WO2020034454A1 - 一种确定污水处理生物膜最适保存温度的方法 - Google Patents

一种确定污水处理生物膜最适保存温度的方法 Download PDF

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WO2020034454A1
WO2020034454A1 PCT/CN2018/114552 CN2018114552W WO2020034454A1 WO 2020034454 A1 WO2020034454 A1 WO 2020034454A1 CN 2018114552 W CN2018114552 W CN 2018114552W WO 2020034454 A1 WO2020034454 A1 WO 2020034454A1
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biofilm
nitrifying
denitrifying
granular sludge
aerobic granular
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French (fr)
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王硕
朱引
李激
王燕
郑凯凯
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江南大学
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Priority claimed from CN201810925421.3A external-priority patent/CN110835157B/zh
Priority claimed from CN201811061392.7A external-priority patent/CN110894101B/zh
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Priority to JP2019564051A priority Critical patent/JP6905088B2/ja
Priority to US16/720,243 priority patent/US20200140301A1/en
Publication of WO2020034454A1 publication Critical patent/WO2020034454A1/zh

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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
    • C02F3/006—Regulation methods for biological 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
    • C02F3/02—Aerobic processes
    • C02F3/10—Packings; Fillings; Grids
    • C02F3/102—Permeable membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/56—Polyamides, e.g. polyester-amides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2676—Centrifugal separation
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/02—Temperature
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/16—Total nitrogen (tkN-N)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the invention relates to a method for determining the optimal storage temperature of a sewage treatment biofilm, and belongs to the technical field of environmental engineering.
  • the low level of organic pollutants in the sewage treatment plant has always been a technical difficulty in restricting the discharge of total nitrogen.
  • a large amount of land has been occupied by reconstruction and expansion projects, and a large amount of organic carbon source substances and phosphorus removal agents have been Adding it to water bodies has significantly increased investment and construction and operating costs.
  • Traditional activated sludge processes have been unable to meet the requirements for pollutant discharge, which has seriously affected the energy conservation and consumption reduction effects of sewage treatment plants. Therefore, wastewater denitrification technology based on land-saving goals and low-carbon source utilization is being increasingly valued.
  • the more effective wastewater treatment processes in wastewater treatment include aerobic granular sludge and nitrification and denitrification biofilm processes.
  • the aerobic granular sludge process has good sedimentation performance, lower operating costs, higher biomass and treatment.
  • the efficiency and great development potential; the nitrification and denitrification biofilm process can achieve simultaneous nitrification and denitrification, and it is also of great significance for the removal of ammonia nitrogen and total nitrogen in sewage.
  • the film formation period of the nitrification and denitrification biofilm is about 30-40 days, and the culture period of the aerobic granular sludge is 70-120 days, and the culture conditions are relatively harsh. If the aerobic granular sludge and the nitrifying and denitrifying biofilm can be cultivated and preserved, it can effectively help the sewage treatment plant with low carbon source and low land resources to start operation in a short period of time and make the pollutants Discharge. Temperature is an important parameter that affects the biofilm activity.
  • Determining the temperature that is most suitable for the preservation of biofilms such as aerobic granular sludge and nitrifying and denitrifying biofilms will help simplify the process of recovering its activity, shorten the start-up time of engineering applications, and save energy. Consumption reduction effect.
  • the determination of the optimal storage temperature by the existing method requires re-inoculation of aerobic granular sludge and nitrifying denitrifying biofilm in the bioreactor, and it takes about 8 to 35 days to determine the aerobic granular sludge and nitrifying denitrifying biofilm.
  • the recovery effect of the active material takes too long and becomes the key to restrict its engineering application.
  • the pollutants of the sewage treatment plant will be discharged within a short period of time, while achieving land-saving and energy-saving effects.
  • the invention is based on the flow cytometry to characterize the cell active state in the biofilm preserved under different temperature conditions, and through the cell active state after the biofilm activity is restored and the effect of removing pollutants, the characterization results of the flow cytometer After verification, a method for determining the optimal storage temperature of biofilms based on flow cytometry was finally established to provide technical support for high-standard pollutant discharge and energy-saving operation of sewage treatment plants.
  • the first object of the present invention is to provide a method for determining the optimal storage temperature of a sewage treatment biofilm.
  • the method is based on measuring the cell activity state of a sewage treatment biofilm based on flow cytometry, and comparing organisms stored at different temperatures. As a result of the measurement of the activity state of the membrane and the biofilm cell before storage, the storage temperature closest to the activity state of the biofilm cell before storage was taken as the optimal storage temperature.
  • the determination of the cell activity state of the biofilm includes determination of the content of live cells, early apoptotic cells, late apoptotic cells, and dead cells.
  • the biofilm includes aerobic granular sludge and a nitrifying and denitrifying biofilm.
  • the step of confirming the optimal temperature by flow cytometry includes:
  • a phosphate buffer is used as the buffer for dilution.
  • the phosphate buffer solution includes sodium dihydrogen phosphate and disodium hydrogen phosphate.
  • the test sample solution is prepared by diluting the aerobic granular sludge with a buffer solution of pH 7.0-8.0.
  • the test sample solution is prepared by diluting the nitrifying and denitrifying biofilm with a buffer solution having a pH of 6.6 to 7.0.
  • the dilution volume ratio of the buffer solution to the biofilm is 8 to 10: 1.
  • a nylon membrane with a pore size of 5 to 15 ⁇ m is used for filtration.
  • a nylon membrane with a pore size of 6 to 8 ⁇ m is selected for filtration.
  • the centrifugal speed is 5000 to 10000 rpm / min.
  • a mixing volume ratio of the sample serum to 10xAnnexinVBindingBindingBuffer is 1: 2 to 4.
  • the flow cytometry is used to determine the cell activity status of each sample by adding 0.5 ⁇ l PI staining agent to the control FITC Annexin V group, and adding 0.5 ⁇ l FITC Annexin V to the control PI group, and detecting the group. Add 0.5 ⁇ l FITC Annexin V and 0.5 ⁇ l PI, mix and incubate in the dark at room temperature, and then test on a flow cytometer.
  • the incubation time is 10-20 minutes.
  • the second object of the present invention is to provide a method for quickly starting the biofilm engineering of sewage treatment.
  • the method is to mature the biofilm in advance, place it in a storage matrix and store it at the optimal storage temperature. After the activity is restored, It can be used for sewage treatment; the optimal temperature is determined by using the above method.
  • COD in the storage matrix of the aerobic granular sludge, is 250-350 mg / L, NH 4 + -N 55-65 mg / L, and PO 4 3- -P 6-10 mg / L. .
  • the recovery of the aerobic granular sludge is to inoculate the aerobic granular sludge into a sequencing batch reactor (SBR), the effective volume of the SBR is 10.0L, and the water is drained.
  • SBR sequencing batch reactor
  • the ratio is 45 ⁇ 60%
  • the reaction period is 2.5 ⁇ h
  • the static water intake period is 1 ⁇ 1.5h
  • the aeration reaction period is 1.5 ⁇ 2.5h
  • the sludge sedimentation period is 2 ⁇ 6min
  • the rapid drainage period is 2 ⁇ 6min
  • SRT is controlled at 25 days.
  • COD in the storage matrix of the nitrifying and denitrifying biofilm, is 180 to 220 mg / L, NH 4 + -N 25 to 35 mg / L, NO 3 - to N 18 to 25 mg / L, PO 4 3- to P 6 to 10 mg / L.
