A method for high-density fermentation of thiobacillus denitrificans by using nitrifying bacteria fermented waste liquid

Abstract

The present application relates to the technical field of microbial fermentation, and proposes a method for high-density fermentation of Thiobacillus denitrificans by using nitrifying bacteria fermentation waste liquid, which comprises the following steps: taking nitrifying bacteria fermentation waste liquid and 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 M 2023992; the nitrifying bacteria fermentation waste liquid is waste liquid produced after co-fermentation of nitrosobacteria, nitrifying bacteria and denitrifying bacteria. When autotrophic nitrifying bacteria is produced by a microbial fermentation method, waste liquid containing high-concentration nitrate, inorganic salt and metal ions is generated, which needs to be treated professionally to reach the discharge standard, thereby increasing the cost of enterprises. The Thiobacillus denitrificans SZG-SAD-004 of the present application can directly utilize the waste liquid for high-density fermentation, without additional treatment, thereby realizing resource utilization of the waste liquid.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, 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 requires no carbon source, produces low sludge, consumes little energy, and produces no secondary pollution. This makes it a promising alternative to traditional heterotrophic denitrification, particularly when treating wastewater with a low carbon-to-nitrogen ratio. The core of the simultaneous denitrification and desulfurization process in sulfur autotrophic denitrification is its functional bacterial community, known as denitrifying and desulfurizing bacteria (NR-SOB). Therefore, achieving highly efficient and active functional bacterial communities through bioaugmentation and other methods is crucial for the development of sulfur autotrophic denitrification processes.

[0003] The NR-SOB that are found to be more common at present mainly 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 pipe corrosion; this bacterium can also be used for natural gas desulfurization of hydrogen sulfide. Microbial desulfurization offers advantages such as low investment, low cost, mild reaction conditions, and minimal pollution. Currently, denitrifying Thiobacillus is mostly cultivated in laboratory shake flasks, requiring CO₂ or N₂ aeration to maintain an anaerobic state. This results in lengthy cultivation times and low bacterial concentrations, hindering mass production and application. For example, patent CN103773706A utilizes an airlift bioreactor to cultivate denitrifying Thiobacillus under aerated conditions for microbial desulfurization of sulfur-containing industrial waste gas. However, after several consecutive aerobic subcultures, denitrifying Thiobacillus loses its ability to grow anaerobically in nitrate solutions. Patent CN110818093A utilizes secondary treatment effluent to adjust nitrate content, inoculating anoxic sludge in a microaerobic environment for denitrifying Thiobacillus cultivation. The required nitrate is a highly explosive substance and requires strict control. However, the low nitrate concentration in secondary wastewater treatment plant water makes it difficult to achieve high-density fermentation.

[0004] Autotrophic nitrifying bacteria is produced by microbial fermentation, and a large amount of waste liquid can be produced in the production process. Since the nitrifying bacteria fermentation liquid tail liquid contains high concentration nitrate, inorganic salts and metal ions, the waste liquid needs professional company processing to reach the discharge requirement, which increases the cost of the production enterprise. Nitrifying bacteria fermentation waste liquid can be turned into treasure, resource recycling, the patent No. is that the invention patent of CN116143343A discloses a method for processing nitrifying bacteria waste liquid and can be used for sulfur autotrophic denitrifying bacteria fermentation, but a large amount of acid is needed to adjust pH in the method processing process, and flocculants, iron-containing materials, catalysts, carbonaceous materials are added afterwards, and nitrifying bacteria fermentation waste liquid is aerated, and process is relatively complicated, and increases energy consumption. Therefore, it is necessary to have a method that can directly utilize nitrifying bacteria fermentation waste liquid to carry out high-density fermentation. 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 wastewater, comprising the following steps:

[0007] Nitrifying bacteria fermentation waste liquid and initial nutrient solution were added into a bioreactor, and denitrifying Thiobacillus SZG-SAD-004 was inoculated and stirred for fermentation under anaerobic environment; the denitrifying Thiobacillus SZG-SAD-004 had a deposit number of CCTCC M2023992.

[0008] Based on the above technical solution, 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 the pH is 6.5-8.0.

[0009] Based on the above technical solution, 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] Based on the above technical solution, preferably, before fermentation, the carrier, nitrifying bacteria fermentation waste liquid and initial nutrient solution are added to 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] Based on 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] Based on the above technical solution, preferably, 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 nitrate concentration in the nitrifying bacteria fermentation waste liquid added during feeding is 180-220 mg / L.

