Method for high-density fermentation of thiobacillus denitrificans by using nitrifying bacteria fermentation waste liquid

Abstract

The invention relates to the technical field of microbial fermentation, and provides a method for high-density fermentation of thiobacillus denitrificans by using nitrifying bacteria fermentation waste liquid, which comprises the following steps: adding the nitrifying bacteria fermentation waste liquid and an initial nutrient solution into a bioreactor, inoculating thiobacillus denitrificans SZG-SAD-004, and fermenting in an anaerobic environment; the preservation number of the thiobacillus denitrificans SZG-SAD-004 is CCTCC (China Center for Type Culture Collection) M 2023992; the nitrifying bacteria fermentation waste liquid is waste liquid generated after co-fermentation of nitrite bacteria, nitrifying bacteria and denitrifying bacteria. When autotrophic nitrifying bacteria are produced through a microbial fermentation method, waste liquid containing high-concentration nitrate, inorganic salt and metal ions can be generated, the waste liquid needs to be specially treated to reach the emission standard, and the enterprise cost is increased. The thiobacillus denitrificans SZG-SAD-004 disclosed by the invention can directly utilize the waste liquid to perform high-density fermentation, additional treatment is not needed, and resource utilization of the waste liquid is realized.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method for high-density fermentation of denitrifying thiobacillus using nitrifying bacteria fermentation waste liquid. Background Art

[0002] Sulfur autotrophic denitrification technology does not require the addition of carbon sources, has low sludge production, low energy consumption, and no secondary pollution. Especially when treating low C / N sewage, it has become one of the best choices to replace traditional heterotrophic denitrification processes. The core of sulfur autotrophic denitrification reaction and simultaneous denitrification and desulfurization is its functional flora, namely denitrifying and desulfurizing bacteria (NR-SOB). Therefore, obtaining high-efficiency and high-activity functional flora through bioaugmentation and other means is of great significance for the development of sulfur autotrophic denitrification process.

[0003] The NR-SOB that have been found to be more numerous include denitrifying Thiobacillus, denitrifying Thiomonas and pantrophic Ryukococcus. Among them, denitrifying Thiobacillus is a typical autotrophic sulfur-oxidizing denitrifying bacterium, and given the characteristics of this bacterium, denitrifying Thiobacillus can coexist with SRB in the same environment and oxidize its metabolite H2S into S and SO4 2- , thereby reducing the corrosion of pipes; the bacteria can also be used for natural gas desulfurization of hydrogen, and microbial desulfurization has the advantages of low investment, low cost, mild reaction conditions, and less pollution. At present, denitrifying thiobacillus is mostly found in laboratory shake flask culture, and it needs to be filled with CO2 or N2 to keep it in an anaerobic state. The culture time is long and the bacterial concentration is low, which is not conducive to mass production and application. For example, the invention patent with patent number CN103773706A uses an airlift bioreactor to cultivate denitrifying thiobacillus under aeration conditions, which is applied to the microbial desulfurization of sulfur-containing industrial waste gas. However, after several consecutive aerobic subcultures, the ability of denitrifying thiobacillus to grow anaerobically in nitrate solution will be lost. The invention patent with patent number CN110818093A uses secondary treated water to adjust the nitrate content, and inoculates anoxic sludge to culture denitrifying thiobacillus in a micro-aerobic environment. The nitrate required is an explosive substance and is strictly controlled. However, the nitrate concentration contained in the secondary water of the sewage treatment plant is low, which is difficult to meet its high-density fermentation.

[0004] Autotrophic nitrifying bacteria are produced by microbial fermentation, and a large amount of waste liquid will be generated during the production process. Since the tail liquid of the nitrifying bacteria fermentation liquid contains high concentrations of nitrates, inorganic salts and metal ions, the waste liquid needs to be processed by a professional company to meet the discharge requirements, increasing the cost of the production enterprise. The nitrifying bacteria fermentation waste liquid can be turned into treasure, and resources are recycled. The invention patent No. CN116143343A discloses a method for treating nitrifying bacteria waste liquid and can be used for sulfur autotrophic denitrifying bacteria fermentation, but the method requires a large amount of acid to adjust the pH during the treatment process, and then adds flocculants, iron-containing materials, catalysts, and carbon-containing materials, and the nitrifying bacteria fermentation waste liquid is aerated, and the process is relatively complicated and increases energy consumption. Therefore, a method for high-density fermentation that can directly utilize nitrifying bacteria fermentation waste liquid is needed. Summary of the invention