  • the restoration of the activity of the nitrifying and denitrifying biofilm is inoculating the nitrifying and denitrifying biofilm in a bioreactor. Based on the anoxic-aerobic process, the HRT is set to 10-15 hours. The filling ratio of nitrification and denitrification is 40% to 60%, and the reflux ratio of the nitrification solution is 70% to 85%.
  • a third object of the present invention is to apply the above method to sewage treatment.
  • the present invention uses flow cytometry to characterize a variety of biofilm live cells, early apoptotic cells, late apoptotic cells and dead cell ratios, confirms the optimal storage temperature within a few hours, and correlates with the characteristic indicators of the biofilm activity recovery process Correlation analysis was performed to establish a method for determining the optimal storage temperature of biofilms based on flow cytometry.
  • the step of recovering biofilm activity can be omitted, which can effectively help the sewage treatment plant that prepares to adopt biofilm process technology to discharge pollutants (ammonia nitrogen, total nitrogen, total phosphorus, etc.) to achieve land saving and energy saving. It can effectively shorten the start-up time of the biofilm process engineering application, maintain the long-term stable operation of the biofilm process, and has high industrial feasibility.
  • Figure 1 shows the sedimentation performance of aerobic granular sludge (sludge volume index SVI);
  • Figure 2 shows the changes of extracellular polymers (PN / PS) of aerobic granular sludge
  • Figure 3 shows the removal rate of total nitrogen (TN) from aerobic granular sludge
  • Figure 4 shows the total phosphorus (TP) removal rate of aerobic granular sludge
  • Figure 5 shows the change of the PN / PS of the extracellular polymer of the nitrifying and denitrifying biofilm
  • Figure 6 shows the removal rate of ammonia nitrogen (AN) from the nitrifying and denitrifying biofilm
  • Fig. 7 shows the removal rate of total nitrogen (TN) in the nitrifying and denitrifying biofilm.
  • the sewage of the sewage treatment plant of the present invention includes domestic water in upstream residential areas and a small part of industrial waste water.
  • the average annual influent is 236 mg / L, ammonia nitrogen 30.1 mg / L, total nitrogen 37.8 mg / L, and total phosphorus 4.5 mg / L , Nitrate nitrogen content is less than 1.0mg / L.
  • Aerobic granular sludge storage conditions Aerobic granular sludge storage conditions:
  • the storage temperature of aerobic granular sludge was set to -20 ° C, 4 ° C and 20 ° C.
  • 900ml of the mud-water mixture in the aerobic granular sludge pilot plant was taken out, and an average of three equal portions were placed in a 1000ml serum bottle with a 500ml storage matrix.
  • the components of the storage matrix are as follows: NaAc 4200mg / L, NH 4 Cl 1100mg / L, K 2 HPO 4 370mg / L, KH 2 PO 4 140mg / L, MgSO 4 440mg / L, KCl 170mg / L, trace element liquid 1ml / L; the trace element liquid composition is as follows: FeCl 3 ⁇ 6H 2 O 1.5g / L, H 3 BO 3 0.15g / L, CuSO 4 ⁇ 5H 2 O 0.03g / L, KI 0.03g / L, MnCl 2 ⁇ 4H 2 O 0.12g / L, Na 2 MoO 4 ⁇ 2H 2 O 0.06g / L, ZnSO 4 ⁇ 7H 2 O 0.12g / L, CoCl 2 ⁇ 6H 2 O 0.15g / L.
  • Serum bottles (three parallel samples at each storage temperature) were placed at -20 ° C, 4 ° C, and 20 ° C, respectively, and stored in a light-shielded state for 3 months.
  • the results of the aerobic granular sludge cell status are shown in Table 1.
  • the proportion of aerobic granular sludge living cells in the pilot system is high, indicating that the pilot system operates well.
  • the aerobic granular sludge stored at -20 ° C has the lowest living cell content and the highest dead cell content, indicating that aerobic granular sludge is not suitable for storage at -20 ° C.
  • the aerobic granular sludge stored at 4 ° C has a ratio of late apoptotic cells and dead cells of about 25.2%, which proves that the 4 ° C condition can be used for the storage of aerobic granular sludge.
  • SBR Sequencing Batch Reactor
  • the stored aerobic granular sludge was placed in R1, R2 and R3, respectively.
  • the effective volume of SBR is 10.0L, the drainage ratio is 50%, the reaction period is 3h, the static water intake period is 60min, the aeration reaction period is 112min, the sludge sedimentation period is 3min, and the rapid drainage period is 5min.
  • the real-time control system controls the air and nitrogen content and ratio to ensure the anaerobic state of the water inlet section and the aerobic state of the reaction section; the SRT is controlled at 25 days.
  • the aerobic granular sludge in R1, R2 and R3 has better performance.
  • Table 2 after the recovery of the aerobic granular sludge activity, the density and particle size of the aerobic granular sludge at different storage temperatures were close to those of the aerobic granular sludge before storage.
  • the biomass of aerobic granular sludge at different storage temperatures is only 91% before storage, this biomass is 37.9% higher on average than the biomass of aerobic granular sludge after storage, indicating aerobic granular sludge Re-adapted to the environment, the biomass increased steadily.
  • the average denitrification rate and phosphorus release rate of activated sludge in sewage treatment plants are 3.0mg / gMLSS ⁇ h and 2.2mg / gMLSS ⁇ h, respectively.
  • domesticated aerobic granular sludge in the pilot plant achieved the same denitrification rate and phosphorus release rate. The time required is 25 and 29d, respectively.
  • the time required for the aerobic granular sludge in R1 to reach the same denitrification rate and phosphorus release rate is 12 and 13 days
  • the aerobic granular sludge after the recovery of activity has a better denitrification and phosphorus removal effect.
  • the aerobic granular sludge stored at 20 ° C has the shortest activity recovery time, which is more suitable for storing aerobic granular sludge.
  • the aerobic granular sludge in R1, R2 and R3 all have good sedimentation performance.
  • the volume of aerobic granular sludge at different storage temperatures The index SVI is lower than 50.0ml / g.
  • the aerobic granular sludge SVI decreased slightly and finally stabilized between 46.1-47.8 ml / g.
  • Extracellular polymer is an important factor in the formation of aerobic granular sludge, and the ratio of protein (PN) to polysaccharide (PS) in the extracellular polymer (PN / PS) is a measure of the structure of aerobic granular sludge An important indicator of stability.
  • the cell status of the aerobic granular sludge was analyzed by flow cytometry (as shown in Table 3).
  • the content of living cells in the aerobic granular sludge of the test system is basically the same, which shows that after the activity recovery, the aerobic granular sludge can play the role of pollutant removal.
  • the proportion of aerobic granular sludge living cells (86.5% ⁇ 3.5%) was the highest in R3, and the proportion of late apoptotic cells (3.8% ⁇ 1.0%) and dead cells (3.3% ⁇ 0.3%) were the lowest.
  • Aerobic granular sludge has the highest cell activity under storage conditions, and is more suitable as a condition for aerobic granular sludge.
  • the aerobic granular sludge denitrification rate has a very high correlation with the phosphorus release rate and the aerobic granular sludge living cell ratio (as shown in Table 4). It shows that using aerobic granular sludge cell ratio as a method to evaluate aerobic granular sludge activity is highly feasible. At the same time, the aerobic granular sludge had the highest viable cell ratio in the stored aerobic granular sludge at 20 ° C, which was consistent with the result of the aerobic granular sludge viable cell ratio in R3 after recovery.
  • 20 ° C is the optimal condition for storing aerobic granular sludge.
