1:0.01-0.04; and the volume of the deionized water is 50-150 mL / g based on the total mass of the absolutely dry bamboo charcoal powder and the absolutely dry bamboo fiber.

7. The bacteria-enzyme synergistically immobilized bioactive functional filler according to claim 1, wherein the bacteria-enzyme synergistically immobilized bioactive functional filler is a bacteria-enzyme synergistically immobilized bioactive functional filler for removing formaldehyde. The fungus enzyme synergistically fixed bioactive functional filler is prepared by the following method: the bamboo fiber-bamboo biochar composite material is added into an inorganic salt solution containing immobilized enzymes, and then soaked at 25°C; the soaking is repeated for 4 times, and the soaking time is 3 hours each time; then the mixture is taken out and dried at 30°C to a constant weight; then the mixture is added into an inorganic salt solution containing a composite microbial agent, and then soaked at 25°C; the soaking is repeated for 4 times, and the soaking time is 3 hours each time; then the mixture is taken out and dried at 30°C to a constant weight, and then a fungus enzyme synergistically fixed bioactive functional filler is obtained; the mass ratio of the bamboo fiber-bamboo biochar composite material to the immobilized enzymes is 1:0.01-0.1; and the mass ratio of the dry weight of the bamboo fiber-bamboo biochar composite material to the composite microbial agent is 1:0.1-1.

8. The use of the bacteria-enzyme synergistically immobilized bioactive functional filler as claimed in claim 1 in the degradation of organic pollutants, characterized in that, The organic pollutants include n-hexane, cyclohexane, butyl acetate, trichloromethane or propyl mercaptan.

9. Use according to claim 8, wherein the compound is ###0002### The application adopts a biological trickling filter tower device to degrade the organic pollutants: the fungus enzyme synergistically fixed bioactive functional filler is loaded into a filler layer of the biological trickling filter tower; fresh air is introduced into the biological trickling filter tower for 24 hours; the organic pollutants are introduced into the biological trickling filter tower in the form of simulated waste gas generated by air bubbling through an air pump; at the same time, the inorganic salt solution is circulated and sprayed to the filler layer; the treated tail gas is introduced into a fume hood; the inorganic salt solution is replaced every 3 days, and thus the degradation of the organic pollutants is realized.

Images