[0005] In view of this, the present invention proposes a method for directly utilizing nitrifying bacteria fermentation wastewater for high-density fermentation of Thiobacillus denitrificans without additional treatment.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a method for high-density fermentation of denitrifying thiobacillus using nitrifying bacteria fermentation waste liquid, comprising the following steps:

[0007] The nitrifying bacteria fermentation waste liquid and the initial nutrient solution are added into the bioreactor, and denitrifying Thiobacillus SZG-SAD-004 is inoculated, and stirred and fermented under anaerobic environment; the deposit number of the denitrifying Thiobacillus SZG-SAD-004 is CCTCC M2023992.

[0008] On the basis of the above technical scheme, preferably, the NO3-N concentration in the nitrifying bacteria fermentation waste liquid is controlled to be 200-700 mg / L; the initial nutrient solution components are: NH4Cl 0.5-1g / L, MgCl2·6H2O0.5-1.5g / L, KH2PO43-5g / L, Na2S2O3·5H2O 8-12g / L, NaHCO31-3g / L, FeSO4·7H2O0.005-0.015g / L, trace elements 0.5-1.5mL / L, and pH is 6.5-8.0.

[0009] On the basis of the above technical scheme, preferably, the trace element components are: Na2-EDTA 40-60 g / L, CaCl2·2H2O 6-8 g / L, FeSO4·7H2O 4-6 g / L, MnCl2·4H2O 2-3 g / L, ZnSO4·7H2O2-3 g / L, (NH4)6Mo7O2·4H2O 0.4-0.6 g / L, CaSO4·5H2O 0.1-0.3 g / L and NaOH 9-12 g / L.

[0010] On the basis of the above technical solution, preferably, the inoculation amount of Thiobacillus denitrificans SZG-SAD-004 is 8-12 v / v%.

[0011] On the basis of the above technical scheme, preferably, before fermentation, the carrier, nitrifying bacteria fermentation waste liquid and initial nutrient solution are added into the bioreactor together, and the carrier is one or more combinations of zeolite powder, mica powder, kaolin and calcium carbonate.

[0012] Based on the above technical solution, preferably, the added amount of the carrier is 1-2 g / L.

[0013] On the basis of the above technical solution, preferably, during fermentation, the dissolved oxygen concentration in the bioreactor is less than 0.5 mg / L, the temperature is 28-32° C., the rotation speed is 30-70 rpm, and the pH value is 7.0-7.5.

[0014] On the basis of the above technical solution, preferably, the volume ratio of nitrifying bacteria fermentation waste liquid: initial nutrient solution is 70-230:1000.

[0015] On the basis of the above technical solution, preferably, feed is added regularly during fermentation to maintain salinity ≤40000 mg / L.

[0016] On the basis of the above technical scheme, preferably, when supplementing, except for the amount of Na2S2O3·5H2O and NaHCO3 in the supplementary nutrient solution which is the same as that of the initial nutrient solution, the other components are 15%-25% of the concentration of the initial nutrient solution, and the concentration of nitrate in the nitrifying bacteria fermentation waste liquid added during supplementing is 180-220 mg / L.

[0017] The method of the present invention for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid has the following beneficial effects compared with the prior art:

[0018] (1) The nitrifying bacteria fermentation waste liquid contains more than 3000 mg / L of nitrate, and also contains elements such as phosphorus, iron, copper, and zinc, which can provide the required nutrition for denitrifying Thiobacillus, and can fully meet the industrial fermentation production of denitrifying Thiobacillus. The present invention utilizes the nitrifying bacteria fermentation waste liquid as a nitrate source to cultivate denitrifying Thiobacillus SZG-SAD-004, realizes the recycling of nitrifying bacteria fermentation waste liquid resources, and reduces the cost of hazardous waste treatment.

[0019] (2) Adding carrier zeolite powder, mica powder, kaolin, and calcium carbonate minerals into the bioreactor can prevent the denitrifying Thiobacillus SZG-SAD-004 from agglomerating, increase the consumption of nitrate by the denitrifying Thiobacillus SZG-SAD-004, and promote the high-density fermentation of the denitrifying Thiobacillus SZG-SAD-004.