  • Flow cytometry can be used as a basis for determining the optimal storage temperature of aerobic granular sludge. Flow cytometry is easy to operate, the data is fast, easy to obtain, accurate and reliable, and the process of recovering aerobic granular sludge can be omitted, which is of great significance for the preservation and recovery of aerobic granular sludge.
  • the storage temperature of the nitrifying and denitrifying biofilm was set to -20 ° C, 4 ° C and 20 ° C. Take out 180 nitrating and denitrifying biofilms in the biochemical reaction tank of the sewage treatment plant, and place an average of three equal parts in 1000ml serum bottles with a 500ml storage matrix. In order to maintain the nitrification and denitrification capabilities of the biofilm, the matrix components are preserved.
  • NaAc 240mg / L NH 4 Cl 110mg / L, KNO 3 80mg / L, K 2 HPO 4 30mg / L, KH 2 PO4 15mg / L, MgSO 4 40mg / L, KCl 70mg / L.
  • the serum bottles (three parallel samples at each storage temperature) were placed at -20 ° C, 4 ° C and 20 ° C, respectively, and stored in a light-shielded still.
  • the nitrifying and denitrifying biofilm is used to determine the state of the nitrifying and denitrifying biofilm cells.
  • the conditions of the flow cytometry test are as follows:
  • filter pore size for sample preparation is particularly important. If the pore size is too large, more biological flocs will be introduced, causing uneven dyeing and affecting the final result. If the pore size is too small, biological flocs cannot be obtained effectively.
  • Table 5 shows the test results of nitrification and denitrification biofilm cells.
  • Stored at -20 ° C for the determination of nitrifying and denitrifying biofilms has the lowest cell content and the highest dead cell content, indicating that -20 ° C is not suitable for storing nitrifying and denitrifying biofilms.
  • the proportion of viable cells in nitrifying and denitrifying biofilms is as high as 59.4%, which is only 12.6% lower than that in nitrifying and denitrifying biofilms stored at 4 ° C.
  • the ratio of total and dead cells is about 31.6%, indicating that the storage conditions at 20 ° C are not suitable for the preservation of nitrifying and denitrifying biofilms. Therefore, 4 ° C was initially determined as the optimal temperature for storing the nitrifying and denitrifying biofilm.
  • Example 4 Verification of test results of nitrification and denitrification biofilms
  • Sequential batch reactor operation mode Nitrogen and denitrification biofilms from different serum bottles are taken and inoculated in the bioreactor (effective volume 10.0L) to restore the activity of the nitrification and denitrification biofilms. Nitrifying and denitrifying biofilms stored at -20 ° C, 4 ° C and 20 ° C were placed in R1, R2 and R3, respectively. Based on the anoxic-oxic (AO) process, the bioreactor achieves simultaneous nitrification and denitrification in a sequential batch reaction. The HRT is set to 12 hours, the filling ratio of the nitrification and denitrification is 50%, and the reflux ratio of the nitrating solution is 80%.
  • AO anoxic-oxic
  • the nitrifying and denitrifying biofilms in R1, R2 and R3 all have better performance.
  • Table 6 after the activity of the nitrifying and denitrifying biofilm is restored, the density and thickness of the nitrifying and denitrifying biofilm stored at 4 ° C and 20 ° C are similar to the nitrifying and denitrifying biofilm before storage, and only stored at -20 The density and thickness of the nitrifying and denitrifying biofilm decreased slightly at °C.
  • the biomass of nitrifying and denitrifying biofilms has been reduced, but after recovery of activity, the biomass of nitrifying and denitrifying biofilms stored at 4 ° C and 20 ° C has reached the level of nitrifying and denitrifying biofilms before storage.
  • the level of biomass indicates that the nitrifying and denitrifying biofilm has re-adapted to the environment and the biomass has increased steadily.
  • the average nitrification rate and denitrification rate of biofilms in sewage treatment are 4.5 and 5.0g NO 3 -- N / m 2 ⁇ d, respectively.
  • Nitrification and denitrification biofilms domesticated by sewage treatment plants reach the same nitrification rate and denitrification rate. It takes 25 and 21 days, respectively. After the stored nitrification and denitrification biofilm is restored to activity, the time required for the nitrification and denitrification biofilm in R1 to reach the same nitrification rate and denitrification rate is 19 and 17 days, respectively. It takes 8 and 6 days for the rate, respectively, and it takes 13 and 10 days for the nitrification and denitrification biofilm in R3 to reach the same nitrification rate and denitrification rate, respectively.
  • the biofilm thickness L of R1 decreased significantly before and after the recovery of activity, but both of R2 and R3 were able to maintain a high thickness, so that oxygen formed a concentration gradient in the biofilm, which was beneficial to denitrification. It shows that the nitrification and denitrification biofilms after recovery have good denitrification effect. Among them, the nitrification and denitrification biofilms stored at 4 °C have the shortest activity recovery time, which is more suitable for preservation of nitrification and denitrification biofilms.
  • Extracellular polymer is an important factor in the formation of nitrifying and denitrifying biofilms, and the ratio of protein (PN) and polysaccharide (PS) substances (PN / PS) in extracellular polymers is a measure of the structure of nitrifying and denitrifying biofilms. An important indicator of stability. During the restoration of the activity of the nitrifying and denitrifying biofilm, the change of the extracellular polymer PN / PS is shown in Fig. 5.
  • the PN / PS is quite different, and the PN / PS of the nitrifying and denitrifying biofilm in R1 is decreasing, indicating that the stability of the nitrifying and denitrifying biofilm stored at -20 ° C is poor, and it is not suitable to store the nitrifying and denitrifying Biofilm;
  • the PN / PS of the nitrifying and denitrifying biofilm in R3 increased slightly, indicating that the nitrifying and denitrifying biofilm stored at 20 ° C can maintain a stable state before storage;
  • the PN / PS of the nitrifying and denitrifying biofilm in R2 increased significantly It can reach more than 4.2, and tends to be stable, indicating that the stability of the nitrifying and denitrifying biofilm stored at 4 ° C gradually increases after the activity is restored, which is suitable as the storage temperature of the nitrifying and denitrifying biofilm.
  • the fastest recovery of the nitrification and denitrification biofilm corresponds to the higher nitrification rate and denitrification rate, which indicates that the condition of 4 ° C is more suitable for the preservation of the nitrification and denitrification biofilm, and has high feasibility in practical applications.
  • the content of living cells in the nitrifying and denitrifying biofilms at different storage temperatures is basically the same as the content of living cells in the nitrifying and denitrifying biofilms of wastewater treatment plants, indicating that after the restoration of activity, the nitrifying and denitrifying biofilms can play a role in pollutant removal.
  • the proportion of living cells (84.0% ⁇ 3.0%) of the nitrifying and denitrifying biofilm in R2 is the highest, and the proportion of late apoptotic cells (6.2% ⁇ 1.5%) and the proportion of dead cells (4.3% ⁇ 0.3%) are the lowest.
  • the cell activity of the nitrification and denitrification biofilm is the highest, which is more suitable as the conditions for the preservation of the nitrification and denitrification biofilm.
  • the nitrification rate of the nitrifying and denitrifying biofilm has a very high correlation with the denitrifying rate and the proportion of living cells of the nitrifying and denitrifying biofilm.
  • the correlation coefficients are 0.9286 and 0.9819, indicating that the use of The ratio of viable cells in nitrifying and denitrifying biofilms is highly feasible as a method to evaluate the activity of nitrifying and denitrifying biofilms.
  • the proportion of viable cells in the nitrifying and denitrifying biofilms was the highest under the storage condition of 4 ° C, which was in agreement with the results of the proportion of viable cells in the nitrifying and denitrifying biofilms in R2 after the recovery of activity.
