Preparation method of biomineralization biological agent for cleaning RO (Reverse Osmosis) membrane

Abstract

The invention discloses a preparation method of a biomineralization biological agent for RO (Reverse Osmosis) membrane cleaning, and relates to the technical field of membrane cleaning. The invention aims to solve the problems that chemical cleaning of a reverse osmosis membrane in the prior art is easy to cause membrane material corrosion and microcrack proliferation to cause performance degradation, chemical residues of cleaning waste liquid can aggravate desalination load and environmental risk of a subsequent process, and existing biological cleaning is not thorough. The method comprises the following steps: 1, expanding culture of a primary seed solution; 2, carrying out scale-up culture in a secondary fermentation tank; 3, thallus separation and pretreatment; 4, constructing a microorganism-carrier composite system; and 5, performing engineering forming on the biological agent. The biomineralization biological agent for cleaning the RO membrane is suitable for efficient regeneration of the RO membrane in the scenes of seawater desalination, industrial wastewater treatment and the like, the service life of the RO membrane is prolonged, efficient scale inhibition and corrosion inhibition are achieved, and the water quality is stable.

Description

Technical Field

[0001] The invention relates to the technical field of membrane cleaning, and in particular to a method for preparing a biomineralization biopharmaceutical for RO membrane cleaning. Background Art

[0002] As the core technology in the fields of water treatment, biopharmaceuticals and chemical separation in the 21st century, membrane separation technology is highly dependent on the development of membrane pollution control and cleaning technology for its efficient operation. In recent years, membrane cleaning research has shown three major characteristics: deepening mechanism, technology integration, and green intelligence. It is leapfrogging from empirical operation to precise control. Physical cleaning is a technology that uses physical methods to clean membranes. It has the advantages of zero chemical addition, ecological compatibility, and intelligent control. It can efficiently remove reversible pollutants on the membrane surface, but it has limited depth of action and is difficult to remove deep retentate. High-frequency and high-voltage pulses cause stress accumulation in membrane materials, resulting in decreased flux stability. Chemical cleaning is a technology that uses chemical methods to clean membranes. Through mechanisms such as acid-base neutralization, redox, and surfactant complexation (covering three types of acid / base / oxidant cleaning processes), although it can target the decomposition of pollutants and penetrate the membrane pores for deep cleaning, it is easy to cause corrosion of membrane materials and microcracks, resulting in performance degradation, and the chemical residues in the cleaning waste liquid will increase the desalination load and environmental risks of subsequent processes.

[0003] As an emerging green membrane regeneration strategy, biological cleaning technology targets pollutants through the biological metabolism of functional bacteria or enzyme systems, and shows unique advantages in solving problems such as secondary pollution from chemical cleaning and insufficient efficiency of physical cleaning. Biological cleaning uses biodegradation as its core mechanism, and the final products are CO2, H2O and small molecular organic matter (such as fatty acids and amino acids), without any toxic by-products. Compared with chemical cleaning, which produces tens of thousands of tons of waste liquid each year, the carbon footprint of biological cleaning technology is reduced by more than 60%. Moreover, functional microorganisms can decompose stubborn pollutants that are difficult to handle by traditional methods through specific enzyme systems.

[0004] The application of biomineralization in reverse osmosis (RO) membrane cleaning is an emerging and promising research direction. Its core principle is to use the mineralization process regulated by microorganisms to improve membrane fouling, improve cleaning efficiency and reduce environmental impact. Therefore, it has broad development prospects and is suitable for reverse osmosis membrane cleaning in high-salt or high-alkali environments. For example, it can deal with carbonate / sulfate scaling problems caused by high-hardness water in seawater desalination, deal with membrane fouling problems of oily and high-organic wastewater in industrial wastewater reuse, and deal with chemical residue problems in drinking water treatment.

[0005] Although reverse osmosis membrane biological cleaning technology is environmentally friendly, it has the following disadvantages: low cleaning efficiency, slow and incomplete decomposition of complex biofilms by biological enzymes or microorganisms; limited applicability, specific enzyme preparations only target some pollutants (such as proteases that decompose proteins), and cannot cope with multiple complex pollution, and enzyme activity is easily affected by factors such as pH and temperature. Summary of the invention

[0006] The purpose of the present invention is to solve the problems in the prior art of chemically cleaning reverse osmosis membranes, which easily cause membrane material corrosion and microcrack proliferation, leading to performance degradation, and the chemical residues in the cleaning waste liquid will aggravate the desalination load and environmental risks of subsequent processes, and the problem that the existing biological cleaning has incomplete cleaning, and to provide a method for preparing a biomineralization biopharmaceutical for RO membrane cleaning.