[0020] (3) During the fermentation process, the salt content in the bioreactor was monitored and fresh nutrient solution was regularly added to promote high-density fermentation of denitrifying Thiobacillus SZG-SAD-004. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The denitrifying Thiobacillus of the present invention is the denitrifying Thiobacillus (Thiobacillus denitrificans) SZG-SAD-004 screened and preserved by Mizunoku, which was deposited in China Center for Type Culture Collection (CCTCC) on June 12, 2023, at Wuhan University, Wuhan, China, with a deposit number of CCTCC M 2023992, and was identified as alive on June 19, 2023. The bacterium is Gram-negative and short rod-shaped under an optical microscope, arranged singly, in pairs or in short chains; it is an obligate inorganic chemoautotrophic type, and can consume thiosulfate and S under aerobic and anaerobic conditions. 0 , S 2- It is used as an energy source for growth. When cultured on a solid plate in a 28°C constant temperature incubator, colonies appear on the surface of the culture medium. They are white, smooth, and round, with a diameter of 0.2 to 1 mm.

[0023] The nitrifying bacteria fermentation waste liquid used in the present invention is the supernatant produced in the process of mixing and fermenting European nitrosomonas AT7 (Nitrosomonas europaeaAT7) and European nitrosomonas C-31 (Nitrosomonas europaea C-31) in a ratio of 1:1, which is overflowed into the nitrifying bacteria tube tank, and the supernatant in the process of culturing Nitrobacter WinogradskyiY3-2 (Nitrobacter WinogradskyiY3-2) in the nitrifying bacteria tank is used. For details, see the fermentation method disclosed in CN108424862A, European nitrosomonas AT7 and European nitrosomonas C-31 convert ammonia nitrogen into nitrite, and the nitrifying bacteria Nitrobacter Winogradskyi converts nitrite into nitrate.

[0024] When European Nitrosomonas AT7 and European Nitrosomonas C-31 are fermented, the fermentation medium components are: 0.2-0.8 g / L of ammonium sulfate, 0.2-0.8 g / L of sodium chloride, 0.01-0.05 g / L of ferrous sulfate, 0.5-2 g / L of sodium dihydrogen phosphate, 0.01-0.05 g / L of magnesium sulfate heptahydrate, and 5-10 g / L of calcium chloride; the components of the metal salt promoter are: 0.1wt% MnCl2·4H2O, 0.1wt% Na2B4O7·10H2O, 0.1wt% ZnSO4·7H2O, 0.1wt% EDTA-Fe, 0.1wt% CoSO4·7H2O, 0.1wt% CuSO4·5H2O, and 0.1wt% NiSO4·6H2O.

[0025] The nitrite-transforming bacteria tank is inoculated with 10% (v / v) nitrite-transforming bacteria, and during the culture process, sodium carbonate solution is added to adjust the pH to 7.5-8.0, the temperature is controlled at 28-30°C, aeration is performed intermittently, aeration is performed for 3 hours, and then stopped for 1 hour, and the dissolved oxygen is controlled at 2-5 mg / L. The culture time is 5-7 days. When the ammonia nitrogen in the culture medium no longer decreases, the cultured nitrite-transforming bacteria are obtained, and the ammonia nitrogen removal rate reaches 92.3%. The bacteria are separated by standing, and the supernatant overflows into the nitrifying bacteria tank.

[0026] The nitrifying bacteria tank is inoculated with 10% (v / v) of the nitrifying bacteria Y3-2. During the culture process, a sodium carbonate solution is added to adjust the pH to 7.5-8.0, the temperature is controlled at 28-30°C, aeration is performed intermittently, aeration is performed for 3 hours, and then stopped for 1 hour. The dissolved oxygen is controlled at 2-5 mg / L, and the culture time is 5-7 days. When the nitrite in the culture medium is consumed, the cultured nitrifying bacteria are obtained; the bacteria are separated by standing, and the supernatant overflows to obtain the nitrifying bacteria fermentation waste liquid used in the present invention.

[0027] When the nitrifying bacteria Y3-2 of Wisconsin are fermented, the components of the fermentation medium are: 1.5g / L sodium nitrite, 0.03g / L magnesium sulfate heptahydrate, 0.01g / L manganese sulfate, 0.8g / L dipotassium hydrogen phosphate, 0.15g / L anhydrous sodium carbonate, 0.13g / L sodium dihydrogen phosphate, and 10mL / L metal salt promoter; the components of the metal salt promoter are: 0.1wt% MnCl2·4H2O, 0.1wt% Na2B4O7·10H2O, 0.1wt% ZnSO4·7H2O, 0.1wt% EDTA-Fe, 0.1wt% CoSO4·7H2O, 0.1wt% CuSO4·5H2O, and 0.1wt% NiSO4·6H2O.

