CN115286118A - Method for treating sewage by using low-temperature-resistant nitrogen and phosphorus removal composite microbial agent - Google Patents

Method for treating sewage by using low-temperature-resistant nitrogen and phosphorus removal composite microbial agent Download PDF

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CN115286118A
CN115286118A CN202210139963.4A CN202210139963A CN115286118A CN 115286118 A CN115286118 A CN 115286118A CN 202210139963 A CN202210139963 A CN 202210139963A CN 115286118 A CN115286118 A CN 115286118A
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CN115286118B (en
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董怡华
王凌潇
陈锋
李亮
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Shenyang University
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/30—Aerobic and anaerobic processes
    • C02F3/302—Nitrification and denitrification treatment
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/30—Aerobic and anaerobic processes
    • C02F3/308—Biological phosphorus removal
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F7/00—Aeration of stretches of water
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/105—Phosphorus compounds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/16—Nitrogen compounds, e.g. ammonia
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/38—Organic compounds containing nitrogen
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

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Abstract

The invention discloses a method for treating sewage by using a low-temperature-resistant nitrogen and phosphorus removal compound microbial agent, and relates to a method for treating sewage by using a microbial agent. The nitrobacteria and the denitrifying bacteria in the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent are matched with each other to remove total nitrogen in the sewage, and the phosphorus accumulating bacteria remove total phosphorus in the sewage, so that the purposes of synergy and synchronous nitrogen and phosphorus removal can be achieved, and the effect of removing nitrogen and phosphorus in the sewage in a low-temperature environment is obviously higher than that of a single bacterial strain. Each strain in the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent is separated from winter sludge and river bottom sludge in northern cold regions, and is an environment-friendly microorganism. The fertilizer is directly put in when in use, does not need a complex engineering process, is convenient to operate, has no secondary pollution, and has good application prospect.

Description

Method for treating sewage by using low-temperature-resistant nitrogen and phosphorus removal composite microbial agent
Technical Field
The invention relates to a method for treating sewage, in particular to a method for treating sewage by using a low-temperature-resistant nitrogen and phosphorus removal compound microbial agent.
Background
With the rapid development of social economy and the continuous improvement of living standard, the continuous discharge of a large amount of urban sewage containing nitrogen and phosphorus pollutants causes excessive water body pollution load, causes eutrophication and black and odorous phenomena of water bodies of rivers, lakes and the like, and further causes serious threats to the ecological environment and human health. Therefore, the research for finding safe, efficient, simple and feasible sewage nitrogen and phosphorus removal technology becomes a hotspot.
At present, the domestic and foreign technologies for removing nitrogen and phosphorus from sewage are mainly classified into physical methods (adsorption and electrodialysis), chemical methods (flocculation precipitation and ion exchange), biological methods and the like. Among them, biological methods usually employ microbial agents for nitrogen and phosphorus removal. The microbial nitrogen and phosphorus removal technology has the advantages of simple operation, low operation cost, good treatment effect, no secondary pollution and the like, and is a method which is most widely applied in various sewage nitrogen and phosphorus removal technologies and has the greatest development prospect. However, the temperature of the urban sewage in winter in cold areas in northern China can be reduced to 8-15 ℃ or even lower than 5 ℃. Most of microorganisms with nitrogen and phosphorus removal functions belong to mesophilic bacteria, the optimal growth temperature is 20-37 ℃, the treatment effect is good at 20-25 ℃, the biological activity and the metabolic efficiency of the microorganisms are rapidly reduced in a low-temperature environment below 15 ℃, the nitrogen and phosphorus removal effect of the functional bacteria is seriously hindered, and the nitrogen and phosphorus removal effect on the domestic sewage is poor in the low-temperature environment. Therefore, the development of the microbial agent which can grow well under low temperature and can efficiently remove nitrogen and phosphorus from the sewage has very important social and economic benefits.
At present, researchers propose that the low-temperature microbial agent can solve the problems of poor cell membrane fluidity, poor permeability, low enzyme activity and the like under the low-temperature condition, so that the effect of removing nitrogen and phosphorus in sewage under the low-temperature environment in winter is improved. However, most of domestic related researches are still in the beginning stage, most of the adopted single low-temperature strains are difficult to achieve the effect of purifying and treating the urban sewage, the development and construction force of the low-temperature-resistant compound microbial agent is weak, and the successful application cases in water pollution treatment are rarely reported.
Disclosure of Invention
The invention aims to provide a method for treating sewage by using a low-temperature-resistant nitrogen and phosphorus removal composite microbial agent, which aims to solve the problems of low microbial activity and poor nitrogen and phosphorus removal effect of microorganisms on urban sewage under a low-temperature condition in winter.
The purpose of the invention is realized by the following technical scheme:
a method for treating sewage by using a low-temperature-resistant nitrogen and phosphorus removal compound microbial agent comprises the following steps:
firstly, preparing the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent, which comprises the following preparation steps:
(1) Preparing heterotrophic nitrification composite bacterial liquid: inoculating 3-4 rings of cold-resistant pseudomonas putida from the slant solid culture medium by using an inoculating loop, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 24-72 hours to obtain cold-resistant pseudomonas putida seed liquid (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml); inoculating 3-4 rings of cold-resistant pseudomonas putida from the slant solid culture medium by using an inoculating ring, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 24-72 hours to obtain cold-resistant pseudomonas putida seed liquid (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml); the cold-resistant pseudomonas putida seed liquid and the cold-resistant pseudomonas putida seed liquid are mixed according to the volume ratio of inoculation amount (1-10): (1-10) inoculating 5-20% of total inoculum size to a heterotrophic nitrifier culture medium at the same time, and controlling the temperatureCarrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 48-96 hours to obtain heterotrophic nitrification compound bacterial liquid; the total viable count of the heterotrophic nitrification composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Colony count/ml;
(2) Preparing aerobic denitrification composite bacterial liquid: inoculating 3-4 rings of cold-resistant pseudomonas veronii from a slant solid culture medium by using an inoculating loop, inoculating into an aerobic denitrifying bacterium culture medium, and performing oscillation culture in a full-temperature oscillation culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 24-72 hours to obtain cold-resistant pseudomonas veronii seed liquid (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml); selecting 3-4 rings of pseudomonas fluorescens from the slant solid culture medium by using an inoculating loop to be inoculated into an aerobic denitrifying bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 24-72 hours to obtain pseudomonas fluorescens seed liquid (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml); the cold-resistant pseudomonas veronii seed solution and the pseudomonas fluorescens are mixed according to the volume ratio of the inoculation amount (1-10): (1-10), inoculating 5-20% of the total inoculum size to an aerobic denitrifying bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 48-96 hours to obtain an aerobic denitrifying composite bacterial liquid; the total viable count of the heterotrophic nitrification composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Colony count/ml;
(3) Preparing phosphorus-accumulating composite bacterial liquid: inoculating 3-4 rings of Arthrobacter antarctica from the slant solid culture medium by using an inoculating ring, and performing shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotation speed of 120-170 r/min for 23-72 hours to obtain the Arthrobacter antarctica (the number of viable bacteria is 1 × 10) 7 ～2×10 9 Number of colonies/ml). Inoculating the solid slant culture medium with 3-4 rings of psychrophilic halophilus bacteria, and vibration culturing in full-temperature vibration culture box at 6-15 deg.c and 120-170 rpm for 24-72 hr to obtain seed liquid of psychrophilic halophilus with viable count of 1 × 10 7 ～2×10 9 Number of colonies/ml). Mixing Arthrobacter antarctica seed solution withThe psychrobacter halophilus seed liquid is prepared by inoculating the following components in a volume ratio of (1-10): (1-10), inoculating 5-20% of the total inoculum size to a phosphorus-accumulating bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 48-96 hours to obtain phosphorus-accumulating composite bacterial liquid; the total viable count of the phosphorus-accumulating composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Colony count/ml;
(4) The preparation of the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent comprises the following steps: mixing heterotrophic nitrification composite bacterial liquid, aerobic denitrification composite bacterial liquid and phosphorus accumulation composite bacterial liquid according to the volume ratio of (1-10): (1-10): (1-20) to obtain the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent; the total viable count of the composite microbial agent is not less than 1 × 10 10 Colony count/ml;
the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent is applied to low-temperature biological sewage treatment and comprises the following steps:
the additive amount in the urban sewage to be treated is 2 multiplied by 10 2 ～5×10 2 The low temperature resistant nitrogen and phosphorus removal composite microbial agent with colony number per square centimeter is aerated for 24 to 48 hours at the aeration rate of 1.0 to 3.5 cubic meters per hour, and is filtered after standing for 20 to 30 minutes.