[0007] A method for preparing a biomineralization biopharmaceutical for RO membrane cleaning is specifically completed in the following steps:

[0008] 1. Primary seed liquid expansion culture:

[0009] ①. Activation of bacterial strains: inoculate the three frozen functional strains on three LB solid culture media respectively, culture them at a constant temperature for a period of time, and pick out single colonies for subsequent amplification culture;

[0010] ② Liquid pre-culture: Select 3 typical bacterial colonies and inoculate them into a certain amount of liquid culture medium, and then place them in a constant temperature shaker for a period of time to obtain 3 primary seed solutions;

[0011] 2. Secondary fermentation tank scale-up culture:

[0012] ①. Transfer the three primary seed solutions to a fully automatic fermenter filled with liquid culture medium according to a certain inoculation amount, adopt a batch feeding culture strategy and control the fermentation conditions;

[0013] ②. Nutrient feeding control:

[0014] Carbon source supplementation: When the residual sugar concentration drops to a certain value, glucose solution is added at a certain rate;

[0015] Nitrogen source regulation: urea concentration is maintained at an appropriate concentration;

[0016] End point determination: When OD600 reaches a certain value, the fermentation is terminated and the fermentation liquid is obtained;

[0017] 3. Bacteria separation and pretreatment:

[0018] ①, Centrifugal harvest: Use a continuous flow centrifuge to process the fermentation liquid and collect the wet bacterial sludge;

[0019] ②. Resuspend the bacterial sludge: Use pre-cooled phosphate buffer to resuspend the bacterial sludge to the final concentration to obtain a bacterial suspension for later use;

[0020] 4. Construction of microorganism-carrier composite system:

[0021] The microorganism-carrier composite system is constructed by using method one or method two;

[0022] The construction of the microorganism-carrier composite system using method 1 is specifically completed in the following steps:

[0023] ①. Carrier pretreatment: modifying diatomaceous earth to obtain modified diatomaceous earth; sieving and grading the modified diatomaceous earth, selecting a certain particle size fraction, and obtaining a carrier;

[0024] ②, the bacterial suspension was mixed with the carrier, and the mixture was adsorbed under constant temperature oscillation for a period of time. During this period, the OD600 value of the supernatant was measured every 30 minutes. When the OD600 value changed by less than 5%, the oscillation adsorption was stopped to obtain the biological-carrier composite system;

[0025] The construction of the microorganism-carrier composite system using method 2 is specifically completed in the following steps:

[0026] (1) Preparation of gel microspheres:

[0027] The bacterial suspension is mixed with the sodium alginate solution, and then a CaCl2 solution is dripped into the microsphere generator, and the mixture is allowed to stand at room temperature for 10 to 30 minutes to obtain solidified microspheres.

[0028] (2) Wash the solidified microspheres 2 to 4 times with sterile saline to obtain a biological-carrier composite system, which is stored at 4°C for future use;

[0029] 5. Engineering Formation of Biological Pharmaceuticals:

[0030] ①, mixing the bio-carrier composite system with an adhesive, and then adding a protective agent to obtain a mixture;

[0031] ②, extrusion granulation: using a twin-screw extruder granulator to granulate the mixture obtained in step 5① to obtain wet mixture granules;

[0032] ③. Spread the wet mixture particles on a fluidized bed dryer and dry them until the water content is less than 10%, then vacuum-package them with an aluminum-plastic composite film, and store them at 4°C away from light to obtain a biomineralization biopharmaceutical for RO membrane cleaning.

[0033] Principle of the present invention:

[0034] The present invention is an environmentally friendly cleaning agent that uses the principle of biomineralization to remove the composite pollution (biofilm + inorganic scale) on the surface of reverse osmosis (RO) membranes. The sulfate-reducing bacteria DB-3 produces sulfide precipitation, and the cyanobacteria Synechocystis PCC6803 and the facultative anaerobic bacteria Klebsiella ATCC13883 produce extracellular carbonic anhydrase to convert CO3 2 -Converted into precipitation to realize the biofilm cleaning function; this agent is specifically suitable for the efficient regeneration of RO membranes in scenarios such as seawater desalination and industrial wastewater treatment, and is a new type of biological agent that can extend the service life of RO membranes, effectively prevent scale and corrosion, and stabilize water quality.

[0035] Beneficial effects of the present invention:

[0036] 1. The present invention prepares a new type of biological agent prepared by the principle of biomineralization. The agent has no chemical scale inhibitor residue, reduces pollution, and has a carrier slow-release bacterial agent with a long continuous action time;

[0037] 2. The biomineralization technology used in the present invention is a new technology that regulates mineral precipitation through the extracellular carbonic anhydrase produced by microorganisms, and has significant effects in inorganic scale layer regulation and flux recovery rate;

[0038] 3. The pH value of the biofilm cleaning agent used in the present invention is neutral (7.0-7.5), which prevents strong acid or strong alkali from hydrolyzing and damaging the reverse osmosis membrane, while traditional cleaning agents (pH 2-12) easily cause membrane rupture;

[0039] 4. The immobilized microbial carrier can slowly release the bacterial agent on the membrane surface and continuously secrete extracellular polymers (EPS) to inhibit Ca 2+ / Redeposition of organic matter. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0041] Specific implementation method 1: This implementation method is a method for preparing a biomineralization biopharmaceutical for RO membrane cleaning, which is specifically completed in the following steps:

[0042] 1. Primary seed liquid expansion culture:

[0043] ①. Activation of bacterial strains: inoculate the three frozen functional strains on three LB solid culture media respectively, culture them at a constant temperature for a period of time, and pick out single colonies for subsequent amplification culture;