[0028] The supernatant overflowed during the fermentation of the Vernix nitrifying bacteria Y3-2 contains more than 3000 mg / L of nitrate, and also contains phosphorus, iron, copper, zinc and other elements that can provide the necessary nutrients for denitrifying Thiobacillus. When used, the fermentation waste liquid is diluted to a usable concentration.

[0029] The reactor for cultivating high-density denitrifying thiobacillus of the present invention is a conventional stirring aeration reactor, wherein a bacterial suspension feeding port, a nutrient solution feeding port and an exhaust gas discharge port are arranged at the upper part of the reactor, a temperature probe, a pH probe and a dissolved oxygen probe are arranged inside the reactor for monitoring temperature, pH value and dissolved oxygen; an air inlet / liquid port, an air aeration pipe and a nutrient solution waste liquid outlet are arranged at the lower part of the reactor, and the air inlet / liquid port and the air aeration pipe are used to adjust the dissolved oxygen content. The temperature probe, the pH probe and the dissolved oxygen probe are operated and controlled by an electric control device.

[0030] Example 1

[0031] The method of high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater in this embodiment comprises the following steps:

[0032] S1, nitrifying bacteria fermentation wastewater was diluted to a NO3-N concentration of 560 mg / L;

[0033] S2, prepare the initial nutrient solution: NH4Cl 0.8g / L, MgCl2·6H2O 1g / L, KH2PO44g / L, Na2S2O3·5H2O10g / L, NaHCO32g / L, FeSO4·7H2O 0.01g / L, 1.0mL / L trace elements. The trace element components are: Na2-EDTA50g / L, CaCl2·2H2O 7.34g / L, FeSO4·7H2O 5.0g / L, MnCl2·4H2O 2.5g / L, ZnSO4·7H2O2.2g / L, (NH4)6Mo7O2·4H2O 0.5g / L, CaSO4·5H2O0.2g / L and NaOH 11.0g / L. The pH of the initial nutrient solution was finally adjusted to 7.3, and the nutrient solution was sterilized before use, with a sterilization temperature of 121°C, a pressure of 0.12 MPa, and a sterilization time of 30 min.

[0034] S3, take 0.5L of nitrifying bacteria fermentation waste liquid and 2.5L of initial nutrient solution and add them into the bioreactor, inoculate denitrifying Thiobacillus SZG-SAD-004 at an inoculation rate of 10v / v%, and perform anaerobically fermentation under the conditions of dissolved oxygen concentration <0.5mg / L, temperature of 28°C, and pH value of 7.3, and detect nitrate consumption at regular intervals.

[0035] Example 2

[0036] The difference between Example 2 and Example 1 is that 2 g / L of carrier (zeolite powder, kaolin, mica powder, calcium carbonate) is added during fermentation, and the nitrifying bacteria fermentation waste liquid and initial nutrient solution are the same as those in Example 1.

[0037] The fermentation method is as follows: 0.5 L of nitrifying bacteria fermentation waste liquid and 2.5 L of initial nutrient solution are added into a bioreactor, 6 g of carrier (zeolite powder, kaolin, mica powder, calcium carbonate) is added, and no carrier is added as a control (CK), and denitrifying Thiobacillus SZG-SAD-004 is inoculated at an inoculation amount of 10 v / v%, and anaerobically fermented under the conditions of dissolved oxygen concentration <0.5 mg / L, temperature of 28°C, and pH of 7.3, and nitrate consumption (mg N / L / h) is monitored at regular intervals. The results are shown in Table 1.

[0038] Table 1 Nitrate consumption after adding carrier to culture of Thiobacillus denitrificans SZG-SAD-004

[0039]

[0040] The test results in Table 1 show that adding zeolite powder, kaolin, mica powder, and calcium carbonate bacteria during fermentation can increase the consumption of nitrate, indicating that zeolite powder, kaolin, mica powder, and calcium carbonate bacteria can accelerate the growth of the denitrifying Thiobacillus SZG-SAD-004 bacterial population and achieve short-term high-density culture. Among the four carriers, the denitrifying Thiobacillus SZG-SAD-004 in the zeolite powder group grew faster, and the nitrate was basically consumed after 48 hours of culture.