The method for treating sewage by using the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent is characterized in that the heterotrophic nitrification compound bacterial liquid is prepared, and cold-resistant pseudomonas pratensis and cold-resistant pseudomonas stutzeri are both sink activated sludge of a sewage treatment plant and are respectively identified as pseudomonas pratensis (pseudomonas (P) pratensis by 16SrDNA sequence analysisPseudomonas poae) And Pseudomonas putida: (A)Pseudomonas peli)。
The method for treating sewage by using the low-temperature-resistant denitrification and dephosphorization composite microbial agent comprises the following steps of preparing a heterotrophic nitrification composite bacterial liquid, wherein a heterotrophic nitrification bacterial culture medium comprises the following components: 1.0 to 5.0 grams/liter of sodium citrate, 0.2 to 1.0 gram/liter of ammonium sulfate, 0.3 to 1.5 grams/liter of monopotassium phosphate, 0.1 to 1.0 gram/liter of magnesium sulfate heptahydrate, 0.1 to 1.0 gram/liter of sodium chloride, 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate, and 0.01 to 0.1 gram/liter of anhydrous calcium chloride. The pH of the heterotrophic nitrifier culture medium is adjusted to 6.5-7.5 by adopting 10% HCl and NaOH.
The method for treating sewage by using the low-temperature-resistant denitrification and dephosphorization composite microbial agent is characterized in that the heterotrophic nitrification composite bacterial liquid is prepared, wherein the cold-resistant pseudomonas veronii and the pseudomonas fluorescens are river water frozen bottom mud, and are respectively identified as pseudomonas veronii (pseudomonas) (pseudomonas) after 16SrDNA sequence analysisPseudomonas veronii) And Pseudomonas fluorescens (Pseudomonas fluorescens)。
The method for treating sewage by using the low-temperature-resistant denitrification and dephosphorization composite microbial agent comprises the following steps of preparing a heterotrophic nitrification composite bacterial liquid and preparing an aerobic denitrification bacterial culture medium: 1.0 to 5.0 grams/liter of sodium citrate, 0.2 to 2.0 grams/liter of sodium nitrate, 0.3 to 1.5 grams/liter of monopotassium phosphate, 0.1 to 1.0 gram/liter of magnesium sulfate heptahydrate, 0.1 to 1.0 gram/liter of sodium chloride, 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate, and 0.01 to 0.1 gram/liter of anhydrous calcium chloride; the pH value of the aerobic denitrifying bacteria culture medium is adjusted to 6.5-7.5 by adopting 10% HCl and NaOH.
The method for treating sewage by using the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent is characterized in that a QHZ-98A type full-temperature oscillation incubator is adopted for culture of the microbial agent.
The method for treating sewage by using the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent is characterized in that an SN210C type vertical pressure steam sterilizer is adopted for high-pressure steam sterilization.
According to the method for treating sewage by using the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent, the culture medium is sterilized by high-pressure steam under the conditions that the temperature is 121 ℃ and the pressure is 0.105 MPa, and the treatment is maintained for 20 to 30 minutes.
The invention has the following significant technical advantages:
1. the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent can normally grow, metabolize and propagate at a lower temperature (6-15 ℃), and can effectively remove nitrogen and phosphorus from sewage.
2. Each strain in the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent is separated from winter sludge and river bottom sludge in northern cold regions, and is an environment-friendly microorganism. The fertilizer is directly put in when in use, does not need complex engineering technology, is convenient to operate, has no secondary pollution, and has good application prospect.
3. The nitrobacteria and the denitrifying bacteria in the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent are matched with each other to remove total nitrogen in the sewage, and the phosphorus accumulating bacteria remove total phosphorus in the sewage, so that the purposes of synergy and synchronous nitrogen and phosphorus removal can be achieved, and the effect of removing nitrogen and phosphorus in the sewage in a low-temperature environment is obviously higher than that of a single bacterial strain.
Drawings
FIG. 1 is an electron micrograph of Pseudomonas praecox according to the present invention;
FIG. 2 is an electron micrograph of Pseudomonas putida of the present invention;
FIG. 3 is an electron micrograph of Pseudomonas veronii according to the present invention;
FIG. 4 is an electron micrograph of Pseudomonas fluorescens of the present invention;
FIG. 5 is an electron micrograph of Arthrobacter antarctica of the present invention;
FIG. 6 is an electron micrograph of the psychrobacter halophilus of the present invention.
Detailed Description
The present invention will be described in detail with reference to the embodiments shown in the drawings.