[0044] ② Liquid pre-culture: Select 3 typical bacterial colonies and inoculate them into a certain amount of liquid culture medium, and then place them in a constant temperature shaker for a period of time to obtain 3 primary seed solutions;

[0045] 2. Secondary fermentation tank scale-up culture:

[0046] ①. Transfer the three primary seed solutions to a fully automatic fermenter filled with liquid culture medium according to a certain inoculation amount, adopt a batch feeding culture strategy and control the fermentation conditions;

[0047] ②. Nutrient feeding control:

[0048] Carbon source supplementation: When the residual sugar concentration drops to a certain value, glucose solution is added at a certain rate;

[0049] Nitrogen source regulation: urea concentration is maintained at an appropriate concentration;

[0050] End point determination: When OD600 reaches a certain value, the fermentation is terminated and the fermentation liquid is obtained;

[0051] 3. Bacteria separation and pretreatment:

[0052] ①, Centrifugal harvest: Use a continuous flow centrifuge to process the fermentation liquid and collect the wet bacterial sludge;

[0053] ②. Resuspend the bacterial sludge: Use pre-cooled phosphate buffer to resuspend the bacterial sludge to the final concentration to obtain a bacterial suspension for later use;

[0054] 4. Construction of microorganism-carrier composite system:

[0055] The microorganism-carrier composite system is constructed by using method one or method two;

[0056] The construction of the microorganism-carrier composite system using method 1 is specifically completed in the following steps:

[0057] ①. Carrier pretreatment: modifying diatomaceous earth to obtain modified diatomaceous earth; sieving and grading the modified diatomaceous earth, selecting a certain particle size fraction, and obtaining a carrier;

[0058] ②, the bacterial suspension was mixed with the carrier, and the mixture was adsorbed under constant temperature oscillation for a period of time. During this period, the OD600 value of the supernatant was measured every 30 minutes. When the OD600 value changed by less than 5%, the oscillation adsorption was stopped to obtain the biological-carrier composite system;

[0059] The construction of the microorganism-carrier composite system using method 2 is specifically completed in the following steps:

[0060] (1) Preparation of gel microspheres:

[0061] The bacterial suspension is mixed with the sodium alginate solution, and then a CaCl2 solution is dripped into the microsphere generator, and the mixture is allowed to stand at room temperature for 10 to 30 minutes to obtain solidified microspheres.

[0062] (2) Wash the solidified microspheres 2 to 4 times with sterile saline to obtain a biological-carrier composite system, which is stored at 4°C for future use;

[0063] 5. Engineering Formation of Biological Pharmaceuticals:

[0064] ①, mixing the bio-carrier composite system with an adhesive, and then adding a protective agent to obtain a mixture;

[0065] ②, extrusion granulation: using a twin-screw extruder granulator to granulate the mixture obtained in step 5① to obtain wet mixture granules;

[0066] ③. Spread the wet mixture particles on a fluidized bed dryer and dry them until the water content is less than 10%, then vacuum-package them with an aluminum-plastic composite film, and store them at 4°C away from light to obtain a biomineralization biopharmaceutical for RO membrane cleaning.

[0067] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is that the functional strains described in step 1① are sulfate-reducing bacteria DB-3 (purchased from China General Microbiological Culture Collection Center), cyanobacteria Synechocystis PCC6803 (purchased from China General Microbiological Culture Collection Center), and facultative anaerobic bacteria Klebsiella ATCC13883 (purchased from Shanghai Luwei Technology Co., Ltd.); the temperature of the constant temperature culture described in step 1① is 28℃~30℃, and the constant temperature culture time is 24h~28h; the formula of the LB solid culture medium described in step 1① is: tryptone 10g / L~16g / L, yeast extract 5g / L~11g / L, NaCl 10g / L~19g / L, agar 10g / L~15g / L. The other steps are the same as the specific embodiment 1.

[0068] In this embodiment, the sulfate-reducing bacteria DB-3, cyanobacterium Synechocystis PCC6803, and facultative anaerobic bacterium Klebsiella ATCC13883 described in step 1① are all existing bacteria. The sulfate-reducing bacteria DB-3 was purchased from the China General Microbiological Culture Collection Administration Center, the cyanobacterium Synechocystis PCC6803 was purchased from the China General Microbiological Culture Collection Administration Center, and the facultative anaerobic bacterium Klebsiella ATCC13883 was purchased from Shanghai Luwei Technology Co., Ltd.

[0069] Specific implementation method three: The difference between this implementation method and specific implementation method one or two is that: the formula of the liquid culture medium described in step one ② is: glucose 10g / L~16g / L, urea 20g / L~26g / L, peptone 5g / L~11g / L, CaCl2·2H2O 1g / L~7g / L, and the solvent is a phosphate buffer solution with a pH of 7.5; the temperature of the constant temperature shaker described in step one ② is 28℃~30℃, and the speed is 120rpm~200rpm; the time of culturing in the constant temperature shaker in step one ② is 24h; the OD600 of the primary seed solution described in step one ② is 0.8~1.2. The other steps are the same as those in specific implementation method one or two.