  • the flow cytometry can be used as the basis for determining the optimal storage temperature of the nitrifying and denitrifying biofilm.
  • Flow cytometry is easy to operate, the data is fast, easy to obtain, accurate and reliable, and the process of recovering the activity of nitrifying and denitrifying biofilms can be omitted, which is of great significance for the preservation and recovery of nitrifying and denitrifying biofilms.
  • Example 5 Testing of optimal storage temperature for nitrifying and denitrifying biofilms under different pH environments
  • the storage temperature of the nitrifying and denitrifying biofilm was set to -20 ° C, 4 ° C and 20 ° C. 180 of the nitrifying and denitrifying biofilms in the biochemical reaction tank of the sewage treatment plant were taken out, and the average three aliquots were placed in 1000ml serum bottles with 500ml storage matrix.
  • the components of the storage matrix are as follows: NaAc 240mg / L, NH 4 Cl 110 mg / L, KNO 3 80 mg / L, K 2 HPO 4 30 mg / L, KH 2 PO 4 15 mg / L, MgSO 4 40 mg / L, and KCl 70 mg / L.
  • the nitrifying and denitrifying biofilm is used to determine the state of the nitrifying and denitrifying biofilm cells.
  • the conditions of the flow cytometry test are as follows:
  • Table 9 shows the results of the nitrification and denitrification biofilm cell state tests.
  • the inventors also investigated the influence of the filter pore size on the detection of the decontamination substance: the nitrification and denitrification biofilm samples prepared with the pore sizes of 8 ⁇ m and 10 ⁇ m were respectively found.
  • the data is reliable; the corresponding data of 10 ⁇ m is not analytically effective and cannot be used to determine the most suitable storage temperature.

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Abstract

一种确定污水处理生物膜最适保存温度的方法，利用流式细胞术测定生物膜的细胞活性状态，以最接近保存前的细胞活性状态时的保存温度作为最适保存温度。运用该方法，可以省略生物膜工艺的活性恢复步骤，有效缩短生物膜工艺工程化应用启动时间；还公开了一种快速启动生物膜工程的方法及一种污水处理方法。

Description

一种确定污水处理生物膜最适保存温度的方法 技术领域
本发明涉及一种确定污水处理生物膜最适保存温度的方法，属于环境工程技术领域。
背景技术
污水处理厂进水有机污染物含量偏低一直是制约总氮达标排放的技术难点，同时随着污水处理排放标准的提高，大量土地被改扩建工程占用，大量有机碳源物质和除磷药剂被投加到水体中，显著增加了投资建设和运营成本，传统的活性污泥工艺已经不能满足污染物排放的要求，进而严重影响污水处理厂的节能降耗效果。因此，基于节地目标和低碳源利用的污水脱氮技术被日益重视。污水处理中较为有效的污水处理工艺包括好氧颗粒污泥和硝化反硝化生物膜工艺等，其中好氧颗粒污泥工艺具有良好的沉降性能，较低的运行成本，较高的生物量和处理效能而极具发展潜力；硝化反硝化生物膜工艺可实现同步硝化反硝化作用，对污水中氨氮和总氮的去除也具有重要意义。
然而，在工程实例中硝化反硝化生物膜的成膜周期约30～40d，好氧颗粒污泥的培养周期则长达70～120d，且培养条件相对苛刻。如果可以将好氧颗粒污泥和硝化反硝化生物膜培养成熟并予以保存，则可以有效帮助进水碳源较低，土地资源紧张的污水处理厂在较短时间内启动运行，并使污染物达标排放。温度是影响生物膜活性的重要参数，确定最适于好氧颗粒污泥和硝化反硝化生物膜等生物膜保存的温度，有助于简化其活性恢复过程，缩短工程化应用启动时间，实现节能降耗效果。但现有方法对于最适保存温度的确定需要将好氧颗粒污泥和硝化反硝化生物膜重新接种于生物反应器中，约需8～35d才能确定好氧颗粒污泥和硝化反硝化生物膜的活性恢复效果，耗时过长，成为制约其工程化应用的关键。
发明内容
为了简化好氧颗粒污泥和硝化反硝化生物膜等生物膜活性的恢复过程，使污水处理厂污染物指标短时间内达标排放，同时实现节地和节能降耗效果。本发明基于流式细胞术对不同温度条件下保存的生物膜中的细胞活性状态进行表征，并通过生物膜活性恢复后的细胞活性状态和对污染物的去除效果，对流式细胞术的表征结果进行验证，最终建立基于流式细胞术的确定生物膜最适保存温度的方法，为污水处理厂高标准污染物排放与节能降耗运行提供技术支撑。
本发明的第一个目的是提供一种确定污水处理生物膜最适保存温度的方法，所述方法是基于流式细胞术对污水处理生物膜的细胞活性状态进行测定，对比不同温度保存的生物膜与保存前生物膜细胞活性状态的测定结果，以最接近保存前生物膜细胞活性状态的保存温度作 为最适保存温度。
本发明的一种实施方式中，所述生物膜的细胞活性状态测定包括活细胞、凋亡早期细胞、凋亡晚期细胞和死细胞的含量测定。