The invention firstly prepares the low temperature resistant nitrogen and phosphorus removal composite microbial agent, and the total viable count of the composite microbial agent is not less than 1 multiplied by 10 10 The colony number/ml comprises low temperature resistant heterotrophic nitrification composite bacteria, low temperature resistant aerobic denitrification composite bacteria and low temperature resistant phosphorus accumulating composite bacteria. Wherein the low temperature resistant heterotrophic nitrification complex bacteria comprise Pseudomonas praecox (Pseudomonas praecox)Pseudomonas poae) And Pseudomonas putida: (A)Pseudomonas peli) The total viable count of the low temperature resistant heterotrophic nitrification complex bacteria is 1 multiplied by 10 8 ～5×10 9 Colony count/ml; the low-temperature-resistant aerobic denitrification compound bacteria comprise pseudomonas veronii (a. Veroni)Pseudomonas veronii) And Pseudomonas fluorescens (Pseudomonas fluorescens) The total viable count of the low-temperature resistant aerobic denitrification composite bacteria is 1 multiplied by 10 8 ～5×10 9 Colony count/ml; the low temperature resistant phosphorus-accumulating composite bacteria include Arthrobacter antarctica (A)Arthrobacter antarcticus) And Haemophilus halophilus: (Psychrobacter cryohalolentis) The low temperature resistant phosphorus-accumulating compositeThe total viable count of the bacteria is 1 × 10 8 ～5×10 9 Number of colonies/ml.
Experiments prove that the compound microbial agent has a reasonable formula, the strains of the pseudomonas herbaceum, the pseudomonas putrescentiae, the pseudomonas veronii, the pseudomonas fluorescens, the arthrobacter antarctica and the psychrophilum halophilum do not generate antagonistic action, the six strains are compounded to realize synergistic effect, and the compound microbial agent has obvious nitrogen and phosphorus removal effects on municipal sewage under the low-temperature condition and has good application prospect.
Pseudomonas aeruginosa (Pseudomonas poae) Pseudomonas putida (b)Pseudomonas peli) Pseudomonas veronii (A)Pseudomonas veronii) Pseudomonas fluorescens (A)Pseudomonas fluorescens) Arthrobacter antarctica (A), (B), (C)Arthrobacter antarcticus) And Acidovorax halophila: (Psychrobacter cryohalolentis) The active sludge of a secondary sedimentation tank of a sewage treatment plant in Shenyang city of Liaoning province and the frozen bottom sludge of the river water in winter are respectively separated and identified as known strains, and all the large strains are preserved in a preservation warehouse.
The preparation method of the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent comprises the following steps:
(1) Preparing heterotrophic nitrification composite bacterial liquid: inoculating 3-4 rings of cold-resistant pseudomonas putida from the slant solid culture medium by using an inoculating loop, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 24-72 hours to obtain cold-resistant pseudomonas putida seed liquid (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml). Inoculating 3-4 rings of cold-resistant pseudomonas putida from the slant solid culture medium by using an inoculating ring, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 24-72 hours to obtain cold-resistant pseudomonas putida seed liquid (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml). The cold-resistant pseudomonas putida seed liquid and the cold-resistant pseudomonas putida seed liquid are mixed according to the volume ratio of inoculation amount (1-10): (1-10) inoculating 5-20% of total inoculum size to a heterotrophic nitrifier culture medium at the same time, and vibrating at the full temperature of 6-15 ℃ and the rotating speed of 120-170 r/minOscillating and culturing in an incubator for 48-96 hours to obtain the heterotrophic nitrification compound bacterial liquid. The total viable count of the heterotrophic nitrification composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Number of colonies/ml.
Wherein cold-resistant Pseudomonas praecox and cold-resistant Pseudomonas putida are both isolated from activated sludge in secondary sedimentation tank of sewage treatment plant in south Shenyang city, liaoning province in 2021 month, and identified as Pseudomonas praecox by 16SrDNA sequence analysisPseudomonas poae) And Pseudomonas putida: (Pseudomonas peli)。
The heterotrophic nitrifier culture medium comprises the following components: 1.0 to 5.0 grams/liter of sodium citrate, 0.2 to 1.0 gram/liter of ammonium sulfate, 0.3 to 1.5 grams/liter of monopotassium phosphate, 0.1 to 1.0 gram/liter of magnesium sulfate heptahydrate, 0.1 to 1.0 gram/liter of sodium chloride, 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate, and 0.01 to 0.1 gram/liter of anhydrous calcium chloride. The pH of the heterotrophic nitrifier culture medium is adjusted to 6.5-7.5 by adopting 10% HCl and NaOH.
(2) Preparing aerobic denitrification composite bacterial liquid: inoculating 3-4 rings of cold-resistant pseudomonas veronii from a slant solid culture medium by using an inoculating loop, inoculating into an aerobic denitrifying bacterium culture medium, and performing oscillation culture in a full-temperature oscillation culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 24-72 hours to obtain cold-resistant pseudomonas veronii seed liquid (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml). Inoculating 3-4 rings of pseudomonas fluorescens from a slant solid culture medium by using an inoculating ring to an aerobic denitrifying bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 24-72 hours to obtain pseudomonas fluorescens seed liquid (the viable count is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml). The cold-resistant pseudomonas veronii seed solution and the pseudomonas fluorescens are mixed according to the volume ratio of the inoculation amount (1-10): (1-10) inoculating 5-20% of the total inoculum size to an aerobic denitrifying bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 48-96 hours to obtain the aerobic denitrifying composite bacterial liquid. The total viable count of the heterotrophic nitrification composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Number of colonies/ml.
Therein, it is resistant toPseudomonas veitchii and Pseudomonas fluorescens were separated from frozen bottom sediment of North canal river, shenyang, liaoning province in 1 month in 2021, and were identified as Pseudomonas veitchii by 16SrDNA sequence analysisPseudomonas veronii) And Pseudomonas fluorescens (Pseudomonas fluorescens)。
The aerobic denitrifying bacteria culture medium comprises: 1.0 to 5.0 grams/liter of sodium citrate, 0.2 to 2.0 grams/liter of sodium nitrate, 0.3 to 1.5 grams/liter of monopotassium phosphate, 0.1 to 1.0 gram/liter of magnesium sulfate heptahydrate, 0.1 to 1.0 gram/liter of sodium chloride, 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate, and 0.01 to 0.1 gram/liter of anhydrous calcium chloride. The pH value of the aerobic denitrifying bacteria culture medium is adjusted to 6.5-7.5 by adopting 10% HCl and NaOH.
