A method for optimizing the cultivation of sulfur autotrophic denitrifying bacteria

Abstract

The present invention discloses an optimized culture method for sulfur autotrophic denitrifying bacteria. The optimized culture method uses anaerobic activated sludge as the original strain, adopts a sulfur autotrophic denitrifying culture medium for constant temperature continuous periodic directional culture, and concentrates the fermented liquid at the end of each period and inoculates it in the next period culture medium; detects the nitrate nitrogen concentration, total phosphorus concentration and / or ammonia nitrogen concentration of the culture liquid at the end of each period, and adjusts the ammonia nitrogen concentration and / or total phosphorus concentration in the next period culture medium according to the detection results, until the nitrate nitrogen removal rate, total phosphorus removal rate and ammonia nitrogen removal rate of the culture liquid meet the standards, and the optimized culture of the sulfur autotrophic denitrifying bacteria is completed. The present invention improves the culture efficiency of the sulfur autotrophic denitrifying bacteria by optimizing the culture method. The improved culture medium and technology make the screening and enrichment of functional microorganisms more efficient, the steps are simple, no specific anaerobic microorganism culture device is required, and the entire screening and separation cycle of the bacteria is shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular is an optimized cultivation method for sulfur autotrophic denitrifying bacteria. Background Art

[0002] Nitrogen is a common pollutant in sewage, and total nitrogen in sewage is one of the most important indicators in sewage discharge standards. Nitrogen removal is necessary in sewage treatment to ensure water quality, ecological balance and public health. In the "Pollutant Discharge Standard for Urban Sewage Treatment Plants" (GB 18918-2002), the first-level A effluent standard requires total nitrogen effluent to be less than 15 mg / L; in the "Surface Water Environmental Quality Standard" (GB3838-02), Class II and Class III (mainly applicable to the first and second level protection areas of centralized drinking water sources) require ammonia nitrogen ≤ 0.5 mg / L.

[0003] Nitrogen in sewage mainly exists in two forms: ammonia nitrogen and nitrate nitrogen. Nitrogen removal usually includes two main steps: nitrification and denitrification. Nitrification is the conversion of ammonia nitrogen into nitrate nitrogen, while denitrification is the reduction of nitrate nitrogen to nitrogen gas or nitrogen oxides and releases them into the atmosphere, completely removing them from sewage.

[0004] Microorganisms are one of the main means of nitrogen removal in sewage treatment. Microorganisms promote nitrogen removal by converting nitrogen compounds into different forms, performing the functions of nitrification and denitrification. Sulfur autotrophic denitrification technology is a treatment method that removes nitrogen compounds through sulfur autotrophic denitrifying microorganisms. It is usually used in wastewater treatment systems, especially under low oxygen or anaerobic conditions. It has the advantages of not requiring an external organic carbon source and low sludge yield, and has attracted much attention.

[0005] However, sulfur autotrophic microorganisms are sensitive to the living environment and have a low growth rate, which makes their cultivation difficult. In addition, conventional sulfur autotrophic denitrifying bacterial flora is cultivated in a reactor using activated sludge containing sulfur autotrophic microorganisms, but there are problems of contamination by foreign bacteria and a low proportion of the target flora. If pure bacterial culture or composite bacterial flora is used, the idea of ​​"enrichment first and then separation" has certain limitations: separation is blind and random, and there is uncertainty in the physiological characteristics of the strains. At the same time, sulfur autotrophic denitrifying bacterial flora is a field that few people have studied, so it is difficult and time-consuming. Summary of the invention

[0006] In order to solve the above problems, the present invention provides an optimized cultivation method for sulfur autotrophic denitrifying bacteria. By improving the culture medium and optimizing the cultivation technology, functional microorganisms are screened and enriched. No purification and no specific anaerobic bacteria cultivation equipment are required, thus shortening the whole cycle of screening and separating bacteria and improving efficiency.

[0007] The invention provides an optimized cultivation method for sulfur autotrophic denitrifying bacteria.

[0008] The optimized culture method uses anaerobic activated sludge as the original bacterial strain, adopts a sulfur autotrophic denitrification culture medium for constant temperature continuous periodic directional culture, and at the end of each period, the fermentation liquid is concentrated and inoculated into the next period culture medium; the nitrate nitrogen concentration, total phosphorus concentration and / or ammonia nitrogen concentration of the culture liquid at the end of each period are detected, and the ammonia nitrogen concentration and / or total phosphorus concentration in the next period culture medium are adjusted according to the test results, until the nitrate nitrogen removal rate, total phosphorus removal rate and ammonia nitrogen removal rate of the culture liquid meet the standards, the optimized culture of the sulfur autotrophic denitrifying bacteria group is completed.