本发明的一种实施方式中，所述生物膜包括好氧颗粒污泥和硝化反硝化生物膜。
本发明的一种实施方式中，所述流式细胞术确认最适温度的步骤包括：
(1)生物膜测试样液的制备：用缓冲液稀释生物膜样品，震荡均匀后，过滤、离心，留用上清液，使用预冷磷酸盐缓冲液吹洗细胞，重复离心和清洗两次，再取上清液作为样品，使用适量10x Annexin V Binding Buffer混匀制得；
(2)置于流式细胞仪测定各样液的细胞活性状态。
在本发明的一种实施方式中，选用磷酸盐缓冲液作为缓冲液进行稀释。
在本发明的一种实施方式中，所述磷酸盐缓冲液包括磷酸二氢钠和磷酸氢二钠。
在本发明的一种实施方式中，所述生物膜为好氧颗粒污泥时，其测试样液制备是利用pH7.0～8.0的缓冲液稀释好氧颗粒污泥制备得到。
在本发明的一种实施方式中，所述生物膜为硝化反硝化生物膜时，其测试样液制备是利用pH 6.6～7.0的缓冲液稀释硝化反硝化生物膜制备得到。
在本发明的一种实施方式中，所述缓冲液与生物膜的稀释体积比为8～10：1。
在本发明的一种实施方式中，所述生物膜为好氧颗粒污泥时，选用5～15μm孔径的尼龙膜进行过滤。
在本发明的一种实施方式中，所述生物膜为硝化反硝化生物膜时，选用6～8μm孔径的尼龙膜进行过滤。
在本发明的一种实施方式中，所述离心速度为5000～10000rpm/min。
在本发明的一种实施方式中，所述样品清液与10x Annexin V Binding Buffer的混合体积比例为1：2～4。
在本发明的一种实施方式中，所述流式细胞仪测定各样液的细胞活性状态是在对照FITC Annexin V组加入0.5μl PI染色剂，对照PI组加入0.5μl FITC Annexin V，检测组加入0.5μl FITC Annexin V和0.5μl PI，混匀后室温下避光孵育，之后于流式细胞仪上机检测。
在本发明的一种实施方式中，所述孵育时间为10～20min。
本发明的第二个目的是提供一种快速启动污水处理生物膜工程化的方法，所述方法是预先将生物膜培养成熟，置于保存基质中在最适保存温度下进行保存，活性恢复后即可用于污水处理；所述最适温度是利用上述方法确定。
在本发明的一种实施方式中，所述好氧颗粒污泥的保存基质中COD 250～350mg/L，NH 4 +-N 55～65mg/L，PO 4 3--P 6～10mg/L。
在本发明的一种实施方式中，所述好氧颗粒污泥的活性恢复是将好氧颗粒污泥接种于序批式反应器(Sequencing Batch Reactor,SBR)中，SBR有效容积10.0L，排水比45～60％，反应周期为2.5～h，静态进水期1～1.5h，曝气反应期1.5～2.5h，污泥沉降期2～6min，快速排水期2～6min；通过实时控制系统控制空气、氮气含量与比例以保证进水段厌氧状态，反应段好氧状态；SRT控制在25天。
在本发明的一种实施方式中，所述硝化反硝化生物膜的保存基质中COD 180～220mg/L，NH 4 +-N 25～35mg/L，NO 3 -～N 18-25mg/L,PO 4 3-～P 6～10mg/L。
在本发明的一种实施方式中，所述硝化反硝化生物膜的活性恢复是将硝化反硝化生物膜接种于生物反应器中，以缺氧-好氧工艺为基础，HRT设置为10～15h，硝化反硝化填充比为40％～60％，硝化液回流比例为70％～85％。
本发明的第三个目的是将上述方法应用于污水处理中。
本发明有益效果
本发明通过流式细胞术对多种生物膜活细胞，凋亡早期细胞，凋亡晚期细胞和死细胞比例进行表征，数小时内确认最适保存温度，并与生物膜活性恢复过程的特征指标进行关联性分析，建立了基于流式细胞术的确定生物膜最适保存温度的方法。运用此方法，可以省略生物膜活性恢复的步骤，有效帮助准备采用生物膜工艺技术进行污染物(氨氮、总氮、总磷等)达标排放的污水处理厂实现节地和节能降耗运行，同时可以有效缩短生物膜工艺工程化应用启动时间，维持生物膜工艺长期稳定运行，具有很高的工业可行性。
附图说明
图1为好氧颗粒污泥的沉降性能(污泥体积指数SVI)；
图2为好氧颗粒污泥胞外聚合物(PN/PS)的变化；
图3为好氧颗粒污泥总氮(TN)的去除率；
图4为好氧颗粒污泥总磷(TP)的去除率；
图5为硝化反硝化生物膜胞外聚合物PN/PS的变化；
图6为硝化反硝化生物膜氨氮(AN)的去除率；
图7为硝化反硝化生物膜总氮(TN)的去除率。
具体实施方式
本发明污水处理厂污水包括上游各居民区的生活用水和少部分工业废水，进水年平均值为COD 236mg/L，氨氮30.1mg/L，总氮37.8mg/L，总磷4.5mg/L，硝态氮含量低于1.0mg/L。
实施例1：确认好氧颗粒污泥的最适保存温度
好氧颗粒污泥保存条件：
好氧颗粒污泥的保存温度设置为-20℃，4℃和20℃。将好氧颗粒污泥中试装置中的泥水混合物900ml取出，平均三等份分别置于装有500ml保存基质的1000ml血清瓶中，保存基质成分如下所示：NaAc 4200mg/L，NH 4Cl 1100mg/L，K 2HPO 4 370mg/L，KH 2PO 4 140mg/L，MgSO 4 440mg/L，KCl 170mg/L，微量元素液1ml/L；微量元素液成分如下所示：FeCl 3·6H 2O 1.5g/L,H 3BO 3 0.15g/L,CuSO 4·5H 2O 0.03g/L,KI 0.03g/L,MnCl 2·4H 2O 0.12g/L,Na 2MoO 4·2H 2O 0.06g/L,ZnSO 4·7H 2O 0.12g/L,CoCl 2·6H 2O 0.15g/L。保存基质COD 300mg/L，NH 4 +-N 60mg/L，PO 4 3--P 8mg/L。将血清瓶(每个保存温度下设置3个平行样)分别置于-20℃，4℃和20℃，静止遮光保存3个月。
保存的好氧颗粒污泥细胞状态表征：
流式细胞术细胞状态测试条件如下：
(1)分别取10ml中试系统中和各温度下保存3个月的好氧颗粒污泥混合物，用pH为7.2的磷酸盐缓冲液稀释至100ml，于涡旋仪漩涡震荡2min，使污泥破碎为絮体并保证均匀分布；
(2)用10μm孔径的尼龙膜过滤破碎后的样品，取1.5ml置于1.5ml尖底离心管中；
(3)样品于8000rpm/min离心5min；
(4)用移液枪吸取离心后的样品上清液，留下约0.1ml样品，使用预冷磷酸盐缓冲液吹洗细胞，重复离心和清洗两次；
(5)离心后的样品用移液枪吸取上清液，留下约0.1ml样品，使用0.3ml 10x Annexin V Binding Buffer混匀；
(6)对照FITC Annexin V组加入0.5μl PI染色剂，对照PI组加入0.5μl FITC Annexin V，检测组加入0.5μl FITC Annexin V和0.5μl PI，混匀后室温下避光孵育15min，之后于流式细胞仪上机检测。
好氧颗粒污泥细胞状态结果如表1所示，中试系统中好氧颗粒污泥活细胞比例较高，表明中试系统运行效果良好。保存于-20℃的好氧颗粒污泥活细胞含量最低，死细胞含量最高， 表明-20℃不适宜保存好氧颗粒污泥。保存于4℃的好氧颗粒污泥其凋亡晚期细胞和死细胞比例约为25.2％，证明4℃条件可以用于好氧颗粒污泥的保存。但是保存温度为20℃时，好氧颗粒污泥活细胞比例高达68.5％，仅比中试系统中好氧颗粒污泥活细胞比例低20.0％，表明20℃的保存条件更加适合保存好氧颗粒污泥，同时由于控制和维持4℃的低温条件需要消耗更多能源，因此，初步确定20℃为保存好氧颗粒污泥的最适温度。