(3) Preparing a phosphorus-accumulating composite bacterial liquid: inoculating 3-4 rings of Arthrobacter antarctica from the slant solid culture medium by using an inoculating ring, and performing shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 23-72 hours to obtain the Arthrobacter antarctica (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml). Inoculating 3-4 rings of psychrophilic halophilus from the slant solid culture medium by using an inoculating loop, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 24-72 hours to obtain a seed liquid of the psychrophilic halophilus (the viable count is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml). The method comprises the following steps of (1) inoculating Arthrobacter antarctica seed liquid and the psychrophilus halophilus seed liquid according to the volume ratio of the inoculum size (1-10): (1-10) inoculating 5-20% of the total inoculum size to a phosphorus-accumulating bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 48-96 hours to obtain the phosphorus-accumulating composite bacterial liquid. The total viable count of the phosphorus-accumulating composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Number of colonies/ml.
Wherein, arthrobacter antarctica and Haemophilus halophilus are both separated from frozen bottom mud of northern canal river of Shenyang city, shenyang, liaoning in 2021 month, and are respectively identified as Arthrobacter antarctica by 16SrDNA sequence analysis(s) ((R))Arthrobacter antarcticus) And Acidovorax halophila: (Psychrobacter cryohalolentis)。
The phosphorus accumulating bacteria culture medium comprises: 1.0 to 5.0 grams of sodium acetate per liter, 0.1 to 2.0 grams of ammonium sulfate per liter, 0.01 to 1.0 gram of dipotassium phosphate per liter, 0.05 to 0.2 gram of magnesium sulfate heptahydrate per liter, 0.01 to 0.1 gram of calcium chloride per liter, and 7.0 to 9.0 grams of 4-hydroxyethyl piperazine ethane sulfonic acid per liter. The pH of the culture medium of the polyphosphate accumulating bacteria is adjusted to 6.5-7.5 by adopting 10 percent HCl and NaOH.
(4) Preparing a low-temperature-resistant nitrogen and phosphorus removal compound microbial agent: mixing heterotrophic nitrification composite bacterial liquid, aerobic denitrification composite bacterial liquid and phosphorus accumulation composite bacterial liquid according to the volume ratio of (1-10): (1-10): (1-20) to obtain the low temperature resistant nitrogen and phosphorus removal composite microbial agent. The total viable count of the composite microbial agent is not less than 1 × 10 10 Number of colonies/ml.
The application process of the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent for sewage treatment comprises the following steps:
the additive amount in the urban sewage to be treated is 2 multiplied by 10 2 ～5×10 2 The low-temperature-resistant nitrogen and phosphorus removal composite microbial agent with the colony number/square centimeter is aerated for 24 to 48 hours at the aeration rate of 1.0 to 3.5 cubic meters/hour, and is filtered after standing for 20 to 30 minutes.
The technical solution of the present invention is further explained by the following embodiments.
Example 1:
(1) Preparing heterotrophic nitrification composite bacterial liquid: inoculating 3 rings of cold-resistant Pseudomonas pratensis from the slant solid culture medium with inoculating loop, and shake culturing in a full-temperature shaking culture box at 6 deg.C and 150 rpm for 48 hr to obtain cold-resistant Pseudomonas pratensis seed solution (viable count of 2 × 10) 7 Number of colonies/ml). Inoculating 3 rings of cold-rot resistant Pseudomonas putida from the slant solid culture medium with inoculating loop, and shake culturing in a full-temperature shake culture box at 6 deg.C and 150 rpm for 48 hr to obtain cold-rot resistant Pseudomonas seed solution (viable count of 3 × 10) 7 Number of colonies/ml). Carrying out inoculation on the cold-resistant pseudomonas palustris seed liquid and the cold-resistant pseudomonas mucida seed liquid according to the volume ratio of 1: 2. inoculating 15% of the total inoculum size to heterotrophic nitrifier culture medium at the same time, and performing full-temperature shaking culture at 6 ℃ and 150 r/minAnd carrying out shake culture in a culture box for 96 hours to obtain the heterotrophic nitrification compound bacterial liquid. The total viable count of the heterotrophic nitrification composite bacterial liquid is 1 multiplied by 10 8 Number of colonies/ml.
Wherein the heterotrophic nitrifier culture medium comprises the following components: 2.5 g/l sodium citrate, 0.5 g/l ammonium sulphate, 0.3 g/l potassium dihydrogen phosphate, 0.1 g/l magnesium sulphate heptahydrate, 0.4 g/l sodium chloride, 0.04 g/l ferrous sulphate heptahydrate, 0.03 g/l anhydrous calcium chloride. The pH of the heterotrophic nitrifier medium was adjusted to 7.0 with 10% HCl and NaOH.
(2) Preparing aerobic denitrification composite bacterial liquid: inoculating 3 rings of cold-resistant Pseudomonas veronii from slant solid culture medium with inoculating ring, and shake culturing in full-temperature shake culture box at 6 deg.C and 150 rpm for 48 hr to obtain cold-resistant Pseudomonas veronii seed solution (viable count of 4 × 10) 8 Number of colonies/ml). Inoculating 3-ring Pseudomonas fluorescens from the slant solid culture medium with an inoculating ring, and performing shake culture in a full-temperature shake culture box at 6 deg.C and 150 rpm for 48 hr to obtain Pseudomonas fluorescens seed solution (viable count is 3 × 10) 7 Number of colonies/ml). Carrying out inoculation on cold-resistant pseudomonas veronii seed solution and pseudomonas fluorescens according to the volume ratio of 2: 1. inoculating 15% of the total inoculum size into an aerobic denitrifying bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at 6 ℃ and 150 r/min for 96 hours to obtain the aerobic denitrifying composite bacterial liquid. The total viable count of the heterotrophic nitrification composite bacterial liquid is 3 multiplied by 10 8 Number of colonies/ml.
The aerobic denitrifying bacteria culture medium comprises: 2.5 g/l sodium citrate, 1.5 g/l sodium nitrate, 0.3 g/l potassium dihydrogen phosphate, 0.1 g/l magnesium sulfate heptahydrate, 0.4 g/l sodium chloride, 0.04 g/l ferrous sulfate heptahydrate, 0.03 g/l anhydrous calcium chloride. The pH of the aerobic denitrifying bacteria culture medium was adjusted to 7.0 using 10% HCl and NaOH.
(3) Preparing a phosphorus-accumulating composite bacterial liquid: selecting 3 rings of Antarctic arthrobacterium from the slant solid culture medium by using an inoculating loop, inoculating into a polyphosphate culture medium, and performing shake culture in a full-temperature shake culture box at 6 ℃ and 150 r/min for 48 hours to obtain Antarctic arthrobacterBacteria (viable count 3X 10) 7 Number of colonies/ml). Inoculating 3 rings of Bacillus halophilus from slant solid culture medium with inoculating loop, and performing shake culture in full-temperature shake culture box at 6 deg.C and 150 rpm for 48 hr to obtain Bacillus halophilus seed solution (viable count is 3 × 10) 7 Number of colonies/ml). Carrying out inoculation on the Arthrobacter antarctica seed liquid and the psychrophilus halophilus seed liquid according to the volume ratio of inoculation amount of 1: 1. inoculating 10% of the total inoculum size into a phosphorus accumulating bacteria culture medium, and performing shaking culture in a full-temperature shaking incubator at 6 ℃ and 150 r/min for 96 hours to obtain a phosphorus accumulating composite bacterial liquid. The total viable count of the phosphorus-accumulating composite bacterial liquid is 2 multiplied by 10 8 Number of colonies/ml.