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

[0018] (1) Nitrifying bacteria fermentation wastewater contains more than 3000 mg / L of nitrate, and also contains elements such as phosphorus, iron, copper, and zinc, which can provide the necessary nutrients for denitrifying Thiobacillus, fully meeting the requirements for industrial fermentation production of denitrifying Thiobacillus. The present invention utilizes nitrifying bacteria fermentation wastewater as a nitrate source for the cultivation of denitrifying Thiobacillus SZG-SAD-004, thereby achieving resource recycling of nitrifying bacteria fermentation wastewater and reducing hazardous waste treatment costs.

[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 Thiobacillus denitrificans 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 embodiments described 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 making creative efforts 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, and was deposited in the China Center for Type Culture Collection (CCTCC) on June 12, 2023, at Wuhan University, Wuhan, China, with the deposit number: CCTCC M 2023992. It 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 chemolithoautotroph and can consume thiosulfate and S under aerobic and anaerobic conditions. 0 、S 2- When cultured on solid plates in a 28°C incubator, white, smooth, round colonies with a diameter of 0.2 to 1 mm appear on the surface of the culture medium.

[0023] The nitrifying bacteria fermentation waste liquid used in the present invention is the supernatant generated during the fermentation process of Nitrosomonas europaea AT7 and Nitrosomonas europaea C-31 in a ratio of 1:1, which is overflowed into a nitrifying bacteria tube tank, and the supernatant is obtained during the cultivation of Nitrobacter Winogradskyi Y3-2 in the nitrifying bacteria tank. For details, see the fermentation method disclosed in CN108424862A. Nitrosomonas europaea AT7 and Nitrosomonas europaea C-31 convert ammonia nitrogen into nitrite, and the nitrifying bacteria Nitrobacter winogradskyi converts nitrite into nitrate.

[0024] During the fermentation of European Nitrosomonas AT7 and European Nitrosomonas C-31, 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: 0.1 wt% MnCl2·4H2O, 0.1 wt% Na2B4O7·10H2O, 0.1 wt% ZnSO4·7H2O, 0.1 wt% EDTA-Fe, 0.1 wt% CoSO4·7H2O, 0.1 wt% CuSO4·5H2O, and 0.1 wt% NiSO4·6H2O.

[0025] The nitrosating bacteria tank is inoculated with nitrosating bacteria at 10% (v / v). 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., intermittent aeration is performed, 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 ammonia nitrogen in the culture medium no longer decreases, the cultured nitrosating 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] A nitrifying bacteria tank is inoculated with 10% (v / v) of 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, cultured nitrifying bacteria are obtained; the culture is allowed to stand to separate the bacteria, and the supernatant overflows to obtain the nitrifying bacteria fermentation waste liquid used in the present invention.

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

[0028] The supernatant overflowing from the fermentation of Nitrifying Bacteria Y3-2 contains over 3000 mg / L of nitrate. It also contains phosphorus, iron, copper, zinc, and other elements that provide nutrients for Thiobacillus denitrificans. When used, dilute the fermentation wastewater to a usable concentration.

[0029] The reactor for cultivating high-density Thiobacillus denitrificans is a conventional stirred-ventilated reactor. The upper portion of the reactor is equipped with a bacterial suspension feed port, a nutrient solution feed port, and an exhaust gas discharge port. A temperature probe, a pH probe, and a dissolved oxygen probe are provided inside the reactor for monitoring temperature, pH, and dissolved oxygen. The lower portion of the reactor is equipped with an air / liquid inlet, an air aeration pipe, and a nutrient solution waste outlet. The air / liquid inlet and air aeration pipe are used to adjust the dissolved oxygen content. The temperature probe, pH probe, and dissolved oxygen probe are operated and controlled by an electronic 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: 0.8 g / L NH4Cl, 1 g / L MgCl2·6H2O, 4 g / L KH2PO4, 10 g / L Na2S2O3·5H2O, 2 g / L NaHCO3, 0.01 g / L FeSO4·7H2O, and 1.0 mL / L trace elements. The trace element composition is: 50 g / L Na2-EDTA, 7.34 g / L CaCl2·2H2O, 5.0 g / L FeSO4·7H2O, 2.5 g / L MnCl2·4H2O, 2.2 g / L ZnSO4·7H2O, 0.5 g / L (NH4)6Mo7O2·4H2O, 0.2 g / L CaSO4·5H2O, and 11.0 g / L NaOH. The pH of the initial nutrient solution was finally adjusted to 7.3, and the nutrient solution was sterilized before use at 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 to a bioreactor, 6 g of carriers (zeolite powder, kaolin, mica powder, calcium carbonate) are added, and no carrier is added as a control (CK). Denitrifying Thiobacillus SZG-SAD-004 is inoculated at an inoculum rate 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. 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] Table 1 shows that the addition of zeolite powder, kaolin, mica powder, and calcium carbonate bacteria during fermentation increased nitrate consumption, indicating that these three factors can accelerate the growth of Thiobacillus denitrificans SZG-SAD-004, enabling high-density cultivation within a short period of time. Among the four carriers, the zeolite powder group exhibited the fastest growth of Thiobacillus denitrificans SZG-SAD-004, with nitrate consumption essentially complete within 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, 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L of carrier zeolite powder were added respectively, and the inoculum amount of denitrifying Thiobacillus SZG-SAD-004 was 10 v / v%. After culturing for 48 hours at 28°C and a pH value of 7.3, the nitrate consumption was detected. The results are shown in Table 2.