表1 保存90d的好氧颗粒污泥细胞活性状态(％)
好氧颗粒污泥 活细胞 凋亡早期细胞 凋亡晚期细胞 死细胞
中试系统 85.6±3.5 8.4±1.1 1.8±0.2 4.2±0.3
保存于-20℃ 41.5±1.8 6.0±1.1 4.5±1.0 48.0±2.5
保存于4℃ 56.8±2.2 18.2±1.5 10.1±1.2 15.1±1.5
保存于20℃ 68.5±2.9 14.1±1.3 11.8±1.2 5.6±0.6
实施例2：好氧颗粒污泥测试结果验证
保存的好氧颗粒污泥的活性恢复条件：
取源于不同血清瓶好氧颗粒污泥，接种于序批式反应器(Sequencing Batch Reactor,SBR)中，用以对好氧颗粒污泥进行活性恢复；于-20℃，4℃和20℃保存的好氧颗粒污泥分别置于R1，R2和R3中。SBR有效容积10.0L，排水比50％，反应周期为3h，静态进水期60min，曝气反应期112min，污泥沉降期3min，快速排水期5min。通过实时控制系统控制空气、氮气含量与比例以保证进水段厌氧状态，反应段好氧状态；SRT控制在25天。
活性恢复后好氧颗粒污泥特性：
经过活性恢复后，R1，R2和R3中的好氧颗粒污泥均具有较好的性能。如表2所示，在好氧颗粒污泥活性恢复后，不同保存温度下好氧颗粒污泥密度和粒径与保存之前的好氧颗粒污泥较为接近。虽然不同保存温度下好氧颗粒污泥的生物量(MLSS)仅为保存之前的91％，但是此生物量比保存之后好氧颗粒污泥的生物量平均高37.9％，说明好氧颗粒污泥重新适应环境，生物量稳定增加。通常污水处理厂活性污泥平均反硝化速率和释磷速率分别为3.0mg/gMLSS·h和2.2mg/gMLSS·h，中试装置中驯化好氧颗粒污泥达到相同反硝化速率和释磷速率的时间分别需要25和29d。将保存的好氧颗粒污泥进行活性恢复后，R1中好氧颗粒污泥达到相同反硝化速率和释磷速率的时间分别需要12和13d，R2中好氧颗粒污泥达到相同反硝化速率和释磷速率的时间分别需要10和11d，R3中好氧颗粒污泥达到相同反硝化速率和释磷速率的时间分别需要8和7d。说明经活性恢复后的好氧颗粒污泥均具有较好的脱氮除磷效果，其中保存于20℃条件的好氧颗粒污泥具有最短的活性恢复时间，较为适宜保存好氧 颗粒污泥。
表2 保存和活性恢复后好氧颗粒污泥的性状
Figure PCTCN2018114552-appb-000001
活性恢复后好氧颗粒污泥的沉降性能和稳定性：
在经过活性恢复后，R1，R2和R3中的好氧颗粒污泥均具有较好的沉降性能，如图1所示，在活性恢复的第10d，不同保存温度下的好氧颗粒污泥体积指数SVI均低于50.0ml/g。随后，好氧颗粒污泥SVI略微降低，最终稳定在46.1-47.8ml/g之间。胞外聚合物是好氧颗粒污泥形成的重要因素，而胞外聚合物中蛋白质类(PN)物质和多糖类(PS)物质的比值(PN/PS)是衡量好氧颗粒污泥结构稳定的重要指标。在好氧颗粒污泥活性恢复过程中，其胞外聚合物PN/PS的变化如图2所示。不同保存温度下，PN/PS差别较大，R1中好氧颗粒污泥PN/PS呈降低趋势，表明保存于-20℃条件的好氧颗粒污泥稳定性较差，不适宜保存好氧颗粒污泥；R2中好氧颗粒污泥PN/PS略微升高，表明保存于4℃条件的好氧颗粒污泥可维持保存前的稳定状态；R3中好氧颗粒污泥PN/PS显著升高并趋于稳定，表明保存于20℃条件的好氧颗粒污泥在活性恢复后其稳定性逐渐升高，适宜于作为好氧颗粒污泥的保存温度。
活性恢复后好氧颗粒污泥对污染物去除效能：
经过活性恢复过程后，不同保存温度下的好氧颗粒污泥对总氮和总磷的去除率均逐渐升高(图3和图4)，其对总氮(TN)和总磷(TP)的去除率均超过70％以上。在活性恢复的第10d，R3中的好氧颗粒污泥对TN和TP的去除效果最好，并一直呈现TN和TP去除率稳定升高的趋势，此结果也同表2中R3内好氧颗粒污泥最快恢复较高的反硝化速率和释磷速率相对应，说明20℃的条件较为适宜保存好氧颗粒污泥，在实际应用中具有很高的可行性。
活性恢复后好氧颗粒污泥特性与污泥细胞状态相关性：
在好氧颗粒污泥活性恢复30d后，采用流式细胞术对好氧颗粒污泥细胞状态进行分析(如表3所示)，不同保存温度下的好氧颗粒污泥中活细胞含量与中试系统好氧颗粒污泥中活细胞含量基本一致，说明经过活性恢复后，好氧颗粒污泥均可发挥污染物去除的作用。其中R3内好氧颗粒污泥活细胞比例(86.5％±3.5％)最高，且凋亡晚期细胞比例(3.8％±1.0％)和死细胞比例(3.3％±0.3％)最低，说明20℃的保存条件下好氧颗粒污泥细胞活性最高，较为适宜作为保存好氧颗粒污泥的条件。
表3 活性恢复后(30d)好氧颗粒污泥细胞活性状态(％)
好氧颗粒污泥 活细胞 凋亡早期细胞 凋亡晚期细胞 死细胞
中试系统 86.5±3.9 6.7±1.4 5.8±0.8 1.0±0.2
保存于-20℃ 82.3±3.8 6.5±1.5 6.2±1.3 5.0±0.2
保存于4℃ 83.1±3.2 6.5±1.3 6.3±1.2 4.1±0.3
保存于20℃ 86.5±3.5 6.4±1.3 3.8±1.0 3.3±0.3
依据Correl相关性分析发现，好氧颗粒污泥反硝化速率与释磷速率和好氧颗粒污泥活细胞比例具有极高的相关性(如表4所示)，相关系数分别为0.9940和0.9954，表明利用好氧颗粒污泥活细胞比例作为评价好氧颗粒污泥活性的方法具有极高的可行性。同时，由于保存的好氧颗粒污泥中，20℃的保存条件下好氧颗粒污泥活细胞比例最高，与活性恢复后R3中好氧颗粒污泥活细胞比例结果相吻合。
表4 活性恢复后好氧颗粒污泥特性与细胞活性状态关联性
Figure PCTCN2018114552-appb-000002
因此，确定20℃是保存好氧颗粒污泥的最适条件，利用流式细胞术可以作为确定好氧颗粒污泥最适保存温度的依据。流式细胞术操作简便，数据快速易得且准确可靠，亦可省略好氧颗粒污泥活性恢复过程，对于好氧颗粒污泥的保存与活性恢复具有重要意义。
实施例3：确认硝化反硝化生物膜的最适保存温度
硝化反硝化生物膜保存培养：
硝化反硝化生物膜的保存温度设置为-20℃，4℃和20℃。将污水处理厂生化反应池中的硝化反硝化生物膜取出180个，平均三等份分别置于装有500ml保存基质的1000ml血清瓶中，为保持生物膜的硝化和反硝化能力，保存基质成分如下所示：NaAc 240mg/L，NH 4Cl 110mg/L，KNO 3 80mg/L,K 2HPO 4 30mg/L，KH 2PO4 15mg/L，MgSO 4 40mg/L，KCl 70mg/L。保存基质COD 200mg/L，NH 4 +-N 30mg/L，NO 3 --N 20mg/L,PO 4 3--P 8mg/L。将血清瓶(每个保存温度下设置3个平行样)分别置于-20℃，4℃和20℃，静止遮光保存。
保存的硝化反硝化生物膜细胞状态测试：
于-20℃，4℃和20℃保存的硝化反硝化生物膜在保存超过120d后，用于测定硝化反硝化生物膜细胞状态，流式细胞术细胞状态测试条件如下：