The phosphorus accumulating bacteria culture medium comprises: 3.5 g/l sodium acetate, 0.8 g/l ammonium sulfate, 0.02 g/l dipotassium hydrogen phosphate, 0.1 g/l magnesium sulfate heptahydrate, 0.02 g/l calcium chloride, 8.5 g/l 4-hydroxyethyl piperazine ethanesulfonic acid. The pH of the medium for the polyphosphates was adjusted to 7.0 using 10% HCl and NaOH.
(4) The preparation of the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent comprises the following steps: mixing heterotrophic nitrification composite bacterial liquid, aerobic denitrification composite bacterial liquid and phosphorus accumulation composite bacterial liquid according to a volume ratio of 3:3:5, and preparing the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent. The total viable count of the composite microbial agent is 2 multiplied by 10 10 Number of colonies/ml.
The application of the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent in sewage comprises the following steps: and (3) carrying out a domestic sewage treatment test under a low-temperature condition by adopting the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent. Domestic sewage is taken from sewage treatment center of university campus, and water quality indexes are shown in Table 1.
TABLE 1 index of quality of domestic sewage
Figure DEST_PATH_IMAGE002
The low-temperature-resistant nitrogen and phosphorus removal compound microbial agent, the pseudomonas herbaceous, the pseudomonas putrefactive, the pseudomonas veronii, the pseudomonas fluorescens, the arthrobacter antarctica and the psychrophilum halophilum which are described in the embodiment 1 are respectively inoculated into 1 liter of domestic sewageThe total inoculation amount of the compound microbial inoculum and a single bacterium is the same (3 multiplied by 10) 2 Colony count/square centimeter), aerating for 24 hours at 8 ℃ with an aeration rate of 1.8 cubic meters per hour, standing for 20 minutes, filtering the effluent, measuring the mass concentration of ammonia nitrogen, nitrate nitrogen, total phosphorus and COD in the effluent, and calculating the removal rate of the ammonia nitrogen, the nitrate nitrogen, the total phosphorus and the COD by taking the domestic sewage without the compound microbial agent as a reference, wherein the results are shown in Table 2.
TABLE 2 treatment effect of complex microbial inoculant and single strain on domestic sewage
Figure DEST_PATH_IMAGE004
The removal rates of the compound microbial agent described in the embodiment 1 on ammonia nitrogen, nitrate nitrogen, total phosphorus and COD in the domestic sewage are respectively 88.4%, 86.7%, 83.2%, 88.5% and 98.2% under the low temperature condition, and compared with the removal rate of a single strain, the removal rate is obviously improved. Therefore, the composite microbial strains formed after compounding can synergistically promote growth and metabolism, and the removal efficiency of nitrogen and phosphorus in sewage is effectively improved.
Example 2:
(1) Preparing heterotrophic nitrification composite bacterial liquid: inoculating 4 rings of cold-resistant Pseudomonas pratensis from the slant solid culture medium with inoculating loop, and shake-culturing in a full-temperature shaking culture box at 10 deg.C and 160 r/min for 48 hr to obtain cold-resistant Pseudomonas pratensis seed solution (viable count of 2 × 10) 8 Number of colonies/ml). Inoculating 4 rings of cold-resistant Pseudomonas putrefaction in a solid culture medium, performing shake culture in a full-temperature shake culture box at 10 deg.C and 160 rpm for 48 hr to obtain cold-resistant Pseudomonas putrefaction seed solution (viable count of 4 × 10) 8 Number of colonies/ml). Carrying out inoculation on the cold-resistant pseudomonas palustris seed liquid and the cold-resistant pseudomonas mucida seed liquid according to the volume ratio of 2: 1. inoculating 15% of the total inoculum size into heterotrophic nitrifier culture medium, and performing shake culture in a full-temperature shake culture box at 10 ℃ and 160 r/minCulturing for 72 hours to obtain heterotrophic nitrification composite bacterial liquid. The total viable count of the heterotrophic nitrification composite bacterial liquid is 3 multiplied by 10 9 Number of colonies/ml.
Wherein the heterotrophic nitrifier culture medium comprises the following components: 3.0 g/l sodium citrate, 0.8 g/l ammonium sulphate, 0.5 g/l potassium dihydrogen phosphate, 0.2 g/l magnesium sulphate heptahydrate, 0.1 g/l sodium chloride, 0.03 g/l ferrous sulphate heptahydrate, 0.02 g/l anhydrous calcium chloride. The pH of the heterotrophic nitrifier medium was adjusted to 7.0 with 10% HCl and NaOH.
(2) Preparing aerobic denitrification composite bacterial liquid: inoculating 4 rings of cold-resistant Pseudomonas veronii from the slant solid culture medium with an inoculating ring, and performing shake culture in a full-temperature shake culture box at 10 deg.C and 160 rpm for 48 hr to obtain cold-resistant Pseudomonas veronii seed solution (viable count of 2 × 10) 8 Number of colonies/ml). Inoculating 4-ring Pseudomonas fluorescens from the slant solid culture medium with an inoculating ring, and performing shake culture in a full-temperature shake culture box at 10 deg.C and 160 rpm for 48 hr to obtain Pseudomonas fluorescens seed solution (viable count of 3 × 10) 8 Number of colonies/ml). And (2) inoculating cold-resistant pseudomonas veronii seed solution and pseudomonas fluorescens in a volume ratio of 3: 2. inoculating 10% of the total inoculum size into an aerobic denitrifying bacteria culture medium, and performing shaking culture in a full-temperature shaking incubator at the temperature of 10 ℃ and the rotating speed of 160 r/min for 72 hours to obtain aerobic denitrifying composite bacterial liquid. The total viable count of the aerobic denitrification composite bacterial liquid is 3 multiplied by 10 9 Number of colonies/ml.
The aerobic denitrifying bacteria culture medium comprises: 3.0 g/l sodium citrate, 1.0 g/l sodium nitrate, 0.5 g/l potassium dihydrogen phosphate, 0.2 g/l magnesium sulfate heptahydrate, 0.1 g/l sodium chloride, 0.03 g/l ferrous sulfate heptahydrate, 0.02 g/l anhydrous calcium chloride. The pH of the aerobic denitrifying bacteria culture medium is adjusted to 7.0 by using 10% HCl and NaOH.