[0041] Example 3

[0042] The effect of different amounts of test carrier on nitrate consumption: using the sterilized nutrient solution and nitrifying bacteria fermentation waste liquid of Example 1, 0.5g / L, 1.0g / L, 1.5g / L, 2.0g / L, 2.5g / L, and 3.0g / L of carrier zeolite powder were added respectively, and the inoculation amount of denitrifying Thiobacillus SZG-SAD-004 was 10v / v%. After culturing for 48h at 28°C and pH 7.3, the nitrate consumption was detected. The results are shown in Table 2.

[0043] Table 2 Nitrate consumption of denitrifying Thiobacillus culture with different amounts of zeolite powder

[0044]

[0045] As shown in Table 2, when zeolite powder of 1.5-2.0 g / L is added during the culture of Denitrifying Thiobacillus, Denitrifying Thiobacillus grows faster and consumes about 500 mg / L of nitrate after 48 hours of culture. The consumption of nitrate will be affected if the amount is less than 1.5 g / L or greater than 2.0 g / L.

[0046] Example 4

[0047] Test the nitrate removal rate of N2, CO2, and natural fermentation of denitrifying thiobacillus SZG-SAD-004 fermentation: prepare the required nutrient solution according to the formula to make the final nitrate concentration of 560 mg / L, add nitrifying bacteria fermentation waste liquid, 1.5 g / L zeolite powder, add it to the bioreactor for sterilization, adjust the pH to 7.3, and the denitrifying thiobacillus inoculation amount is 10%. Fill N2 and CO2 respectively, stop aeration when the dissolved oxygen is below 0.5 mg / L, start fermentation culture, and use the control group to ferment naturally without gas filling. The bioreactor seals the valve and does not introduce air. After the fermentation culture is completed, take a certain amount of fermentation liquid to test the nitrate removal rate. The results are shown in Table 3.

[0048] Table 3 Nitrate removal rates of N2, CO2, and natural fermentation of denitrifying Thiobacillus SZG-SAD-004

[0049] Anaerobic Control Nitrate removal rate (mgN / L / h) <![CDATA[Fill with N2]]> 190.53 <![CDATA[Charge with CO2]]> 198.12 Natural (CK) 189.65

[0050] As shown in Table 3, when Thiobacillus denitrificans was cultured in a bioreactor and N2 and CO2 were added to make it quickly reach an anaerobic state, or it was allowed to naturally ferment and consume the oxygen in the reactor to reach an anaerobic state, there was no obvious effect on the fermentation, and the nitrate removal rate of the fermentation broth was about 190 mg N / L / h.

[0051] Example 5

[0052] The nitrate removal rate of denitrifying Thiobacillus SZG-SAD-004 fermentation was tested under different rotation speed conditions: zeolite powder was selected as the carrier, and the addition amount was 1.5 g / L. The nitrifying bacteria fermentation waste liquid and the initial nutrient solution were the same as those in Example 2.

[0053] The difference between Example 5 and Example 2 is that no gas is introduced during fermentation, the fermentation is stirred, and the rotation speed is adjusted to 0 rpm, 30 rpm, 50 rpm, 70 rpm, 100 rpm, and 150 rpm, respectively. After the fermentation culture is completed, a certain amount of fermentation liquid is taken to test the nitrate removal rate, and the results are shown in Table 4.

[0054] Table 4 Nitrate removal rate of SZG-SAD-004 fermentation with different speeds

[0055] Speed Nitrate removal rate (mgN / L / h) 0rpm 188.97 30rpm 230.78 50rpm 270.45 70rpm 260.87 100rpm 203.46 150rpm 154.81

[0056] As shown in Table 4, in the bioreactor, the stirring speed is preferably 30-70 rpm. At this speed, the mass transfer and temperature of the nutrient solution are uniform, which is conducive to the better growth of denitrifying Thiobacillus SZG-SAD-004, and the nitrate removal rate of the fermentation broth is above 230 mg N / L / h. Too low stirring speed or complete static stirring may lead to uneven mass transfer, while too high stirring speed may cause mechanical damage to the bacteria, thereby reducing the nitrate removal rate.

