Method for immobilizing salt-tolerant nitrifying bacteria

Abstract

The invention belongs to the technical field of salt-tolerant nitrifying bacteria biological membrane preparation, and particularly relates to a salt-tolerant nitrifying bacteria immobilization method. The method comprises the following steps: concentrating the salt-tolerant nitrifying bacteria sludge, sucking the concentrated sludge into a polyurethane sponge filler, and standing at room temperature to realize drying and water loss, so that the concentration of alginate-like exopolysaccharide ALE and the concentration of calcium ions in the sludge are improved, and a cross-linking effect is promoted to quickly form a salt-tolerant nitrifying bacteria biological membrane; due to the interception effect of the sponge carrier, the loss of the sludge is synergistically reduced, and the immobilization rate of the salt-resistant nitrifying bacteria biological membrane is improved. Compared with the traditional microbial immobilization technology, the scheme provided by the invention has the characteristics of short time, simplicity in operation, high stability and high immobilization rate.

Description

Technical Field

[0001] The invention belongs to the technical field of salt-tolerant nitrifying bacteria biofilm preparation, and in particular relates to a salt-tolerant nitrifying bacteria immobilization method. Background Art

[0002] With the improvement of living standards, people's demand for seafood is also increasing year by year. Although the marine aquaculture industry has developed rapidly and brought huge economic benefits to society, it has also brought significant impacts on the environment. In the large-scale marine aquaculture process, uneaten feed, dead bodies of aquatic organisms, and feces produced by metabolism and other wastes will produce ammonia nitrogen (NH4 + -N) and nitrite nitrogen (NO2 - -N).

[0003] Biofilm of salt-tolerant nitrifying bacteria is an important factor in controlling NH4 in marine aquaculture + -N and NO2 - -N's main microbial technology. Salt-tolerant nitrifying bacteria can form biofilms through microbial immobilization technology. This technology refers to fixing microorganisms in a specific space by chemical or physical means to keep them active and reused repeatedly. Salt-tolerant nitrifying bacteria can achieve microbial immobilization through natural aggregation and embedding methods. Natural aggregation refers to the natural attachment of microorganisms to a carrier, slow growth, and enrichment to form a biofilm. The biofilm has the characteristics of stable community structure, strong resistance to pH fluctuations, and strong fixation, but it takes a long time. It takes about 21 to 124 days from the formation of the microbial film to the maturity of the biofilm. The embedding method uses a gelling agent to fix the microorganisms on the carrier to achieve microbial immobilization. This method requires a short time and has a high microbial density, but the physical structure stability is poor. The gel is prone to swelling during use, damaging the microorganisms. Summary of the invention

[0004] Based on the deficiencies of the above-mentioned technologies, the object of the present invention is to provide a method for immobilizing salt-tolerant nitrifying bacteria, which is not only simple to operate and highly efficient, but also has a biofilm with high activity, high stability and high solidification rate.

[0005] The present invention provides a method for preparing a salt-tolerant nitrifying bacteria biofilm, comprising the following steps:

[0006] The salt-tolerant nitrifying bacteria sludge is concentrated to obtain concentrated sludge;

[0007] Adsorbing the concentrated sludge into a sponge carrier to obtain an adsorbed sponge carrier;

[0008] The adsorbed sponge carrier is placed to dry at room temperature to fix the salt-tolerant nitrifying bacteria;

[0009] The salinity of the salt-tolerant nitrifying bacteria sludge is 25‰ to 30‰.

[0010] Preferably, the concentration of the salt-tolerant nitrifying bacteria sludge is 700-1000 mg / L.

[0011] Preferably, the static drying time is 1 to 5 hours; the room temperature is 26 to 30°C.

[0012] Preferably, the concentration process includes:

[0013] The salt-tolerant nitrifying bacteria sludge is subjected to a first centrifugation to obtain a first centrifuge;

[0014] re-dissolving the first centrifugal material and performing a second centrifugation to obtain a second centrifugal material;

[0015] re-dissolving the second centrifugal material and then performing a third centrifugation to obtain a third centrifugal material;

[0016] The third centrifugal product is redissolved and then subjected to a fourth centrifugation to obtain the concentrated sludge;

[0017] The solvent used for the re-dissolution is saline; the salinity of the saline is 25‰ to 30‰.