(3) Preparing phosphorus-accumulating composite bacterial liquid: selecting 4 rings of Arthrobacter antarctica from the slant solid culture medium by using an inoculating loop, inoculating into a phosphorus accumulating bacteria culture medium, and oscillating in a full-temperature oscillation incubator at the temperature of 10 ℃ and the rotating speed of 160 r/minCulturing for 48 hours to obtain the Arthrobacter antarctica (viable count is 2 multiplied by 10) 8 Number of colonies/ml). Inoculating 4 rings of the psychrophilic halophilus from the slant solid culture medium by using an inoculating loop, and performing shaking culture in a full-temperature shaking culture box at the temperature of 10 ℃ and the rotating speed of 160 r/min for 48 hours to obtain a seed solution (the viable count is 3 multiplied by 10) of the psychrophilic halophilus 8 Number of colonies/ml). Carrying out inoculation on the Arthrobacter antarctica seed liquid and the psychrophilus halophilus seed liquid according to the volume ratio of inoculation amount of 1: 1. inoculating 10% of the total inoculum size into a phosphorus-accumulating bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 10 ℃ and the rotating speed of 160 r/min for 72 hours to obtain the phosphorus-accumulating composite bacterial liquid. The total viable count of the phosphorus-accumulating composite bacterial liquid is 2 multiplied by 10 9 Number of colonies/ml.
The phosphorus accumulating bacteria culture medium comprises: 4.0 g/l sodium acetate, 1.0 g/l ammonium sulfate, 0.03 g/l dipotassium hydrogen phosphate, 0.05 g/l magnesium sulfate heptahydrate, 0.01 g/l calcium chloride, 7.5 g/l 4-hydroxyethyl piperazine ethanesulfonic acid. The pH of the medium for the polyphosphates was adjusted to 7.0 using 10% HCl and NaOH.
(4) The preparation of the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent comprises the following steps: mixing heterotrophic nitrification composite bacterial liquid, aerobic denitrification composite bacterial liquid and phosphorus accumulation composite bacterial liquid according to a volume ratio of 5:5:12, and preparing the low-temperature-resistant nitrogen and phosphorus removal composite microbial inoculum. The total viable count of the composite microbial agent is 3 × 10 10 Number of colonies/ml.
The application of the low-temperature-resistant nitrogen and phosphorus removal composite bacterial agent in sewage comprises the following steps: the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent is adopted to carry out a domestic sewage treatment test under a low-temperature condition. Domestic sewage is taken from sewage treatment center of university campus, and water quality indexes are shown in Table 3.
TABLE 3 index of quality of domestic sewage
Figure DEST_PATH_IMAGE006
The low-temperature-resistant nitrogen and phosphorus removal compound microbial agent, the pseudomonas herbaceum, the pseudomonas putrefaciens, the pseudomonas veroniae, the pseudomonas fluorescens, the arthrobacter antarctica and the pseudomonas solanacearum which are described in the embodiment 2 are addedThe halophilus is respectively inoculated into 1 liter of domestic sewage, the total bacterial quantity of the composite microbial agent and the single bacterial are the same (5 multiplied by 10) 2 Colony count/square centimeter), aerating for 36 hours at 10 ℃ with an aeration rate of 2.1 cubic meter/hour, standing for 20 minutes, filtering the effluent, measuring the mass concentration of ammonia nitrogen, nitrate nitrogen, total phosphorus and COD in the effluent, and calculating the removal rate of the ammonia nitrogen, the nitrate nitrogen, the total phosphorus and the COD by taking the domestic sewage without the compound microbial agent as a reference, wherein the results are shown in Table 4.
TABLE 4 treatment effect of complex microbial inoculant and single strain on domestic sewage
Figure DEST_PATH_IMAGE008
The removal rates of ammonia nitrogen, nitrate nitrogen, total phosphorus and COD in the domestic sewage by the composite microbial agent in the embodiment 2 are respectively 98.6%, 96.5%, 98.5%, 96.2% and 100% under the low-temperature condition, and compared with the removal rate of a single bacterial strain, the removal rate is obviously improved. Therefore, the composite microbial strains formed after compounding can synergistically promote growth and metabolism, and the removal efficiency of nitrogen and phosphorus in sewage is effectively improved.
Example 3:
(1) Preparing heterotrophic nitrification composite bacterial liquid: inoculating 4 rings of cold-resistant pseudomonas stutzeri from the slant solid culture medium with an inoculating ring, and performing shake culture in a full-temperature shake culture box at 15 deg.C and 140 rpm for 24 hr to obtain cold-resistant pseudomonas stutzeri seed solution (viable count of 2 × 10) 9 Number of colonies/ml). Inoculating 4 rings of cold-rot resistant pseudomonas in a slant solid culture medium by using an inoculating ring, and performing shake culture in a full-temperature shake culture box at 15 ℃ and 140 r/min for 24 hours to obtain cold-rot resistant pseudomonas seed solution (viable count is 3 multiplied by 10) 9 Colony number/ml) is added into cold-resistant pseudomonas herbaceus seed liquid and cold-resistant pseudomonas mucida seed liquid according to the volume ratio of inoculation amount of 1: 1. inoculating 20% of the total inoculum size into heterotrophic nitrifier culture medium at 14 deg.CAnd carrying out shake culture in a full-temperature shake culture box at 140 r/min for 48 hours to obtain the heterotrophic nitrification composite bacterial liquid. The total viable count of the heterotrophic nitrification composite bacterial liquid is 3 multiplied by 10 10 Number of colonies/ml.
The heterotrophic nitrifier culture medium comprises the following components: 3.5 g/l sodium citrate, 1.5 g/l ammonium sulfate, 0.5 g/l potassium dihydrogen phosphate, 0.3 g/l magnesium sulfate heptahydrate, 0.2 g/l sodium chloride, 0.05 g/l ferrous sulfate heptahydrate, 0.05 g/l anhydrous calcium chloride. The pH of the heterotrophic nitrifier culture medium was adjusted to 7.0 using 10% HCl and NaOH.
(2) Preparing aerobic denitrification composite bacterial liquid: inoculating 4 rings of cold-resistant pseudomonas veronii from the slant solid culture medium by using an inoculating ring, and performing shake culture in a full-temperature shake culture box at the temperature of 15 ℃ and the rotating speed of 140 r/min for 24 hours to obtain cold-resistant pseudomonas veronii seed liquid (the viable count is 4 multiplied by 10) 9 Number of colonies/ml). Inoculating 4-ring Pseudomonas fluorescens from the slant solid culture medium by using an inoculating loop, and performing shake culture in a full-temperature shake culture box at 15 deg.C and 140 r/min for 24 hr to obtain Pseudomonas fluorescens seed solution (viable count is 2 × 10) 9 Number of colonies/ml). Carrying out inoculation on cold-resistant pseudomonas veronii seed solution and pseudomonas fluorescens according to the volume ratio of 1: 1. inoculating 20 percent of the total inoculum size into an aerobic denitrifying bacteria culture medium, and performing shaking culture in a full-temperature shaking incubator at the temperature of 15 ℃ and the rotating speed of 140 r/min for 48 hours to obtain aerobic denitrifying composite bacterial liquid. The total viable count of the aerobic denitrification composite bacterial liquid is 4 multiplied by 10 10 Number of colonies/ml.