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

[0044]

[0045] As shown in Table 2, when zeolite powder of 1.5-2.0 g / L was added during the culture of Thiobacillus denitrificans, Thiobacillus denitrificans grew faster and consumed about 500 mg / L of nitrate after 48 h of culture. Nitrate consumption was affected by a concentration less than 1.5 g / L or greater than 2.0 g / L.

[0046] Example 4

[0047] Nitrate removal rates were tested using N₂, CO₂, and naturally fermented Thiobacillus denitrificans SZG-SAD-004. A nutrient solution was prepared according to the formula to achieve a final nitrate concentration of 560 mg / L. Wastewater from nitrifying bacteria fermentation and 1.5 g / L of zeolite powder were added to the bioreactor for sterilization. The pH was adjusted to 7.3, and the inoculum of Thiobacillus denitrificans was 10%. N₂ and CO₂ were aerated to a level below 0.5 mg / L, at which point aeration was stopped and fermentation was initiated. A control group, which was allowed to ferment naturally without aeration, served as the control. The bioreactor valve was sealed, preventing air from entering. After the fermentation, a certain amount of fermentation broth was collected to measure the nitrate removal rate. The results are shown in Table 3.

[0048] Table 3 Nitrate removal rates of N2, CO2, and natural fermentation of Thiobacillus denitrificans 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 nitrogen and CO were added to quickly reach an anaerobic state, or when it was allowed to ferment naturally and consume oxygen in the reactor to reach an anaerobic state, there was no significant effect on the fermentation. The nitrate removal rate of the fermentation broth was around 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] Example 5 differs from Example 2 in that no gas was introduced during fermentation, the fermentation was stirred, and the rotational speed was adjusted to 0 rpm, 30 rpm, 50 rpm, 70 rpm, 100 rpm, and 150 rpm, respectively. After the fermentation was completed, a certain amount of the fermentation broth was collected to measure the nitrate removal rate. The results are shown in Table 4.

[0054] Table 4 Nitrate removal rate of Thiobacillus denitrificans SZG-SAD-004 fermentation at 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] Table 4 shows that within the bioreactor, an optimal stirring speed of 30 to 70 rpm maintains uniform nutrient solution mass transfer and temperature, promoting optimal growth of Thiobacillus denitrificans SZG-SAD-004 and achieving a nitrate removal rate exceeding 230 mg N / L / h. A stirring speed that is too low or completely static can lead to uneven mass transfer, while a stirring speed that is too high can mechanically damage the bacteria, thereby reducing the nitrate removal rate.

[0057] Example 6

[0058] In Example 6, zeolite powder was selected as the carrier, added in an amount of 1.5 g / L. The nitrifying bacteria fermentation wastewater and initial nutrient solution were the same as in Example 1. No gas was introduced during fermentation, and the fermentation was stirred at a rotation speed of 50 rpm. Four batches of feeds were added during fermentation. The nitrate concentration in the nitrifying bacteria fermentation wastewater added to the feed nutrient solution was 200 mg / L. The concentrations of Na2S2O3·5H2O and NaHCO3 remained the same as in the initial nutrient solution. Other components were 20% of the concentrations in the initial nutrient solution. Sampling was performed after each fed-batch fermentation. After the entire fermentation culture was completed, a certain amount of the fermentation broth was collected to test 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] Table 5 shows that each feeding increase in the bioreactor increased the fermentation rate, with the third feeding achieving the highest nitrate removal rate. The nitrate removal rate 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 and indicated that the feeding strategy required salinity control.

[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 was diluted to a NO3-N concentration of 700 mg / L;

[0065] S2: Prepare the initial nutrient solution: 1 g / L NH4Cl, 1.5 g / L MgCl2·6H2O, 5 g / L KH2PO4, 12 g / L Na2S2O3·5H2O, 3 g / L NaHCO3, 0.015 g / L FeSO4·7H2O, and 1.5 mL / L trace elements. The trace element composition is: 60 g / L Na2-EDTA, 8 g / L CaCl2·2H2O, 6 g / L FeSO4·7H2O, 3 g / L MnCl2·4H2O, 3 g / L ZnSO4·7H2O, 0.6 g / L (NH4)6Mo7O2·4H2O, 0.3 g / L CaSO4·5H2O, and 12 g / L NaOH. The pH of the initial nutrient solution was finally adjusted to 7.3, and the nutrient solution was sterilized before use at a sterilization temperature of 121° C., a pressure of 0.12 MPa, and a sterilization time of 30 min.