[0070] Specific embodiment 4: The difference between this embodiment and specific embodiments 1 to 3 is that: the inoculation amount of the three primary seed solutions described in step 2① is 10% (v / v); the formula of the liquid culture medium described in step 2① is glucose 10g / L, sodium lactate 3.5g / L, sodium sulfate 4.5g / L, ammonium chloride 1.0g / L, potassium dihydrogen phosphate 0.5g / L, sodium chloride 1.0g / L, yeast extract 1.0g / L, ascorbic acid 0.1g / L, ferrous sulfate heptahydrate 0.05g / L, trace element solution 10mL / L; CaCl2 0.01g / L, MgSO4 0.2g / L, MnSO4·4H2O in the trace element solution 0.05 g / L, ZnSO4·7H2O0.2 g / L; the fermentation conditions described in step 2① are: temperature 28°C to 30°C, pH value 7.5 to 7.7, dissolved oxygen concentration 30% to 35%. The other steps are the same as those in the first to third embodiments.

[0071] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that: in step 2②, when the residual sugar concentration drops to 2g / L, 50% glucose solution is added at a rate of 0.5g / (L·h) until the residual sugar concentration is 4g / L; in step 2②, the urea concentration is maintained at 15mmol / L to 19mmol / L; in step 2②, the fermentation is terminated when OD600 reaches 2.0. The other steps are the same as those of specific embodiments 1 to 4.

[0072] Specific embodiment 6: The difference between this embodiment and specific embodiments 1 to 5 is that: the speed of the continuous flow centrifuge described in step 2① is 4000rpm, and the centrifugation is carried out at 4°C; the temperature of the precooled phosphate buffer described in step 2② is 0-4°C, the concentration is 10mmol / L, and the pH value is 7.4; the concentration of the bacterial suspension described in step 2② is 1×10 9 CFU / mL; the bacterial suspension described in step 2② is stored at 4°C for ≤12h. The other steps are the same as those in specific embodiments 1 to 5.

[0073] Specific embodiment 7: The difference between this embodiment and specific embodiments 1 to 6 is that: the preparation method of the modified diatomite described in step 4 ① is: immerse the diatomite in sulfuric acid with a concentration of 0.5 mol / L for 8 to 12 hours, then centrifuge and dry to obtain the modified diatomite; the particle size of the carrier described in step 4 ① is 50 μm to 100 μm; the mass ratio of the bacterial suspension to the carrier described in step 4 ② is 1:3; the temperature of the constant temperature oscillation adsorption in step 4 ② is 25°C to 30°C, the time is 3h to 5h, and the speed is 200rpm. The other steps are the same as those of specific embodiments 1 to 6.

[0074] Specific implementation eight: This implementation differs from specific implementations one to seven in that: the mass fraction of the sodium alginate solution described in step (1) is 3%; the mass fraction of the CaCl2 solution described in step (1) is 2%; the volume ratio of the bacterial suspension described in step (1) to the sodium alginate solution is 1:4; the volume ratio of the bacterial suspension described in step (1) to the CaCl2 solution is 1:(5-10); the needle diameter of the microsphere generator described in step (1) is 0.4 mm; the size of the microspheres after solidification in step (1) is 0.5-3 mm, the wall thickness is 50-300 μm, and the porosity is 50%-90%. The other steps are the same as specific implementations one to seven.

[0075] Specific embodiment 9: This embodiment differs from specific embodiments 1 to 8 in that: the binder in step 5① is sodium carboxymethyl cellulose; the protective agent in step 5① is a mixture of trehalose and glycerol, wherein the mass ratio of trehalose to glycerol is 3:1; the mass ratio of the biological-carrier composite system to the binder in step 5① is 99:1; the mass fraction of the protective agent in the mixture in step 5① is 5%. The other steps are the same as those in specific embodiments 1 to 8.

[0076] Specific embodiment 10: The difference between this embodiment and specific embodiments 1 to 9 is that: the rotation speed of the twin-screw extruder granulator described in step 5 ② is 40rpm, and the die head aperture is 2.5mm; the particle size distribution D50 of the mixture particles described in step 5 ② is 2.6mm; the air inlet temperature of the fluidized bed dryer described in step 5 ③ is 40℃, and the wind speed is 1.2m / s; the viable bacterial count retention rate of the biomineralization biopharmaceutical used for RO membrane cleaning described in step 5 ③ is greater than 85%. The other steps are the same as specific embodiments 1 to 9.

[0077] The following examples are used to verify the beneficial effects of the present invention:

[0078] Example 1: A method for preparing a biomineralization biopharmaceutical for RO membrane cleaning is specifically completed according to the following steps:

[0079] 1. Primary seed liquid expansion culture:

[0080] ①. Activation of bacterial strains: inoculate the three frozen functional strains on three LB solid culture media respectively, culture them at a constant temperature for a period of time, and pick out single colonies for subsequent amplification culture;

[0081] The functional strains described in step 1① are sulfate-reducing bacteria DB-3, cyanobacteria Synechocystis PCC6803, and facultative anaerobic bacteria Klebsiella ATCC13883;