[0009] In some embodiments of the present invention, in the above method, the sulfur autotrophic denitrification culture medium components per liter include:

[0010] KNO3 1~ 2g,

[0011] NaHCO3 1~ 2g,

[0012] KH2PO4 0.3~ 2g,

[0013] MgCl2·6H2O 0.2~0.80g,

[0014] NH4Cl 0.1~0.5g,

[0015] FeSO4·7H2O 0.01~0.03g,

[0016] Na2S2O3·5H2O 3~6.00g.

[0017] In some embodiments of the present invention, the method for preparing the sulfur autotrophic denitrification culture medium comprises the following steps:

[0018] (1) Weigh KNO3, NaHCO3, MgCl2·6H2O, NH4Cl and Na2S2O3·5H2O, fully dissolve them, and sterilize them to obtain sterile solution A;

[0019] (2) Weigh KH2PO4, fully dissolve it, and sterilize it to obtain sterile solution B;

[0020] (3) Sterile solution A and sterile solution B are mixed, FeSO4·7H2O is added, and a pH adjuster is added dropwise to adjust the pH of the solution to 7.5±0.1 to obtain a sulfur autotrophic denitrification medium.

[0021] In some embodiments of the present invention, the anaerobic activated sludge is taken from one or more of the anaerobic process section of a domestic sewage treatment system, river sediment, filter tank, and UASB reactor.

[0022] In some preferred embodiments of the present invention, the anaerobic activated sludge is taken from:

[0023] At least one of the following: anaerobic process section sludge of the sewage treatment system of the AAO process; anaerobic sludge of the river with DO < 0.5 mg / L and ammonia nitrogen > 5 mg / L; the middle elevation of the biological filter; and the sludge bed layer of the UASB reactor.

[0024] In some embodiments of the present invention, anaerobic activated sludge is inoculated in a sulfur autotrophic denitrification medium for a first cycle of culture, the suspended solids concentration of the anaerobic activated sludge is 8000-12000 mg / L, the dissolved oxygen concentration is 0.5 mg / L, and the inoculation ratio of the anaerobic activated sludge is 5%-20% of the sulfur autotrophic denitrification medium.

[0025] In some embodiments of the present invention, the fermentation liquid is concentrated at the end of each phase and then inoculated into the next phase culture medium. Specifically, after each phase, the upper liquid in the culture liquid is centrifuged to obtain concentrated strains, which are inoculated into the next phase culture medium for cultivation.

[0026] In some embodiments of the present invention, the inoculation ratio of the concentrated bacteria is 10% to 20% of the sulfur autotrophic denitrification culture medium.

[0027] In some embodiments of the present invention, the concentration of ammonia nitrogen and / or total phosphorus in the next phase of the culture medium is adjusted according to the test results as follows:

[0028] If the nitrate nitrogen removal rate reaches more than 70% in at least one cycle, the unit concentration of total phosphorus in the culture medium in the next period will be lowered;

[0029] and / or

[0030] If the nitrate nitrogen removal rate reaches more than 70% and the total phosphorus removal rate reaches 55%~90% in at least one cycle, the unit concentration of ammonia nitrogen in the culture medium for the next period will be lowered.

[0031] In some embodiments of the present invention, the unit concentration of total phosphorus is reduced by 20% to 85%; the unit concentration of ammonia nitrogen is reduced by 20% to 80%.

[0032] In some embodiments of the present invention, the nitrate nitrogen removal rate, total phosphorus removal rate and ammonia nitrogen removal rate meet the standards as follows:

[0033] The nitrate nitrogen removal rate reaches more than 80%;

[0034] The total phosphorus removal rate reaches 75%~90%;

[0035] The ammonia nitrogen removal rate reaches 75%~90%.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention improves the cultivation efficiency of sulfur autotrophic denitrifying bacteria by optimizing the cultivation method. The improved culture medium and technology make the screening and enrichment of functional microorganisms more efficient, the steps are simple, no specific anaerobic microorganism cultivation device is required, and the entire screening and separation cycle of the bacteria is shortened.