[0018] Preferably, during re-dissolution, the amount of the solvent used is calculated based on the volume of the salt-tolerant nitrifying bacteria sludge, and the volume ratio of the salt-tolerant nitrifying bacteria sludge to the solvent is 1:1 to 1.25.

[0019] Preferably, the time of the first centrifugation, the second centrifugation, the third centrifugation and the fourth centrifugation are all 4 to 6 minutes, and the speed is all 4000 to 4500 rpm.

[0020] Preferably, the sponge comprises polyurethane sponge filler.

[0021] The present invention provides application of the salt-tolerant nitrifying bacteria immobilization method described in the above technical solution in immobilizing salt-tolerant nitrifying bacteria and / or preparing salt-tolerant nitrifying bacteria biofilm.

[0022] Beneficial effects:

[0023] The present invention performs a concentration treatment on salt-tolerant nitrifying bacteria sludge with a salinity of 25‰ to 30‰, adsorbs it to a sponge carrier, and performs a standing drying treatment at room temperature, thereby immobilizing the salt-tolerant nitrifying bacteria. During the fixation process, drying and water loss are achieved by standing at room temperature, and the reduction of water content increases the concentration of alginate-like exopolysaccharides (ALE) and calcium ion concentration in the sludge. Since ALE has a high amount of polyguluronic acid (G) blocks, it can form a rigid and non-deformable gel by cross-linking with calcium ions, thus playing an important role in the formation of biofilms; in addition, the interception effect of the sponge carrier itself synergistically reduces the loss of sludge and improves the immobilization rate of the salt-tolerant nitrifying bacteria biofilm.

[0024] Compared with traditional microbial immobilization technology, the advantages of the present invention are:

[0025] 1) The film-forming time is short. After the concentrated sludge containing salt-tolerant nitrifying bacteria is sucked into the sponge, it only takes 1 hour to dry to form a biofilm. 2) The polyurethane sponge has high physical stability and will not expand. During the drying process, no gelling agent or coagulant is used to fix the sludge. 3) The operation is simple and fast, and is less affected by external factors. It only needs to suck the sludge containing salt-tolerant nitrifying bacteria into the sponge and dry it.

[0026] Based on the above technical advantages, the present invention also provides the application of the salt-tolerant nitrifying bacteria immobilization method described in the above technical solution in immobilizing salt-tolerant nitrifying bacteria and / or preparing salt-tolerant nitrifying bacteria biofilm. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0028] Figure 1 A flow chart of the method for immobilizing salt-tolerant nitrifying bacteria provided by the present invention;

[0029] Figure 2 The biofilm characteristics of salt-tolerant nitrifying bacteria prepared at different drying times provided by the present invention;

[0030] Figure 3 A schematic diagram of a reactor inoculated with a salt-tolerant nitrifying bacteria biofilm provided by the present invention;

[0031] Figure 4 The present invention provides an operating effect of a reactor inoculated with a salt-tolerant nitrifying bacteria biofilm. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments.

[0033] In the present invention, unless otherwise specified, the materials and methods used are conventional methods.

[0034] In the present invention, there are no special requirements for the specifications and adsorption methods of the sponge carrier, as long as the concentrated sludge can be completely adsorbed in the carrier.

[0035] Example 1

[0036] A method for immobilizing salt-tolerant nitrifying bacteria (see process Figure 1 ), the steps are:

[0037] 1) Sample: Prepare salt-tolerant nitrifying bacteria sludge with a salinity of 30‰ and a concentration of 820 mg / L;

[0038] Take 300g NaCl and dissolve it in 1000mL water to prepare a brine with a salinity of 30‰;