[0057] Example 6

[0058] The carrier of Example 6 is zeolite powder, and the addition amount is 1.5g / L. The nitrifying bacteria fermentation waste liquid and the initial nutrient solution are the same as those of Example 1. No gas is introduced during fermentation, and the fermentation is stirred and the speed is adjusted to 50rpm. Batch feeding is performed during fermentation, and a total of 4 times are added. The concentration of nitrate in the nitrifying bacteria fermentation waste liquid added to the feeding nutrient solution is 200mg / L, and the concentration of Na2S2O3·5H2O and NaHCO3 is consistent with the initial nutrient solution. The other components are 20% of the concentration of the initial nutrient solution. Samples are taken after each feeding fermentation, and a certain amount of fermentation liquid is taken after the entire fermentation culture is completed to detect the nitrate removal rate. The results are shown in Table 5.

[0059] Table 5 Nitrate removal rate of batch fed-batch fermentation of Thiobacillus denitrificans SZG-SAD-004

[0060] Nitrate removal rate (mgN / L / h) No refill 270.21 The 1st feeding 320.58 2nd feeding 396.74 The 3rd feeding 489.89 The 4th feeding 306.57

[0061] As shown in Table 5, each feeding can increase the fermentation rate in the bioreactor, and the nitrate removal rate is the highest in the third feeding. The nitrate removal rate of the fermentation broth decreased after the fourth feeding, indicating that the accumulated salinity in the bioreactor was high after the fourth feeding, which inhibited the growth of Thiobacillus denitrificans, indicating that the feeding strategy needs to control the salinity.

[0062] Example 7

[0063] The method of high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater in this embodiment comprises the following steps:

[0064] S1, nitrifying bacteria fermentation wastewater diluted to a NO3-N concentration of 700 mg / L;

[0065] S2, prepare the initial nutrient solution: NH4Cl 1g / L, MgCl2·6H2O 1.5g / L, KH2PO45g / L, Na2S2O3·5H2O12g / L, NaHCO33g / L, FeSO4·7H2O 0.015g / L, trace elements 1.5mL / L. Trace element components are: Na2-EDTA 60g / L, CaCl2·2H2O 8g / L, FeSO4·7H2O 6g / L, MnCl2·4H2O 3g / L, ZnSO4·7H2O 3g / L, (NH4)6Mo7O2·4H2O 0.6g / L, CaSO4·5H2O0.3g / L and NaOH 12g / L. The pH of the initial nutrient solution was finally adjusted to 7.3, and the nutrient solution was sterilized before use, with a sterilization temperature of 121°C, a pressure of 0.12 MPa, and a sterilization time of 30 min.

[0066] S3, 0.56L of nitrifying bacteria fermentation waste liquid and 2.44L of initial nutrient solution were added to the bioreactor, 1g / L of kaolin was added, and then denitrifying Thiobacillus SZG-SAD-004 was inoculated at an inoculum of 12v / v%, and anaerobic fermentation was performed under the conditions of dissolved oxygen concentration <0.5mg / L, temperature of 32°C, rotation speed of 70rpm, and pH value of 7.5. During the fermentation, the feed was regularly supplemented 3 times to maintain the salinity ≤40000mg / L.

[0067] During feeding, the concentration of nitrate in the added nitrifying bacteria fermentation waste liquid was 220 mg / L. Except for the amount of Na2S2O3·5H2O and NaHCO3 in the feeding nutrient solution which was the same as that in the initial nutrient solution, the other components were 25% of the concentration of the initial nutrient solution.

[0068] Example 8

[0069] The method of high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater in this embodiment comprises the following steps:

[0070] S1, nitrifying bacteria fermentation wastewater diluted to a NO3-N concentration of 200 mg / L;

[0071] S2, prepare the initial nutrient solution: NH4Cl 0.5g / L, MgCl2·6H2O 0.5g / L, KH2PO43g / L, Na2S2O3·5H2O 8g / L, NaHCO31g / L, FeSO4·7H2O 0.005g / L, trace elements 0.5mL / L. Trace element components are: Na2-EDTA 40g / L, CaCl2·2H2O 6g / L, FeSO4·7H2O 4g / L, MnCl2·4H2O 2g / L, ZnSO4·7H2O 2g / L, (NH4)6Mo7O2·4H2O 0.4g / L, CaSO4·5H2O0.1g / L and NaOH 9g / L. The pH of the initial nutrient solution was finally adjusted to 6.5, and the nutrient solution was sterilized before use, with a sterilization temperature of 121°C, a pressure of 0.12 MPa, and a sterilization time of 30 min.