(1)取10ml硝化反硝化生物膜，用pH为7.0的磷酸盐缓冲液稀释至100ml，于涡旋仪漩涡震荡2min，使生物膜破碎为絮体并保证均匀分布；
(2)用6μm孔径的尼龙膜过滤破碎后的样品，取1.5ml置于1.5ml尖底离心管中；
(3)样品于8000rpm/min离心5min；
(4)用移液枪吸取离心后的样品上清液，留下约0.1ml样品，使用预冷磷酸盐缓冲液吹洗细胞，重复离心和清洗两次；
(5)离心后的样品用移液枪吸取上清液，留下约0.1ml样品，使用0.3ml 10x Annexin V Binding Buffer混匀；
(6)对照FITC Annexin V组加入0.5μl PI染色剂，对照PI组加入0.5μl FITC Annexin V，检测组加入0.5μl FITC Annexin V和0.5μl PI，混匀后室温下避光孵育15min，之后于流式细胞仪上机检测。
制备样品是过滤孔径的选择尤为重要，孔径过大，会引入更多生物絮体，造成染色不均匀问题，影响最终结果；孔径过小，又无法有效获得生物絮体。
硝化反硝化生物膜细胞状态测试结果如表5所示。污水处理厂生化反应池中硝化反硝化生物膜活细胞比例较高，表明污水处理厂运行效果良好。保存于-20℃的用于测定硝化反硝化生物膜细胞含量最低，死细胞含量最高，表明-20℃不适宜保存硝化反硝化生物膜。保存于4℃的硝化反硝化生物膜其活细胞比例最高，达到68.0％，凋亡晚期细胞和死细胞比例约为19.8％，证明4℃条件可以用于硝化反硝化生物膜的保存。保存温度为20℃时，硝化反硝化生物膜活细胞比例高达59.4％，仅比4℃保存的硝化反硝化生物膜活细胞比例低12.6％，但其的硝化反硝化生物膜其凋亡晚期细胞和死细胞比例约为31.6％，表明20℃的保存条件亦不适于保存硝化反硝化生物膜。因此，初步确定4℃为保存硝化反硝化生物膜的最适温度。
表5 保存120d的硝化反硝化生物膜细胞活性状态(％)
硝化反硝化生物膜 活细胞 凋亡早期细胞 凋亡晚期细胞 死细胞
生化反应池 85.0±3.2 9.7±1.0 4.5±0.2 0.8±0.2
保存于-20℃ 40.7±2.0 24.6±1.8 23.4±1.5 11.3±1.5
保存于4℃ 68.0±2.9 12.2±1.2 12.4±0.9 7.4±1.2
保存于20℃ 59.4±2.5 13.9±1.1 17.7±1.0 9.0±1.6
实施例4：硝化反硝化生物膜测试结果验证
保存的硝化反硝化生物膜的活性恢复条件：
选用序批式反应器运行方式：取源于不同血清瓶硝化反硝化生物膜，接种于生物反应器(有效容积10.0L)中，用以对硝化反硝化生物膜进行活性恢复。于-20℃，4℃和20℃保存的硝化反硝化生物膜分别置于R1，R2和R3中。生物反应器以anoxic-oxic(AO)工艺为基础，在序批式反应中实现了同步硝化反硝化，HRT设置为12h，硝化反硝化填充比为50％，硝化液回流比例为80％。
活性恢复后硝化反硝化生物膜特性：
经过活性恢复后，R1，R2和R3中的硝化反硝化生物膜均具有较好的性能。如表6所示，在硝化反硝化生物膜活性恢复后，保存于4℃和20℃温度下硝化反硝化生物膜密度和厚度与保存之前的硝化反硝化生物膜较为接近，只有保存在-20℃的硝化反硝化生物膜密度和厚度略有下降。不同保存温度下硝化反硝化生物膜的生物量均有所降低，但经过活性恢复后，4℃和20℃温度下保存的硝化反硝化生物膜的生物量已达到保存前硝化反硝化生物膜的生物量水平，说明硝化反硝化生物膜重新适应环境，生物量稳定增加。通常生物膜在污水处理中的平均硝化速率和反硝化速率分别为4.5和5.0gNO 3 --N/m 2·d，污水处理厂驯化的硝化反硝化生物膜达到相同硝化速率和反硝化速率的时间分别需要25和21d。将保存的硝化反硝化生物膜进行活性恢复后，R1中硝化反硝化生物膜达到相同硝化速率和反硝化速率的时间分别需要19和17d，R2中硝化反硝化生物膜达到相同硝化速率和反硝化速率的时间分别需要8和6d，R3中硝化反硝化生物膜达到相同硝化速率和反硝化速率的时间分别需要13和10d。且R1的生物膜厚度L在活性恢复前后均有明显减小，但在R2、R3中均能够保持较高的厚度，使得氧气在生物膜中形成浓度梯度，有利于反硝化进行。说明经活性恢复后的硝化反硝化生物膜均具有较好的脱氮效果，其中保存于4℃条件的硝化反硝化生物膜具有最短的活性恢复时间，较为适宜保存硝化反硝化生物膜。
表6 保存和活性恢复后硝化反硝化生物膜的性状
Figure PCTCN2018114552-appb-000003
Figure PCTCN2018114552-appb-000004
活性恢复后硝化反硝化生物膜的稳定性：
胞外聚合物是硝化反硝化生物膜形成的重要因素，而胞外聚合物中蛋白质类(PN)物质和多糖类(PS)物质的比值(PN/PS)是衡量硝化反硝化生物膜结构稳定的重要指标。在硝化反硝化生物膜活性恢复过程中，其胞外聚合物PN/PS的变化如图5所示。不同保存温度下，PN/PS差别较大，R1中硝化反硝化生物膜PN/PS呈降低趋势，表明保存于-20℃条件的硝化反硝化生物膜稳定性较差，不适宜保存硝化反硝化生物膜；R3中硝化反硝化生物膜PN/PS略微升高，表明保存于20℃条件的硝化反硝化生物膜可维持保存前的稳定状态；R2中硝化反硝化生物膜PN/PS显著升高，可达4.2以上，并趋于稳定，表明保存于4℃条件的硝化反硝化生物膜在活性恢复后其稳定性逐渐升高，适宜于作为硝化反硝化生物膜的保存温度。
活性恢复后硝化反硝化生物膜对污染物去除效能：
经过活性恢复过程后，不同保存温度下的硝化反硝化生物膜对氨氮和总氮的去除率均逐渐升高(图6和图7)，其对氨氮和总氮的去除率分别超过90％和80％。在活性恢复的第8d，R2中的硝化反硝化生物膜对氨氮和总氮的去除效果最好，并一直呈现氨氮和总氮去除率稳定升高的趋势，此结果也同表6中R2内硝化反硝化生物膜最快恢复较高的硝化速率和反硝化速率相对应，说明4℃的条件较为适宜保存硝化反硝化生物膜，在实际应用中具有很高的可行性。
活性恢复后硝化反硝化生物膜特性与污泥细胞状态相关性：
在硝化反硝化生物膜活性恢复后，采用流式细胞术对硝化反硝化生物膜细胞状态进行分析，结果如表7所示。不同保存温度下的硝化反硝化生物膜中活细胞含量与污水处理厂硝化反硝化生物膜中活细胞含量基本一致，说明经过活性恢复后，硝化反硝化生物膜均可发挥污染物去除的作用。其中R2内硝化反硝化生物膜活细胞比例(84.0％±3.0％)最高，且凋亡晚期细胞比例(6.2％±1.5％)和死细胞比例(4.3％±0.3％)最低，说明4℃的保存条件下硝化反 硝化生物膜细胞活性最高，较为适宜作为保存硝化反硝化生物膜的条件。
表7 活性恢复后硝化反硝化生物膜细胞活性状态(％)
硝化反硝化生物膜 活细胞 凋亡早期细胞 凋亡晚期细胞 死细胞
生化反应池 85.1±3.0 8.9±0.7 5.0±0.2 1.0±0.1
保存于-20℃ 82.5±3.5 6.5±1.8 6.5±1.3 4.5±0.5
保存于4℃ 84.0±3.0 5.5±1.7 6.2±1.5 4.3±0.3
保存于20℃ 83.0±3.1 5.8±1.7 6.8±1.3 4.4±0.7