[0082] The sulfate-reducing bacteria DB-3, cyanobacterium Synechocystis PCC6803, and facultative anaerobic bacteria Klebsiella ATCC13883 described in step 1① are all existing bacteria. The sulfate-reducing bacteria DB-3 was purchased from the China General Microbiological Culture Collection Administration Center, the cyanobacterium Synechocystis PCC6803 was purchased from the China General Microbiological Culture Collection Administration Center, and the facultative anaerobic bacteria Klebsiella ATCC13883 was purchased from Shanghai Luwei Technology Co., Ltd.;

[0083] The constant temperature culture temperature in step 1① is 28°C, and the constant temperature culture time is 24h;

[0084] The formula of the LB solid medium described in step 1① is: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L;

[0085] ② Liquid pre-culture: Select 3 typical bacterial colonies and inoculate them into a certain amount of liquid culture medium, and then place them in a constant temperature shaker for a period of time to obtain 3 primary seed solutions;

[0086] The formula of the liquid culture medium described in step 1② is: glucose 10g / L, urea 20g / L, peptone 5g / L, CaCl2·2H2O 1g / L, and the solvent is phosphate buffer with a pH of 7.5;

[0087] The temperature of the constant temperature shaker described in step 1② is 28°C and the rotation speed is 120 rpm;

[0088] In step 1②, the culture time in a constant temperature shaker is 24 hours;

[0089] The OD600 of the primary seed solution described in step 1② is 0.8;

[0090] 2. Secondary fermentation tank scale-up culture:

[0091] ①. According to a certain inoculation volume, the three primary seed solutions were transferred to a fully automatic fermentation tank filled with liquid culture medium (initial liquid volume 10L). A batch feeding strategy was adopted and the fermentation conditions were controlled.

[0092] The inoculation amount of the three primary seed solutions described in step 2① is 10% (v / v);

[0093] The formula of the liquid culture medium described in step 2① is glucose 10g / L, sodium lactate 3.5g / L, sodium sulfate 4.5g / L, ammonium chloride 1.0g / L, potassium dihydrogen phosphate 0.5g / L, sodium chloride 1.0g / L, yeast extract 1.0g / L, ascorbic acid 0.1g / L, ferrous sulfate heptahydrate 0.05g / L, trace element solution 10mL / L; the trace element solution contains CaCl2 0.01g / L, MgSO4 0.2g / L, MnSO4·4H2O 0.05g / L, and ZnSO4·7H2O 0.2g / L;

[0094] The fermentation conditions described in step 2① are: temperature 28°C, pH 7.5, dissolved oxygen concentration (DO) 30%;

[0095] ②. Nutrient feeding control:

[0096] Carbon source supplementation: When the residual sugar concentration drops to a certain value, glucose solution is added at a certain rate;

[0097] Nitrogen source regulation: urea concentration is maintained at an appropriate concentration:

[0098] End point determination: When OD600 reaches a certain value, the fermentation is terminated and the fermentation liquid is obtained;

[0099] In step 2②, when the residual sugar concentration drops to 2 g / L, 50% glucose solution is added at a rate of 0.5 g / (L·h) until the residual sugar concentration reaches 4 g / L;

[0100] In step 2②, the urea concentration is maintained at 15mmol / L;

[0101] In step 2②, the fermentation was terminated when OD600 reached 2.0 (corresponding to a bacterial dry weight of about 8.5 g / L);

[0102] 3. Bacteria separation and pretreatment:

[0103] ①, Centrifugal harvest: Use a continuous flow centrifuge to process the fermentation liquid and collect the wet bacterial sludge;

[0104] The continuous flow centrifuge described in step 2① rotates at 4000 rpm and centrifuges at 4°C;

[0105] ②. Resuspend the bacterial sludge: Use pre-cooled phosphate buffer to resuspend the bacterial sludge to the final concentration to obtain a bacterial suspension for later use;

[0106] The temperature of the precooled phosphate buffer described in step 2② is 4°C, the concentration is 10 mmol / L, and the pH value is 7.4;

[0107] The concentration of the bacterial suspension described in step 2② is 1×10 9 CFU / mL;

[0108] The storage time of the bacterial suspension described in step 2② at 4°C is ≤12h;

[0109] 4. Construction of microorganism-carrier composite system:

[0110] ①. Carrier pretreatment: modifying diatomaceous earth to obtain modified diatomaceous earth; sieving and grading the modified diatomaceous earth, selecting a certain particle size fraction, and obtaining a carrier;

[0111] The preparation method of the modified diatomite described in step 4① is: immerse the diatomite in 0.5 mol / L sulfuric acid for 8 h (the specific surface area is increased to 215 m 2 / g), centrifuged and dried to obtain modified diatomaceous earth;

[0112] The particle size of the carrier described in step 4① is 50 μm to 100 μm;

[0113] ②, the bacterial suspension was mixed with the carrier, and the mixture was adsorbed under constant temperature oscillation for a period of time. During this period, the OD600 value of the supernatant was measured every 30 minutes. When the OD600 value changed by less than 5%, the oscillation adsorption was stopped to obtain the biological-carrier composite system;

[0114] The mass ratio of the bacterial suspension described in step 4② to the carrier is 1:3;

[0115] In step 4②, the temperature of constant temperature oscillation adsorption is 25°C, the time is 4h, and the speed is 200rpm;