[0038] 2. The present invention continuously adjusts the culture medium during the optimization culture process to make it closer to the native habitat of the sulfur autotrophic denitrifying bacteria, thereby improving the adaptation speed of microorganisms to the environment and increasing the success rate of enriching the sulfur autotrophic bacteria. At the same time, reducing the concentration of nitrogen and phosphorus nutrients also reduces secondary risks in the environment, which helps to reduce pollution load.

[0039] 3. The sulfur autotrophic bacteria enrichment of the present invention can accelerate the acclimation process of the sulfur autotrophic denitrification reactor / process section, and significantly shorten the acclimation biofilm period. This has practical significance for engineering applications and provides practical technical support for the effective operation of denitrification process sections such as reactors and wetlands. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0041] Figure 1 Schematic diagram of the process for optimizing the cultivation of sulfur-autotrophic denitrifying bacteria.

[0042] Figure 2 Schematic diagram of the constant temperature culture method.

[0043] Figure 3 This is the periodic removal rate curve of Example 3.

[0044] Figure 4 This is the microbial genus composition diagram of Example 3.

[0045] Figure 5 This is the periodic removal rate curve of Example 4.

[0046] Figure 6 This is the microbial genus composition diagram of Example 4.

[0047] Figure 7 This is an analysis diagram of the NO3-N removal capacity of Example 5.

[0048] Figure 8 This is a TN removal capacity analysis diagram of Example 5. DETAILED DESCRIPTION

[0049] The invention discloses an optimized cultivation method for sulfur autotrophic denitrifying bacteria.

[0050] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0051] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0052] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0053] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0054] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0055] Unless otherwise clearly stated, it should be understood that all percentages described in the present invention are by mass percentages.

[0056] The optimized culture method of the invention adopts a sulfur autotrophic denitrification culture medium for constant temperature continuous periodic directional culture, and at the end of each period, the fermentation liquid is concentrated and treated and then inoculated into the culture medium of the next period.

[0057] The nitrate nitrogen concentration, total phosphorus concentration and / or ammonia nitrogen concentration of the culture medium at the end of each period are tested, and the ammonia nitrogen concentration and / or total phosphorus concentration in the next period of culture medium are adjusted according to the test results. When the nitrate nitrogen removal rate, total phosphorus removal rate and ammonia nitrogen removal rate of the culture medium meet the standards, the optimized cultivation of sulfur-autotrophic denitrifying bacteria is completed.

[0058] In some embodiments of the present invention, in the above method, the sulfur autotrophic denitrification culture medium components per liter include:

[0059] KNO3 1~ 2g,

[0060] NaHCO3 1~ 2g,

[0061] KH2PO4 0.3~ 2g,

[0062] MgCl2·6H2O 0.2~0.80g,

[0063] NH4Cl 0.1~0.5g,

[0064] FeSO4·7H2O 0.01~0.03g,

[0065] Na2S2O3·5H2O 3~6.00g.

[0066] In a preferred embodiment, the solvent of the sulfur autotrophic denitrification culture medium is sterile water.

[0067] Among them, KNO3 (potassium nitrate), as a nitrogen source of nitrate nitrogen, provides the necessary elements for microorganisms to reduce nitrate nitrogen. Preferably, the concentration range in the culture medium is 1~2g / L to ensure sufficient nitrate nitrogen supply while avoiding adverse effects caused by excessive concentration.

[0068] NaHCO3 (sodium bicarbonate), as an inorganic carbon source, provides carbon metabolism for microorganisms. Preferably, the concentration range in the culture medium is 1-2 g / L to ensure sufficient carbon source supply to promote the growth and metabolic activities of microorganisms.

[0069] KH2PO4 (potassium dihydrogen phosphate), as a source of phosphorus, is essential for the growth and metabolic process of microorganisms. Preferably, the concentration range in the culture medium is 0.5~2g / L to ensure the supply of an appropriate amount of phosphorus while avoiding excessive concentration of phosphorus.

[0070] MgCl2·6H2O (magnesium chloride hexahydrate) provides magnesium ions, which are an important component of microbial growth and enzyme activity. Preferably, the concentration range in the culture medium is 0.2~0.80g / L to ensure that an appropriate amount of magnesium is provided to maintain the normal physiological conditions required for microbial growth.

[0071] NH4Cl (ammonium chloride), as a nitrogen source of ammonia nitrogen, provides available nitrogen elements for microorganisms. The concentration range in the culture medium is 0.1~0.5g / L, ensuring sufficient ammonia nitrogen supply while avoiding the negative effects of excessive concentration.