依据Correl相关性分析发现，如表8所述，硝化反硝化生物膜硝化速率与反硝化速率和硝化反硝化生物膜活细胞比例具有极高的相关性，相关系数分别为0.9286和0.9819，表明利用硝化反硝化生物膜活细胞比例作为评价硝化反硝化生物膜活性的方法具有极高的可行性。同时，由于保存的硝化反硝化生物膜中，4℃的保存条件下硝化反硝化生物膜活细胞比例最高，与活性恢复后R2中硝化反硝化生物膜活细胞比例结果相吻合。
表8 活性恢复后硝化反硝化生物膜特性与细胞活性状态关联性
Figure PCTCN2018114552-appb-000005
因此，确定4℃是保存硝化反硝化生物膜的最适条件，利用流式细胞术可以作为确定硝化反硝化生物膜最适保存温度的依据。流式细胞术操作简便，数据快速易得且准确可靠，亦可省略硝化反硝化生物膜活性恢复过程，对于硝化反硝化生物膜的保存与活性恢复具有重要意义。
实施例5：对不同pH环境下的硝化反硝化生物膜进行最适保存温度测试
硝化反硝化生物膜保存培养：
硝化反硝化生物膜的保存温度设置为-20℃，4℃和20℃。将污水处理厂生化反应池中的硝化反硝化生物膜取出180个，平均三等份分别置于装有500ml保存基质的1000ml血清瓶中，保存基质成分如下所示：NaAc 240mg/L，NH 4Cl 110mg/L，KNO 3 80mg/L,K 2HPO 4 30mg/L，KH 2PO 4 15mg/L，MgSO 4 40mg/L，KCl 70mg/L。保存基质COD 200mg/L，NH 4 +-N 30mg/L，NO 3 --N 20mg/L,PO4 3 --P 8mg/L。将血清瓶(每个保存温度下设置3个平行样)分别置于-20℃，4℃和20℃，静止遮光保存。
保存的硝化反硝化生物膜细胞状态测试：
于-20℃，4℃和20℃保存的硝化反硝化生物膜在保存超过120d后，用于测定硝化反硝化生物膜细胞状态，流式细胞术细胞状态测试条件如下：
(1)取10ml硝化反硝化生物膜，用pH为7.2的磷酸盐缓冲液稀释至100ml，于涡旋仪漩涡震荡2min，使生物膜破碎为絮体并保证均匀分布；
(2)用6μm孔径的尼龙膜过滤破碎后的样品，取1.5ml置于1.5ml尖底离心管中；
(3)样品于8000rpm/min离心5min；
(4)用移液枪吸取离心后的样品上清液，留下约0.1ml样品，使用预冷磷酸盐缓冲液吹洗细胞，重复离心和清洗两次；
(5)离心后的样品用移液枪吸取上清液，留下约0.1ml样品，使用0.3ml 10x Annexin V Binding Buffer混匀；
(6)对照FITC Annexin V组加入0.5μl PI染色剂，对照PI组加入0.5μl FITC Annexin V，检测组加入0.5μl FITC Annexin V和0.5μl PI，混匀后室温下避光孵育15min，之后于流式细胞仪上机检测。
硝化反硝化生物膜细胞状态测试结果如表9所示。
表9 保存120d的硝化反硝化生物膜细胞活性状态(pH 7.2)
硝化反硝化生物膜 活细胞 凋亡早期细胞 凋亡晚期细胞 死细胞
保存于-20℃ 47.0±2.0 14.6±1.5 22.5±1.9 15.9±1.7
保存于4℃ 48.0±2.4 15.1±1.1 23.5±2.0 13.4±1.8
保存于20℃ 49.1±2.1 14.9±1.1 21.7±2.1 14.3±1.1
由表9结果发现，选用pH为7.2的磷酸盐缓冲液制备的样品进行测试，各保存温度生存状态下的细胞状态比例较为接近，无法获得有益结果用于分析，数据可靠性较差。
此外，发明人还考察了过滤孔径对去污物质对样品检测的影响：分别将利用8μm和10μm孔径制得的硝化反硝化生物膜样品，结果发现8μm孔径制备的样品分析结果与验证实验一致，数据可靠；而10μm相应数据不具备分析效力，无法用来确定最适合保存温度。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 一种确定污水处理生物膜最适保存温度的方法，其特征在于，所述方法是基于流式细胞术对污水处理生物膜的细胞活性状态进行测定，对比不同温度保存的生物膜与保存前生物膜细胞活性状态的测定结果，以最接近保存前生物膜细胞活性状态的保存温度作为最适保存温度；所述细胞活性状态测定包括活细胞、凋亡早期细胞、凋亡晚期细胞和死细胞的含量测定。
  2. 根据权利要求1所述方法，其特征在于，所述污水处理生物膜包括好氧颗粒污泥和硝化反硝化生物膜中。
  3. 根据权利要求1所述方法，其特征在于，所述流式细胞术确认最适温度的步骤包括：
    (1)污水处理生物膜测试样液的制备：用缓冲液稀释生物膜样品，震荡均匀后，过滤、离心，留用上清液，使用预冷磷酸盐缓冲液吹洗细胞，重复离心和清洗两次，再取上清液作为样品，使用适量10x Annexin V Binding Buffer混匀制得；
    (2)置于流式细胞仪测定各样液的细胞活性状态。
  4. 根据权利要求3所述方法，其特征在于，所述生物膜为好氧颗粒污泥时，选用5～15μm孔径的尼龙膜进行过滤。
  5. 根据权利要求3所述方法，其特征在于，所述生物膜为硝化反硝化生物膜时，选用6～8μm孔径的尼龙膜进行过滤。
  6. 根据权利要求3所述方法，其特征在于，所述生物膜为好氧颗粒污泥时，是利用pH 7.0～8.0的缓冲液稀释好氧颗粒污泥制备得到测试样液。
  7. 根据权利要求3所述方法，其特征在于，所述生物膜为硝化反硝化生物膜时，是利用pH 6.6～7.0的缓冲液稀释硝化反硝化生物膜制备得到测试样液。
  8. 根据权利要求3所述方法，其特征在于，缓冲液与生物膜的稀释体积比为8～10：1。
  9. 一种快速启动生物膜工程的方法，其特征在于，是利用权利要求1～8任一所述方法确定生物膜的最适保存温度，包括：预先将生物膜培养成熟，然后将其置于保存基质中在最适保存温度下进行保存，活性恢复后即可进行工程化应用。
  10. 根据权利要求9所述方法，其特征在于，所述好氧颗粒污泥的保存基质中COD 250～350mg/L，NH 4 +-N 55～65mg/L，PO 4 3--P 6～10mg/L。
  11. 根据权利要求9或10所述方法，其特征在于，所述好氧颗粒污泥的活性恢复是将好氧颗粒污泥接种于序批式反应器中，排水比45～60％，反应周期为2.5～4h，静态进水期1～1.5h，曝气反应期1.5～2.5h，污泥沉降期2～6min，快速排水期2～6min。
  12. 根据权利要求11所述方法，其特征在于，所述序批式反应器通过实时控制系统控制空气、氮气含量与比例以保证进水段厌氧状态，反应段好氧状态。
  13. 根据权利要求9所述方法，其特征在于，所述硝化反硝化生物膜的保存基质中COD180～220mg/L，NH 4 +-N 25～35mg/L，NO 3 --N 18～25mg/L,PO 4 3--P 6～10mg/L。
  14. 根据权利要求9或13所述方法，其特征在于，所述硝化反硝化生物膜的活性恢复是将硝化反硝化生物膜接种于生物反应器中，以缺氧-好氧工艺为基础，设置时间为10～15h，硝化反硝化生物膜填充比为40％～60％，硝化液回流比例为70％～85％。
  15. 一种污水处理方法，其特征在于，是利用权利要求9所述方法。
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