[0116] 5. Engineering Formation of Biological Pharmaceuticals:

[0117] ①, mixing the bio-carrier composite system with an adhesive, and then adding a protective agent to obtain a mixture;

[0118] The adhesive described in step 5① is sodium carboxymethyl cellulose;

[0119] The protective agent described in step 5① is a mixture of trehalose and glycerol, wherein the mass ratio of trehalose to glycerol is 3:1;

[0120] The mass ratio of the biological-carrier composite system to the adhesive described in step 5① is 99:1;

[0121] The mass fraction of the protective agent in the mixture described in step 5① is 5%;

[0122] ②, extrusion granulation: using a twin-screw extruder granulator to granulate the mixture obtained in step 5① to obtain wet mixture granules;

[0123] The speed of the twin-screw extruder granulator described in step 5② is 40 rpm, and the die head aperture is 2.5 mm;

[0124] The particle size distribution of the mixture particles described in step 5② is D50=2.6 mm;

[0125] Mechanical strength test: Particle compressive strength> 20N / cm 2 (Comply with GB / T 15445.2-2017 standard);

[0126] ③, spreading the wet mixture particles on a fluidized bed dryer and drying them until the moisture content is less than 10% (determined by weight loss method, refer to GB / T 6435-2014), and then vacuum packaging them with an aluminum-plastic composite film and storing them at 4°C in the dark to obtain a biomineralization biopharmaceutical for RO membrane cleaning;

[0127] The inlet air temperature of the fluidized bed dryer described in step 5③ is 40°C, the wind speed is 1.2m / s, and the moisture content of the material is monitored in real time (near infrared online detection);

[0128] The viable bacterial count retention rate of the biomineralization biopharmaceutical used for RO membrane cleaning described in step 5③ is >85% (plate count method vs. qPCR absolute quantification).

[0129] The biomineralization biopharmaceutical obtained in Example 1 for RO membrane cleaning is vacuum packaged with aluminum-plastic composite film (oxygen permeability < 0.5 cm 3 / (m 2 ·24h·0.1MPa));

[0130] Accelerated storage test (40°C, RH75%): after 6 months, the viable bacterial count decay rate is less than 0.5 log CFU / g (the shelf life calculated by the Arrhenius model at room temperature is greater than 24 months);

[0131] Cold chain storage and transportation requirements: Under 4℃ dark conditions, the half-life of the bacterial agent is t1 / 2 = 210 days (first-order kinetic model fitting R 2 >0.99).

[0132] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 1 has an effect on Ca 2+ (90%) and Mg 2+ (50%) rapid precipitation, and due to the rapid precipitation of metal cations in the solution, the conductivity of the solution is also reduced. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 75%, 78%, 66% and 56%, respectively.

[0133] Example 2: The difference between this example and Example 1 is that the fermentation conditions described in step 2① are: temperature 28.5°C, pH 7.6, dissolved oxygen concentration (DO) 35%. Other steps and parameters are the same as those in Example 1.

[0134] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 2 has an effect on Ca 2+ (95%) and Mg 2+ (60%) rapid precipitation, and due to the rapid precipitation of metal cations in the solution, the conductivity of the solution is also reduced. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 80%, 75%, 68% and 60% respectively.

[0135] Example 3: The difference between this example and Example 1 is that the formula of the LB solid medium described in step 1① is: tryptone 12g / L, yeast extract 7g / L, NaCl 12g / L, agar 15g / L;

[0136] The formula of the liquid culture medium described in step 1② is: glucose 12g / L, urea 22g / L, peptone 7g / L, CaCl2·2H2O 3g / L, and the solvent is a phosphate buffer with a pH of 7.5; the OD600 of the primary seed solution described in step 1② is 1.2. The other steps and parameters are the same as those in Example 1.

[0137] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 3 has an effect on Ca 2 +(80%) and Mg 2+ (40%) rapid precipitation, and due to the rapid precipitation of metal cations in the solution, the conductivity of the solution is also reduced. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 70%, 72%, 54% and 50%, respectively.

[0138] Embodiment 4: The difference between this embodiment and embodiment 1 is:

[0139] The formula of the LB solid medium described in step 1① is: tryptone 14g / L, yeast extract 9g / L, NaCl 17g / L, agar 10g / L;

[0140] The formula of the liquid culture medium described in step 1② is: glucose 14g / L, urea 24g / L, peptone 9g / L, CaCl2·2H2O 5g / L, and the solvent is phosphate buffer with a pH of 7.5;

[0141] The OD600 of the primary seed solution described in step 1② is 1.2.

[0142] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 4 has an effect on the Ca 2+ (90%) and Mg 2+ (40%) rapid precipitation, and due to the rapid precipitation of metal cations in the solution, the conductivity of the solution is also reduced. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 60%, 52%, 64% and 60%, respectively.

[0143] Embodiment 5: The difference between this embodiment and embodiment 1 is:

[0144] The formula of the LB solid medium described in step 1① is: tryptone 16g / L, yeast extract 11g / L, NaCl 19g / L, agar 10g / L;

[0145] The formula of the liquid culture medium described in step 1② is: glucose 16g / L, urea 26g / L, peptone 11g / L, CaCl2·2H2O 7g / L, and the solvent is phosphate buffer with a pH of 7.5;

[0146] The OD600 of the primary seed solution described in step 1② is 1.2.