[0072] FeSO4·7H2O (ferrous sulfate heptahydrate) provides trace iron, which plays an important role in the growth and metabolism of microorganisms. The concentration range in the culture medium is 0.01~0.03g / L, ensuring the provision of appropriate trace elements.

[0073] Na2S2O3·5H2O (sodium thiosulfate pentahydrate), as a source of sulfur, provides necessary energy and electrons for sulfur autotrophic denitrifying microorganisms. The concentration range in the culture medium is 3~6.00g / L, ensuring sufficient sulfur source to maintain the normal metabolism of sulfur autotrophic microorganisms.

[0074] The culture medium of the present invention is a comprehensive nutritional condition required for the growth and metabolism of microorganisms. By optimizing the combination and concentration of culture medium raw materials, the quality and stability of the culture medium can be ensured, thereby improving the culture effect of sulfur autotrophic denitrifying bacteria.

[0075] In some embodiments of the present invention, the method for preparing the sulfur autotrophic denitrification culture medium comprises the following steps:

[0076] (1) Weigh KNO3, NaHCO3, MgCl2·6H2O, NH4Cl and Na2S2O3·5H2O, fully dissolve them, and sterilize them to obtain sterile solution A;

[0077] (2) Weigh KH2PO4, fully dissolve it, and sterilize it to obtain sterile solution B;

[0078] (3) Sterile solution A and sterile solution B are mixed, FeSO4·7H2O is added, and a pH adjuster is added dropwise to adjust the pH of the solution to 7.5±0.1 to obtain a sulfur autotrophic denitrification medium.

[0079] Among them, the sum of the solution masses of sterile solution A and sterile solution B is equal to the total mass of the final culture medium.

[0080] The present invention optimizes the preparation steps of the culture medium, avoids adverse reactions of the components during high-temperature moist heat sterilization, avoids oxidation of Fe²⁺ and formation of magnesium phosphate precipitation by Mg²⁺ and phosphate, etc. This optimization ensures that the effective components of the culture medium are not damaged, and maximizes the quality and stability of the culture medium.

[0081] In some specific embodiments, KNO3, NaHCO3, MgCl2·6H2O, NH4Cl and Na2S2O3·5H2O are weighed in a conical flask, fully dissolved in water and sterilized. KH2PO4 is weighed in a separate conical flask, fully dissolved in water and sterilized. The total liquid volume of the two conical flasks is 1L.

[0082] In some specific embodiments, the sterilization method is high-pressure moist heat sterilization, the temperature is 115-125° C., and the sterilization time is 20-30 min.

[0083] In some specific embodiments, after the sterile solution A and the sterile solution B are cooled to room temperature, the sterile solution B is added to the sterile solution A for mixing.

[0084] In some preferred embodiments, the pH adjuster is a NaOH solution, and the concentration of the NaOH solution is 20 g / L.

[0085] In some embodiments of the present invention, the oxygen activated sludge is taken from one or more of the anaerobic process section sludge of the sewage treatment system, river sludge, filter tank, and UASB reactor.

[0086] In some preferred embodiments of the present invention, the oxygen activated sludge is taken from one or more of the anaerobic process section sludge of the AAO process sewage treatment system; the river anaerobic sludge with DO < 0.5 mg / L and ammonia nitrogen > 5 mg / L; the middle elevation of the biological filter; and the sludge bed layer of the UASB reactor.

[0087] In some preferred embodiments of the present invention, the oxygen activated sludge is taken from the anaerobic process section sludge of the AAO process sewage treatment system; the anaerobic sludge of the river with DO < 0.5 mg / L and ammonia nitrogen > 5 mg / L; the middle elevation of the biological filter; and the sludge bed of the UASB reactor. The oxygen activated sludge in different environments contains various types of microorganisms, which increases the microbial diversity of the sulfur autotrophic denitrification culture, thereby improving the adaptability and tolerance of the bacterial community.

[0088] In some embodiments of the present invention, anaerobic activated sludge is inoculated in a sulfur autotrophic denitrification medium for a first cycle of cultivation.

[0089] In some specific embodiments, the inoculation ratio of anaerobic activated sludge is 5% to 20% of the sulfur autotrophic denitrification culture medium, wherein the 5% to 20% specifically refers to the mass percentage of anaerobic activated sludge to the mass percentage of the entire mixture being 5% to 20%.

[0090] In some preferred embodiments, the inoculation ratio of anaerobic activated sludge is 10% to 20% of the sulfur autotrophic denitrification culture medium.