[0039] 2) Sample pretreatment: Take 40 mL of the sludge in step 1), put it into a 50 mL centrifuge tube, centrifuge at 4500 rpm for 5 min, pour out the supernatant after centrifugation to obtain a centrifuge; add 40 mL of saline with a salinity of 30‰ to the centrifuge and centrifuge for a second time (centrifuge at 4500 rpm for 5 min) to obtain a second centrifuge; add 40 mL of saline with a salinity of 30‰ to the second centrifuge and centrifuge for a third time (centrifuge at 4500 rpm for 5 min) to obtain a third centrifuge; add 40 mL of saline with a salinity of 30‰ to the third centrifuge and centrifuge for a fourth time (centrifuge at 4500 rpm for 5 min) to obtain concentrated sludge;

[0040] 3) Inhaling concentrated sludge containing salt-tolerant nitrifying bacteria: by repeatedly squeezing and loosening a polyurethane sponge (the specification of the polyurethane sponge is 20 mm×20 mm×20 mm), all the concentrated sludge in step 2) is absorbed into the polyurethane sponge to obtain a sponge carrier for sludge absorption;

[0041] 4) Drying: The sponge that absorbs the sludge in step 3) is placed indoors (the indoor temperature is 26-30°C) for static drying treatment to achieve the immobilization of the salt-tolerant nitrifying bacteria. During the immobilization process, the salt-tolerant nitrifying bacteria biofilm dried for 1 hour (recorded as 1 hour), the salt-tolerant nitrifying bacteria biofilm dried for 2 hours (recorded as 2 hours), the salt-tolerant nitrifying bacteria biofilm dried for 3 hours (recorded as 3 hours), the salt-tolerant nitrifying bacteria biofilm dried for 4 hours (recorded as 4 hours) and the salt-tolerant nitrifying bacteria biofilm dried for 5 hours (recorded as 5 hours) are obtained.

[0042] The five salt-tolerant nitrifying bacteria biofilms prepared in Example 1 were taken for 1 h, 2 h, 3 h, 4 h and 5 h, and the AOB activity, NOB activity, immobilization rate and salinity change of the different biofilms were tested. The results are shown in Figure 2 .

[0043] Figure 2 The results showed that salt-tolerant nitrifying bacteria sludge with an initial salinity of 30‰ could form a salt-tolerant nitrifying bacteria biofilm after 1 hour of drying and dehydration. The biofilm formed after 1 hour of drying had a specific ammonia oxidation rate of 10.20 mg N / (g MLSS h) for AOB and a specific nitrite oxidation rate of 9.60 mg N / (g MLSS h) for NOB. In addition, the biofilm had an immobilization rate of 92.19% when subjected to shear force at 100 rpm on a shaker for 24 hours.

[0044] Embodiment 2:

[0045] Inoculate the biofilm of salt-tolerant nitrifying bacteria into the reactor and run it for 14 days to observe the operation effect of the reactor and the stability of the biofilm. The steps are as follows:

[0046] Preparation: Artificial water is used as raw water for reactor operation. The raw water uses water as solvent and also includes the following solute components: 0.10g MgSO4·7H2O, 0.42g KH2PO4, 0.40g CaCl2, 12g NaHCO3, 1.11g Na2HPO4·12H2O, 0.01g FeCl3·6H2O, 0.38g NH4Cl (10mg / L NH + 4-N) and 300 g of NaCl (30‰ salinity).

[0047] A total of 2 L of salt-tolerant nitrifying bacteria sludge (concentration of 935 mg / L, salinity of 30‰) was taken to prepare a salt-tolerant nitrifying bacteria biofilm. The specific preparation process was the same as the preparation method of the salt-tolerant nitrifying bacteria biofilm in Example 1, thereby obtaining a salt-tolerant nitrifying bacteria biofilm dried for 1 hour;

[0048] Inoculate salt-tolerant nitrifying bacteria biofilm into the reactor. The schematic diagram of the reactor is as follows Figure 3 As shown, the inlet ammonia nitrogen concentration of the reactor is 10 mg / L, the working volume of the reactor is 2 L, the sponge filling rate is 9.25%, the hydraulic retention time is 2 h, the dissolved oxygen concentration is >5 mg / L, and the pH value is 8.0-8.5.