[0147] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 5 has an effect on Ca in water. 2+ (90%) and Mg 2+ (40%) rapid precipitation, and due to the rapid precipitation of metal cations in the solution, the conductivity of the solution was also reduced. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 50%, 56%, 54% and 62%, respectively.

[0148] Example 6: The difference between this example and Example 1 is that the urea concentration in step 2② is maintained at 16 mmol / L. The other steps and parameters are the same as those in Example 1.

[0149] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 6 has an effect on the Ca 2+ (80%) and Mg 2+ (42%) rapid precipitation, and due to the rapid precipitation of metal cations in the solution, the conductivity of the solution was also reduced. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 70%, 75%, 60% and 54% respectively.

[0150] Example 7: The difference between this example and Example 1 is that the urea concentration in step 2② is maintained at 17 mmol / L. The other steps and parameters are the same as those in Example 1.

[0151] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 7 has an effect on the Ca content in water. 2 + (85%) and Mg (47%) were rapidly precipitated, and the conductivity of the solution was also reduced due to the rapid precipitation of metal cations in the solution. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 68%, 66%, 65% and 55%, respectively.

[0152] Example 8: The difference between this example and Example 1 is that the urea concentration in step 2② is maintained at 18 mmol / L. The other steps and parameters are the same as those in Example 1.

[0153] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 8 has an effect on the Ca 2 +(85%) and Mg 2+ (44%) rapid precipitation, and due to the rapid precipitation of metal cations in the solution, the conductivity of the solution was also reduced. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 70%, 74%, 67% and 54%, respectively.

[0154] Example 9: The difference between this example and Example 1 is that the urea concentration in step 2② is maintained at 19 mmol / L. The other steps and parameters are the same as those in Example 1.

[0155] Implementation effect: The biomineralization biopharmaceutical for RO membrane cleaning obtained in Example 9 has an effect on the Ca 2 +(88%) and Mg 2+ (46%) rapid precipitation, and due to the rapid precipitation of metal cations in the solution, the conductivity of the solution was also reduced. In addition, the 7-day removal rates of aerobic organic pollutants (in terms of COD), total phosphorus (TP), total nitrogen (TN) and ammonia nitrogen (NH3-N) were 64%, 68%, 70% and 50%, respectively.

Claims

1. A method for preparing a biomineralization biopharmaceutical for RO membrane cleaning, characterized in that The preparation method is specifically completed according to the following steps:

1. Primary seed liquid expansion culture: ①. Activation of strains: Inoculate the three frozen functional strains on three LB solid culture media respectively, culture them at a constant temperature for a period of time, and pick out single colonies for subsequent amplification culture; ② Liquid pre-culture: Select 3 typical bacterial colonies and inoculate them into a certain amount of liquid culture medium, and then place them in a constant temperature shaker for a period of time to obtain 3 primary seed solutions; 2. Secondary fermentation tank scale-up culture: ①. Transfer the three primary seed solutions to a fully automatic fermenter filled with liquid culture medium according to a certain inoculation amount, adopt a batch feeding culture strategy and control the fermentation conditions; ②. Nutrient feeding control: Carbon source supplementation: When the residual sugar concentration drops to a certain value, glucose solution is added at a certain rate; Nitrogen source regulation: urea concentration is maintained at an appropriate concentration; End point determination: When OD600 reaches a certain value, the fermentation is terminated and the fermentation liquid is obtained; 3. Bacteria separation and pretreatment: ①, Centrifugal harvest: Use a continuous flow centrifuge to process the fermentation liquid and collect the wet bacterial sludge; ②. Resuspend the bacterial sludge: Use pre-cooled phosphate buffer to resuspend the bacterial sludge to the final concentration to obtain a bacterial suspension for later use; 4. Construction of microorganism-carrier composite system: The microorganism-carrier composite system is constructed by using method one or method two; The construction of the microorganism-carrier composite system using method 1 is specifically completed in the following steps: ①. Carrier pretreatment: modifying diatomaceous earth to obtain modified diatomaceous earth; sieving and grading the modified diatomaceous earth, selecting a certain particle size fraction, and obtaining a carrier; ②, the bacterial suspension was mixed with the carrier, and the mixture was adsorbed under constant temperature oscillation for a period of time. During this period, the OD600 value of the supernatant was measured every 30 minutes. When the OD600 value changed by less than 5%, the oscillation adsorption was stopped to obtain the biological-carrier composite system; The construction of the microorganism-carrier composite system using method 2 is specifically completed in the following steps: (1) Preparation of gel microspheres: The bacterial suspension is mixed with the sodium alginate solution, and then a CaCl2 solution is dripped into the microsphere generator, and the mixture is allowed to stand at room temperature for 10 to 30 minutes to obtain solidified microspheres. (2) Wash the solidified microspheres 2 to 4 times with sterile saline to obtain a biological-carrier composite system, which is stored at 4°C for future use; 5. Engineering Formation of Biological Pharmaceuticals: ①, mixing the bio-carrier composite system with an adhesive, and then adding a protective agent to obtain a mixture; ②, extrusion granulation: using a twin-screw extruder granulator to granulate the mixture obtained in step 5① to obtain wet mixture granules; ③. Spread the wet mixture particles on a fluidized bed dryer and dry them until the water content is less than 10%, then vacuum-package them with an aluminum-plastic composite film, and store them at 4°C away from light to obtain a biomineralization biopharmaceutical for RO membrane cleaning.

2. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that The functional strains described in step 1① are sulfate-reducing bacteria DB-3, cyanobacteria Synechocystis PCC6803, and facultative anaerobic bacteria Klebsiella ATCC13883; the temperature of the constant temperature culture described in step 1① is 28°C to 30°C, and the constant temperature culture time is 24h to 28h; the formula of the LB solid culture medium described in step 1① is: tryptone 10g / L to 16g / L, yeast extract 5g / L to 11g / L, NaCl 10g / L to 19g / L, and agar 10g / L to 15g / L.

3. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that The formula of the liquid culture medium described in step 1② is: glucose 10g / L~16g / L, urea 20g / L~26g / L, peptone 5g / L~11g / L, CaCl2·2H2O 1g / L~7g / L, and the solvent is a phosphate buffer with a pH of 7.5; the temperature of the constant temperature shaker described in step 1② is 28°C~30°C, and the rotation speed is 120rpm~200rpm; the time of culturing in the constant temperature shaker in step 1② is 24h; the OD600 of the primary seed solution described in step 1② is 0.8~1.

2.

4. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that The inoculation amount of the three primary seed solutions described in step 2① is 10% (v / v); the formula of the liquid culture medium described in step 2① is 10g / L glucose, 3.5g / L sodium lactate, 4.5g / L sodium sulfate, 1.0g / L ammonium chloride, 0.5g / L potassium dihydrogen phosphate, 1.0g / L sodium chloride, 1.0g / L yeast extract, 0.1g / L ascorbic acid, 0.05g / L ferrous sulfate heptahydrate, and 10mL / L trace element solution; the trace element solution contains 0.01g / L CaCl2, 0.2g / L MgSO4, 0.05g / L MnSO4·4H2O, and 0.2g / L ZnSO4·7H2O; the fermentation conditions described in step 2① are: temperature 28°C to 30°C, pH 7.5 to 7.7, and dissolved oxygen concentration 30% to 35%.

5. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that In step 2②, when the residual sugar concentration drops to 2 g / L, 50% glucose solution is added at a rate of 0.5 g / (L·h) until the residual sugar concentration is 4 g / L; in step 2②, the urea concentration is maintained at 15 mmol / L to 19 mmol / L; in step 2②, the fermentation is terminated when OD600 reaches 2.

0.

6. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that The speed of the continuous flow centrifuge described in step 2① is 4000rpm, and the centrifugation is carried out at 4°C; the temperature of the precooled phosphate buffer described in step 2② is 0-4°C, the concentration is 10mmol / L, and the pH value is 7.4; the concentration of the bacterial suspension described in step 2② is 1×10 9 CFU / mL; the storage time of the bacterial suspension described in step 2② at 4°C is ≤12h.

7. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that The preparation method of the modified diatomaceous earth described in step 4 ① is: immersing the diatomaceous earth in sulfuric acid with a concentration of 0.5 mol / L for 8 to 12 hours, then centrifuging and drying to obtain the modified diatomaceous earth; the particle size of the carrier described in step 4 ① is 50 μm to 100 μm; the mass ratio of the bacterial suspension to the carrier described in step 4 ② is 1:3; the temperature of the constant temperature oscillation adsorption in step 4 ② is 25°C to 30°C, the time is 3h to 5h, and the rotation speed is 200rpm.

8. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that The mass fraction of the sodium alginate solution described in step (1) is 3%; the mass fraction of the CaCl2 solution described in step (1) is 2%; the volume ratio of the bacterial suspension described in step (1) to the sodium alginate solution is 1:4; the volume ratio of the bacterial suspension described in step (1) to the CaCl2 solution is 1:(5-10); the needle diameter of the microsphere generator described in step (1) is 0.4 mm; the size of the microspheres after solidification in step (1) is 0.5-3 mm, the wall thickness is 50-300 μm, and the porosity is 50%-90%.

9. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that The adhesive described in step 5① is sodium carboxymethyl cellulose; the protective agent described in step 5① is a mixture of trehalose and glycerol, wherein the mass ratio of trehalose to glycerol is 3:1; the mass ratio of the biological-carrier composite system described in step 5① to the adhesive is 99:1; the mass fraction of the protective agent in the mixture described in step 5① is 5%.

10. The method for preparing a biomineralization biopharmaceutical for RO membrane cleaning according to claim 1, characterized in that The rotation speed of the twin-screw extruder granulator described in step 5② is 40rpm, and the die head aperture is 2.5mm; the particle size distribution D50 of the mixture particles described in step 5② is 2.6mm; the inlet air temperature of the fluidized bed dryer described in step 5③ is 40℃, and the wind speed is 1.2m / s; the viable bacteria retention rate of the biomineralization biopharmaceutical used for RO membrane cleaning described in step 5③ is >85%.