[0091] In some specific embodiments, the suspended solids concentration of the anaerobic activated sludge is 8000-12000 mg / L, and the suspended solids refer to solid particles suspended in the liquid. The suspended solids concentration described in the present invention refers to the concentration of suspended particles in the anaerobic activated sludge.

[0092] In some specific embodiments, the dissolved oxygen concentration is 0.5 mg / L.

[0093] In some embodiments of the present invention, during each fermentation culture process, when the culture medium changes from clear and transparent to yellow-brown turbid, and the pH does not decrease within 2 hours, the fermentation of one stage is terminated.

[0094] In some embodiments of the present invention, the fermentation liquid is concentrated at the end of each phase and then inoculated into the next phase culture medium. Specifically, after each phase, the upper liquid in the culture liquid is centrifuged to obtain concentrated strains, which are inoculated into the next phase culture medium for cultivation.

[0095] In some specific embodiments, the centrifugal treatment conditions are 4000-8000 rpm, 5-10 min.

[0096] In some preferred embodiments, the centrifugation ratio is about 20% to 40% of the total volume, where the percentage refers to volume percentage.

[0097] In some preferred embodiments, after each phase of inoculation of anaerobic activated sludge or concentrated bacterial solution inoculated in the culture medium, the reaction system environment is deoxygenated to create a more suitable anaerobic environment, which is conducive to the growth of sulfur autotrophic denitrifying bacteria and maintains the stability of the culture. In some preferred embodiments, the deoxygenation treatment is achieved by ventilation deoxygenation, preferably the gas introduced is N2 or CO2, and the time is 15 to 25 minutes.

[0098] In some embodiments of the present invention, the inoculation ratio of the concentrated bacteria is 10% to 20% of the sulfur autotrophic denitrification culture medium.

[0099] In some embodiments of the present invention, the constant temperature culture is a constant temperature shaking culture, the temperature is 25-35° C., and the shaking speed of the shaker is 100-200 r / min.

[0100] In some embodiments of the present invention, the nitrate nitrogen concentration (NO3 - -N), total phosphorus concentration (TP), dissolved oxygen concentration (DO) and / or ammonia nitrogen concentration (NH4 + -N), including but not limited to further testing of chemical oxygen demand (COD).

[0101] In some embodiments of the present invention, the detection method of the above indicators is:

[0102] NO3 - -N: Ultraviolet spectrophotometry HJ / T 346─2007;

[0103] NH4 + -N: Nessler's reagent spectrophotometry HJ 535-2009;

[0104] TP: Ammonium molybdate spectrophotometry GB 11893-89;

[0105] DO: Electrochemical probe method GB 11913-89;

[0106] COD: Rapid digestion spectrophotometry HJ / T 399—2007.

[0107] In some embodiments of the present invention, the concentration of ammonia nitrogen and / or total phosphorus in the next phase of the culture medium is adjusted according to the test results as follows:

[0108] If the nitrate nitrogen removal rate reaches more than 70% in at least one cycle, the unit concentration of total phosphorus in the culture medium in the next period will be lowered;

[0109] and / or

[0110] If the nitrate nitrogen removal rate reaches more than 70% and the total phosphorus removal rate reaches 55%~90% in at least one cycle, the unit concentration of ammonia nitrogen in the culture medium for the next period will be lowered.

[0111] In some preferred embodiments of the present invention, the unit concentration of total phosphorus is reduced by 20% to 85%; the unit concentration of ammonia nitrogen is reduced by 20% to 80%. The unit concentration of total phosphorus refers to the maximum amount of total phosphorus added to the culture medium in the previous culture period, and the unit concentration of ammonia nitrogen refers to the maximum amount of ammonia nitrogen added to the culture medium in the previous culture period.

[0112] In some embodiments of the present invention, the nitrate nitrogen removal rate, total phosphorus removal rate and ammonia nitrogen removal rate meet the standards as follows:

[0113] The nitrate nitrogen removal rate reaches more than 80%;

[0114] The total phosphorus removal rate reaches 75%~90%;

[0115] The ammonia nitrogen removal rate reaches 75%~90%.