[0066] In step S3, 0.56 L of nitrifying bacteria fermentation wastewater and 2.44 L of initial nutrient solution were added to the bioreactor. 1 g / L of kaolin was then inoculated with Thiobacillus denitrificans SZG-SAD-004 at a 12 v / v inoculum. Anaerobic fermentation was carried out under conditions of dissolved oxygen concentration <0.5 mg / L, temperature 32°C, rotation speed 70 rpm, and pH 7.5. Feeds were added regularly three times during fermentation to maintain salinity ≤40,000 mg / L.

[0067] During feeding, the concentration of nitrate in the added nitrifying bacteria fermentation wastewater 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 all 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 as follows: 0.5 g / L NH4Cl, 0.5 g / L MgCl2·6H2O, 3 g / L KH2PO4, 8 g / L Na2S2O3·5H2O, 1 g / L NaHCO3, 0.005 g / L FeSO4·7H2O, and 0.5 mL / L trace elements. The trace element composition is: 40 g / L Na2-EDTA, 6 g / L CaCl2·2H2O, 4 g / L FeSO4·7H2O, 2 g / L MnCl2·4H2O, 2 g / L ZnSO4·7H2O, 0.4 g / L (NH4)6Mo7O2·4H2O, 0.1 g / L CaSO4·5H2O, and 9 g / L NaOH. The pH of the initial nutrient solution was finally adjusted to 6.5, and the nutrient solution was sterilized before use at a sterilization temperature of 121° C., a pressure of 0.12 MPa, and a sterilization time of 30 min.

[0072] In step S3, 0.2 L of nitrifying bacteria fermentation wastewater and 2.8 L of initial nutrient solution were added to the bioreactor. 0.5 g / L of mica powder was then inoculated with Thiobacillus denitrificans SZG-SAD-004 at an inoculum rate of 8 v / v%. Anaerobic fermentation was carried out under conditions of dissolved oxygen concentration <0.5 mg / L, temperature 28°C, rotation speed 30 rpm, and pH 7.0. Feeds were added regularly three times during fermentation to maintain salinity ≤40,000 mg / L.

[0073] During feeding, the concentration of nitrate in the added nitrifying bacteria fermentation wastewater 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 all 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, the absence of either carrier or feed affects the culture of SZG-SAD-004, reducing the total colony count, which is consistent with the nitrate removal rate of SZG-SAD-004. This demonstrates that the technical solution of the present invention can achieve high-density fermentation of SZG-SAD-004 using nitrifying bacteria fermentation wastewater.

[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 scope of protection 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: The waste liquid from nitrifying bacteria fermentation and the initial nutrient solution were added to the bioreactor, inoculated with denitrifying Thiobacillus SZG-SAD-004, and stirred and fermented under anaerobic conditions; The accession number of the Thiobacillus denitrificans SZG-SAD-004 is CCTCC M 2023992; The nitrifying bacteria fermentation waste liquid contains more than 3000 mg / L of nitrate, as well as phosphorus, iron, copper and zinc; Before fermentation, a carrier, nitrifying bacteria fermentation waste liquid, and initial nutrient solution are added to the bioreactor together, wherein the carrier is one of zeolite powder and calcium carbonate; The added amount of the zeolite powder is 1.5-2 g / L, and the added amount of the calcium carbonate is 2 g / L.

2. The method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater according to claim 1, wherein: 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 wastewater 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)6Mo7O2·4H2O 0.4-0.6g / L, CaSO4·5H2O 0.1-0.3g / L and NaOH 9-12g / L.

4. The method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater according to claim 1, wherein: The inoculum amount of Thiobacillus denitrificans SZG-SAD-004 was 8-12 v / v%.

5. The method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater according to claim 1, wherein: 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.

6. The method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater according to claim 1, wherein: The volume ratio of nitrifying bacteria fermentation waste liquid: initial nutrient solution is 1 to 20:

100.

7. The method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater according to claim 2, wherein: During fermentation, feed the fermentation solution regularly to maintain salinity ≤40000 mg / L.

8. A method for high-density fermentation of denitrifying Thiobacillus using nitrifying bacteria fermentation wastewater as claimed in claim 7, 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.

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