The aerobic denitrifying bacteria culture medium comprises: 3.5 g/l sodium citrate, 1.0 g/l sodium nitrate, 0.5 g/l potassium dihydrogen phosphate, 0.3 g/l magnesium sulfate heptahydrate, 0.2 g/l sodium chloride, 0.05 g/l ferrous sulfate heptahydrate, 0.05 g/l anhydrous calcium chloride. The pH of the aerobic denitrifying bacteria culture medium was adjusted to 7.0 using 10% HCl and NaOH.
(3) Preparing phosphorus-accumulating composite bacterial liquid: selecting 4-ring Antarctic Arthrobacter from the slant solid culture medium by using an inoculating ring, inoculating into a phosphorus accumulating bacteria culture medium, and performing inoculation at the temperature of 15 ℃ and the rotating speed of 14 DEG CCarrying out shake culture in a full-temperature shake culture box at the speed of 0 r/min for 24 hours to obtain the Arthrobacter antarctica (the viable count is 2 multiplied by 10) 8 Number of colonies/ml). Inoculating 4 rings of psychrobacter salmonellae from the slant solid culture medium with inoculating loop, and performing shake culture in a full-temperature shake culture box at 10 deg.C and 140 rpm for 24 hr to obtain seed solution (viable count of 4 × 10) of psychrobacter salmonellae 8 Number of colonies/ml). Carrying out inoculation on the Arthrobacter antarctica seed liquid and the psychrophilus halophilus seed liquid according to the volume ratio of the inoculation amount of 2: 1. inoculating 15% of the total inoculum size into a phosphorus-accumulating bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 15 ℃ and the rotating speed of 140 r/min for 48 hours to obtain the phosphorus-accumulating composite bacterial liquid. The total viable count of the phosphorus-accumulating composite bacterial liquid is 3 multiplied by 10 9 Number of colonies/ml.
The phosphorus accumulating bacteria culture medium comprises: 4.5 g/l sodium acetate, 1.5 g/l ammonium sulfate, 1.0 g/l dipotassium hydrogen phosphate, 0.05 g/l magnesium sulfate heptahydrate, 0.03 g/l calcium chloride, 8.5 g/l 4-hydroxyethyl piperazine ethanesulfonic acid. The pH of the medium for the polyphosphate was adjusted to 7.0 using 10% HCl and NaOH.
(4) Preparing a low-temperature-resistant nitrogen and phosphorus removal compound microbial agent: mixing heterotrophic nitrification composite bacterial liquid, aerobic denitrification composite bacterial liquid and phosphorus accumulation composite bacterial liquid according to a volume ratio of 1:1:20, and preparing the low-temperature-resistant nitrogen and phosphorus removal composite microbial inoculum. The total viable count of the composite microbial agent is 3 × 10 10 Number of colonies/ml.
The application of the low-temperature-resistant nitrogen and phosphorus removal composite bacterial agent in sewage comprises the following steps: the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent is adopted to carry out a domestic sewage treatment test under a low-temperature condition. Domestic sewage is taken from sewage treatment center of university campus, and water quality indexes are shown in Table 5.
TABLE 5 index of quality of domestic sewage
Figure DEST_PATH_IMAGE010
The low-temperature-resistant nitrogen and phosphorus removal compound microbial agent, the pseudomonas herbaceum, the pseudomonas putrefactive soil, the pseudomonas veronii, the pseudomonas palustris and the like which are described in the embodiment 3 are added,Pseudomonas fluorescens, arthrobacter antarctica and psychrophilus halophilus are respectively inoculated into 1 liter of domestic sewage, and the total inoculum size of the compound microbial inoculum is the same as that of a single bacterium (all of which are 4 multiplied by 10) 2 Colony count/square centimeter), aerating for 24 hours at 13 ℃ with an aeration rate of 2.4 cubic meters per hour, standing for 20 minutes, filtering the effluent, measuring the mass concentration of ammonia nitrogen, nitrate nitrogen, total phosphorus and COD in the effluent, and calculating the removal rate of the ammonia nitrogen, the nitrate nitrogen, the total phosphorus and the COD by taking the domestic sewage without the compound microbial agent as a reference, wherein the results are shown in Table 6.
TABLE 6 treatment effect of composite microbial inoculum and single strain on domestic sewage
Figure DEST_PATH_IMAGE012
The removal rates of the compound microbial agent described in example 3 on ammonia nitrogen, nitrate nitrogen, total phosphorus and COD in the domestic sewage are respectively 99.1%, 98.8%, 99.0%, 97.3% and 100% under the low temperature condition, and compared with the removal rate of a single strain, the removal rate is obviously improved. Therefore, the composite microbial strains formed after compounding can synergistically promote growth and metabolism, and the removal efficiency of nitrogen and phosphorus in sewage is effectively improved.