[0072] S3, 0.2L of nitrifying bacteria fermentation waste liquid and 2.8L of initial nutrient solution were added to the bioreactor, 0.5g / L of mica powder was added, and then denitrifying Thiobacillus SZG-SAD-004 was inoculated at an inoculum of 8v / v%, and anaerobic fermentation was performed under the conditions of dissolved oxygen concentration <0.5mg / L, temperature of 28°C, rotation speed of 30rpm, and pH value of 7.0. During the fermentation, the feed was regularly supplemented 3 times to maintain the salinity ≤40000mg / L.

[0073] During feeding, the concentration of nitrate in the nitrifying bacteria fermentation waste liquid added was 180 mg / L. Except for the amount of Na2S2O3·5H2O and NaHCO3 in the feeding nutrient solution which was the same as that in the initial nutrient solution, the other components were 15% of the concentration of the initial nutrient solution.

[0074] Comparative Example 1

[0075] The difference between Comparative Example 1 and Example 7 is that no carrier is included, and the rest of the contents are the same.

[0076] Comparative Example 2

[0077] The difference between Comparative Example 2 and Example 7 is that no feed is added, and the rest of the contents are the same.

[0078] Table 6 Total colony counts of different fermentation schemes

[0079] Total colony count after 48h fermentation (CFU / mL) Example 7 <![CDATA[6.8*10 12 ]]> Example 8 <![CDATA[7.4*10 12 ]]> Comparative Example 1 <![CDATA[3.1*10 8 ]]> Comparative Example 2 <![CDATA[1.7*10 9 ]]>

[0080] As shown in Table 6, if no carrier is added or no feed is added, the culture of SZG-SAD-004 will be affected, and the total number of colonies will be reduced, which is consistent with the result of the nitrate removal rate of SZG-SAD-004. This proves that the technical solution of the present invention can achieve the purpose of high-density fermentation of SZG-SAD-004 by using nitrifying bacteria fermentation waste liquid.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater, characterized in that: The following steps are involved: Take the waste liquid from nitrifying bacteria fermentation and the initial nutrient solution and add them into the bioreactor, inoculate denitrifying Thiobacillus SZG-SAD-004, and stir and ferment under anaerobic environment; The accession number of the denitrifying Thiobacillus SZG-SAD-004 is CCTCC M 2023992.

2. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 1, characterized in that: The NO3-N concentration in the nitrifying bacteria fermentation waste liquid is controlled to be 200-700 mg / L; the initial nutrient solution components are: NH4Cl 0.5-1g / L, MgCl2·6H2O 0.5-1.5g / L, KH2PO4 3-5g / L, Na2S2O3·5H2O 8-12g / L, NaHCO3 1-3g / L, FeSO4·7H2O 0.005-0.015g / L, trace elements 0.5-1.5mL / L, and the pH is 6.5-8.

0.

3. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 2, characterized in that: The trace element components are: Na2-EDTA 40-60g / L, CaCl2·2H2O 6-8g / L, FeSO4·7H2O 4-6g / L, MnCl2·4H2O 2-3g / L, ZnSO4·7H2O 2-3g / L, (NH4)6Mo7O2·4H2O0.4-0.6g / L, CaSO4·5H2O0.1-0.3g / L and NaOH 9-12g / L.

4. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 1, characterized in that: The inoculation amount of Thiobacillus denitrificans SZG-SAD-004 was 8-12 v / v%.

5. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 1, characterized in that: Before fermentation, the carrier, nitrifying bacteria fermentation waste liquid and initial nutrient solution are added into the bioreactor together, and the carrier is one or more combinations of zeolite powder, mica powder, kaolin and calcium carbonate.

6. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 5, characterized in that: The added amount of the carrier is 1-2 g / L.

7. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 1, characterized in that: During fermentation, the dissolved oxygen concentration in the bioreactor is less than 0.5 mg / L, the temperature is 28-32° C., the rotation speed is 30-70 rpm, and the pH value is 7.0-7.

5.

8. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 1, characterized in that: The volume ratio of nitrifying bacteria fermentation waste liquid: initial nutrient solution is 1-20:

100.

9. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 2, characterized in that: During fermentation, feed is added regularly to maintain salinity ≤40000mg / L.

10. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation waste liquid as claimed in claim 9, characterized in that: During feeding, except for the amount of Na2S2O3·5H2O and NaHCO3 in the feeding nutrient solution which is the same as that of the initial nutrient solution, the other components are 15%-25% of the concentration of the initial nutrient solution, and the concentration of nitrate in the nitrifying bacteria fermentation waste liquid added during feeding is 180-220 mg / L.

Images