[0049] The sludge discharged from the salt-tolerant nitrifying bacteria biofilm reactor was collected. When the water bucket was drained every day, the bucket was left to stand for one hour, and then the supernatant was filtered with two layers of 400-mesh filters. The filter was repeatedly rinsed with salt water with a salinity of 30‰, so that the sludge attached to the filter was washed back into the water bucket. All the sludge discharged from the reactor within 14 days was collected and its total mass was measured.

[0050] Combination Figure 4 After the operation of the reactor, it was found that after the biofilm provided by the present invention was inoculated, the reactor could be successfully started on the 5th day of operation. + -N concentration is 10mg / L, the effluent NH4 + -N concentration and NO2 - -N concentration is less than 0.5mg / L, which meets the water quality requirements for the survival of white shrimp. Stable NH4 + -N and NO2 - -N control, all meet the water quality requirements for the survival of white shrimp.

[0051] According to statistics, after 14 days of operation, the reactor still retained 99.75% of the sludge, which shows that the reactor has a good sludge retention effect. In addition, the biofilm is highly stable, as shown by the fact that after 14 days of operation, the biofilm shedding rate under the shear force of the shaker is only 0.00% to 2.29%.

[0052] In summary, the technical solution provided by the present invention can quickly form a salt-tolerant nitrifying bacteria biofilm only through the drying and dehydration immobilization technology, which is simple to operate and saves time and cost; in addition, the biofilm provided by the present invention can retain the activity of AOB and NOB, and the reactor can be started in only 5 days after inoculation into the reactor, so that its effluent meets the water quality requirements for the survival of white shrimp. Therefore, the technical solution provided by the present invention not only quickly starts the reactor, but also has a good sludge retention effect in the reactor.

[0053] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for immobilizing salt-tolerant nitrifying bacteria, characterized in that: The following steps are involved: The salt-tolerant nitrifying bacteria sludge is concentrated to obtain concentrated sludge; Adsorbing the concentrated sludge into a sponge carrier to obtain an adsorbed sponge carrier; The adsorbed sponge carrier is placed to dry at room temperature to fix the salt-tolerant nitrifying bacteria; The salinity of the salt-tolerant nitrifying bacteria sludge is 25‰ to 30‰.

2. The method for immobilizing salt-tolerant nitrifying bacteria according to claim 1, characterized in that: The concentration of the salt-tolerant nitrifying bacteria sludge is 700-1000 mg / L.

3. The method for immobilizing salt-tolerant nitrifying bacteria according to claim 1, characterized in that: The static drying time is 1 to 5 hours; the room temperature is 26 to 30°C.

4. The method for immobilizing salt-tolerant nitrifying bacteria according to claim 1, characterized in that: The concentration process includes: The salt-tolerant nitrifying bacteria sludge is subjected to a first centrifugation to obtain a first centrifuge; re-dissolving the first centrifugal material and performing a second centrifugation to obtain a second centrifugal material; re-dissolving the second centrifugal material and then performing a third centrifugation to obtain a third centrifugal material; The third centrifugal product is redissolved and then subjected to a fourth centrifugation to obtain the concentrated sludge; The solvent used for the re-dissolution is saline; the salinity of the saline is 25‰ to 30‰.

5. The method for immobilizing salt-tolerant nitrifying bacteria according to claim 4, characterized in that: During re-dissolution, the amount of the solvent used is calculated based on the volume of the salt-tolerant nitrifying bacteria sludge, and the volume ratio of the salt-tolerant nitrifying bacteria sludge to the solvent is 1:1 to 1.

25.

6. The method for immobilizing salt-tolerant nitrifying bacteria according to claim 4, characterized in that: The time of the first centrifugation, the second centrifugation, the third centrifugation and the fourth centrifugation are all 4 to 6 minutes, and the speed is all 4000 to 4500 rpm.

7. The method for immobilizing salt-tolerant nitrifying bacteria according to claim 1, characterized in that: The sponge includes polyurethane sponge filler.

8. Use of the method for immobilizing salt-tolerant nitrifying bacteria according to any one of claims 1 to 7 in immobilizing salt-tolerant nitrifying bacteria and / or preparing salt-tolerant nitrifying bacteria biofilm.

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