Claims (8)

1. A method for treating sewage by using a low-temperature-resistant nitrogen and phosphorus removal compound microbial agent is characterized by comprising the following steps of:
firstly, preparing the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent comprises the following preparation steps:
(1) Preparing heterotrophic nitrification composite bacterial liquid: inoculating 3-4 rings of cold-resistant pseudomonas pratensis from the slant solid culture medium by using an inoculating ring, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 24-72 hours to obtain cold-resistant pseudomonas pratensis seed liquid (the viable count is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml); inoculating 3-4 rings of cold-resistant sludge-resistant pseudomonas in an inclined solid culture medium by using an inoculating ring to obtain a mixtureIn the bacteria culture medium, the bacteria is cultured for 24 to 72 hours in a full-temperature oscillation culture box with the temperature of 6 to 15 ℃ and the rotating speed of 120 to 170 revolutions per minute to obtain cold-rot-resistant pseudomonas seed liquid (the viable count is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml); the cold-resistant pseudomonas putida seed liquid and the cold-resistant pseudomonas putida seed liquid are mixed according to the volume ratio of the inoculation amount (1-10): (1-10) inoculating 5-20% of the total inoculum size to a heterotrophic nitrifying bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 48-96 hours to obtain heterotrophic nitrifying composite bacterial liquid; the total viable count of the heterotrophic nitrification composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Colony count/ml;
(2) Preparing aerobic denitrification composite bacterial liquid: inoculating 3-4 rings of cold-resistant pseudomonas veronii from a slant solid culture medium by using an inoculating loop, inoculating into an aerobic denitrifying bacterium culture medium, and performing oscillation culture in a full-temperature oscillation culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 24-72 hours to obtain cold-resistant pseudomonas veronii seed liquid (the number of viable bacteria is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml); inoculating 3-4 rings of pseudomonas fluorescens from a slant solid culture medium by using an inoculating ring to an aerobic denitrifying bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 24-72 hours to obtain pseudomonas fluorescens seed liquid (the viable count is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml); the cold-resistant pseudomonas veronii seed solution and the pseudomonas fluorescens are mixed according to the volume ratio of the inoculation amount (1-10): (1-10) inoculating 5-20% of the total inoculum size to an aerobic denitrifying bacteria culture medium, and performing shaking culture in a full-temperature shaking culture box with the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 48-96 hours to obtain an aerobic denitrifying composite bacterial liquid; the total viable count of the heterotrophic nitrification composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Colony count/ml;
preparing phosphorus-accumulating composite bacterial liquid: selecting 3-4 rings of Antarctic Arthrobacter from the slant solid culture medium by using an inoculating ring, inoculating into a polyphosphate culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 23-72 hours to obtain the Antarctic ArthrobacterArthrobacter (viable count 1X 10) 7 ～2×10 9 Number of colonies/ml);
inoculating 3-4 rings of psychrophilic halophilus from the slant solid culture medium by using an inoculating loop, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 r/min for 24-72 hours to obtain a seed liquid of the psychrophilic halophilus (the viable count is 1 multiplied by 10) 7 ～2×10 9 Number of colonies/ml);
the method comprises the following steps of (1) inoculating Arthrobacter antarctica seed liquid and the psychrophilus halophilus seed liquid according to the volume ratio of the inoculum size (1-10): (1-10), inoculating 5-20% of the total inoculum size to a phosphorus-accumulating bacteria culture medium, and carrying out shake culture in a full-temperature shake culture box at the temperature of 6-15 ℃ and the rotating speed of 120-170 rpm for 48-96 hours to obtain phosphorus-accumulating composite bacterial liquid; the total viable count of the phosphorus-accumulating composite bacterial liquid is 1 multiplied by 10 8 ～5×10 9 Colony count/ml;
(3) The preparation of the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent comprises the following steps: mixing heterotrophic nitrification composite bacterial liquid, aerobic denitrification composite bacterial liquid and phosphorus accumulation composite bacterial liquid according to the volume ratio of (1-10): (1-10): (1-20) to obtain the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent; the total viable count of the composite microbial agent is not less than 1 × 10 10 Colony count/ml;
the low-temperature-resistant nitrogen and phosphorus removal compound microbial agent is applied to low-temperature biological sewage treatment and comprises the following steps:
the additive amount in the urban sewage to be treated is 2 multiplied by 10 2 ～5×10 2 The low temperature resistant nitrogen and phosphorus removal composite microbial agent with colony number per square centimeter is aerated for 24 to 48 hours at the aeration rate of 1.0 to 3.5 cubic meters per hour, and is filtered after standing for 20 to 30 minutes.
2. The method for treating sewage by using the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent as claimed in claim 1, wherein the heterotrophic nitrification composite bacterial liquid is prepared by using cold-resistant pseudomonas solanacearum and cold-resistant pseudomonas putida as the activated sludge in the sedimentation tank of the sewage treatment plant, and the prepared bacteria are respectively identified as pseudomonas herbaceus (Pseudomonas aeruginosa) by 16SrDNA sequence analysisPseudomonas poae) And Pseudomonas putida: (Pseudomonas peli)。
3. The method for treating sewage by using the low-temperature-resistant composite microbial agent for nitrogen and phosphorus removal as claimed in claim 1, wherein the heterotrophic nitrification composite bacterial liquid is prepared, and the culture medium of the heterotrophic nitrification bacteria comprises the following components: 1.0 to 5.0 grams/liter of sodium citrate, 0.2 to 1.0 gram/liter of ammonium sulfate, 0.3 to 1.5 grams/liter of monopotassium phosphate, 0.1 to 1.0 gram/liter of magnesium sulfate heptahydrate, 0.1 to 1.0 gram/liter of sodium chloride, 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate, and 0.01 to 0.1 gram/liter of anhydrous calcium chloride;
the pH of the heterotrophic nitrifier culture medium is adjusted to 6.5-7.5 by adopting 10% HCl and NaOH.
4. The method for treating sewage by using the low-temperature-resistant composite microbial agent for nitrogen and phosphorus removal as claimed in claim 1, wherein the heterotrophic nitrification composite bacterial liquid is prepared, and the cold-resistant pseudomonas veronii and the pseudomonas fluorescens are respectively river water frozen substrate sludge which is respectively identified as pseudomonas veronii (pseudomonas aeruginosa), (pseudomonas fluorescens) through 16SrDNA sequence analysisPseudomonas veronii) And Pseudomonas fluorescens (Pseudomonas fluorescens)。
5. The method for treating sewage by using the low-temperature-resistant composite microbial agent for nitrogen and phosphorus removal as claimed in claim 1, wherein the heterotrophic nitrification composite bacterial liquid is prepared by using an aerobic denitrification bacterial culture medium comprising: 1.0 to 5.0 grams/liter of sodium citrate, 0.2 to 2.0 grams/liter of sodium nitrate, 0.3 to 1.5 grams/liter of monopotassium phosphate, 0.1 to 1.0 gram/liter of magnesium sulfate heptahydrate, 0.1 to 1.0 gram/liter of sodium chloride, 0.01 to 0.1 gram/liter of ferrous sulfate heptahydrate, and 0.01 to 0.1 gram/liter of anhydrous calcium chloride; the pH value of the aerobic denitrifying bacteria culture medium is adjusted to 6.5-7.5 by adopting 10% HCl and NaOH.
6. The method for treating sewage by using the low-temperature-resistant nitrogen and phosphorus removal compound microbial inoculant according to claim 1, wherein a QHZ-98A type full-temperature oscillation incubator is used for culturing the microbial inoculant.
7. The method for treating sewage by using the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent as claimed in claim 1, wherein an SN210C type vertical pressure steam sterilizer is adopted for high-pressure steam sterilization.
8. The method for treating sewage by using the low-temperature-resistant nitrogen and phosphorus removal composite microbial agent as claimed in claim 1, wherein the culture medium is sterilized by high-pressure steam under the conditions that the temperature is 121 ℃ and the pressure is 0.105 MPa, and the temperature is maintained for 20 to 30 minutes.
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