[0116] like Figure 1 As shown, in a specific embodiment, a method for optimizing the cultivation of sulfur autotrophic denitrifying bacteria comprises the following steps:

[0117] (1) Inoculate the anaerobic activated sludge into a conical flask containing culture medium, introduce N2 into the conical flask, stop aeration after 20 min, seal the conical flask, and place it in a water bath for constant temperature culture;

[0118] (2) The culture cycle is 3 to 5 days. During the fermentation process, when the culture medium changes from clear to brown and turbid, and the pH does not drop within 2 hours, the first stage of fermentation ends. At the end of the cycle, the nitrate nitrogen concentration (NO3 --N), total phosphorus concentration (TP), dissolved oxygen concentration (DO), ammonia nitrogen concentration (NH4 + -N) and chemical oxygen demand (COD) concentration, centrifuge the liquid at the middle height of the culture solution to obtain concentrated strains;

[0119] (3) Re-prepare new culture medium, inoculate the concentrated bacteria into the new culture medium for the next cycle of cultivation, and detect the nitrate nitrogen concentration (NO3 - -N), total phosphorus concentration (TP), dissolved oxygen concentration (DO), ammonia nitrogen concentration (NH4 + -N) and chemical oxygen demand (COD) concentration, centrifuge the liquid at the middle height of the culture solution to obtain concentrated strains;

[0120] (4) The same method as step (3) is used to carry out cultivation in each cycle. When the nitrate nitrogen removal rate of at least one cycle reaches more than 70%, a new culture medium is prepared, and the total phosphorus unit concentration of the culture medium is reduced by 20-85%. The concentrated strain obtained in the previous cycle is inoculated into the new culture medium for cultivation in the next cycle.

[0121] (5) Continue to cultivate each cycle in the same manner as step (3). When the nitrate nitrogen removal rate of at least one cycle reaches more than 70% and the total phosphorus removal rate reaches 55% to 90%, re-prepare a new culture medium, lower the unit concentration of ammonia nitrogen in the culture medium to 20% to 80%, and adjust the unit concentration of total phosphorus in the culture medium to the total phosphorus unit concentration optimized in the previous cycle. Inoculate the concentrated strain obtained in the previous cycle into the new culture medium for cultivation in the next cycle.

[0122] (6) Continue to cultivate each cycle in the same manner as in step (3). When the nitrate nitrogen removal rate of the culture solution reaches more than 70%, the total phosphorus removal rate reaches 75% to 90%, and the ammonia nitrogen removal rate reaches 75% to 90%, stop the cultivation to optimize the cultivation of the sulfur autotrophic denitrifying bacteria.

[0123] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0124] Example 1 Preparation of sulfur autotrophic denitrification culture medium

[0125] Prepare 1L sulfur autotrophic denitrification culture medium, the culture medium formula is:

[0126] KNO32g / L, NaHCO32g / L, KH2PO41.5g / L, MgCl2·6H2O 0.5g / L, NH4Cl 0.3g / L, FeSO4·7H2O 0.02g / L, Na2S2O3·5H2O 5.00g / L.

[0127] 1) According to the above formula, weigh KNO3, NaHCO3, MgCl2·6H2O, NH4Cl, and Na2S2O3·5H2O, dissolve in 800 ml of pure water, and place in a 1 L stoppered conical flask;

[0128] 2): Weigh KH2PO4 and dissolve it in 200 ml of pure water, and place it in a 1L stoppered conical flask;

[0129] 3): Sterilize two conical flasks at 121 ° C for 30 min, cool to room temperature in a water bath, and mix the solutions;

[0130] 4) Weigh FeSO4·7H2O and add it to the mixed solution to fully dissolve it. Add NaOH solution dropwise to adjust the pH of the solution to 7.5±0.1 to obtain 1 L of sulfur autotrophic denitrification culture solution.

[0131] Example 2 Preparation of sulfur autotrophic denitrification culture medium

[0132] Prepare 1L sulfur autotrophic denitrification culture medium, the culture medium formula is:

[0133] KNO32g / L, NaHCO31g / L, KH2PO40.5g / L, MgCl2·6H2O 0.3g / L, NH4Cl 0.5g / L, FeSO4·7H2O 0.01g / L, Na2S2O3·5H2O 5.00g / L.

[0134] 1) According to the above formula, weigh KNO3, NaHCO3, MgCl2·6H2O, NH4Cl, and Na2S2O3·5H2O, dissolve in 800 ml of pure water, and place in a 1 L stoppered conical flask;

[0135] 2): Weigh KH2PO4 and dissolve it in 200 ml of pure water, and place it in a 1L stoppered conical flask;

[0136] 3): Sterilize two conical flasks at 121 ° C for 30 min, cool to room temperature in a water bath, and mix the solutions;

[0137] 4) Weigh FeSO4·7H2O and add it to the mixed solution to fully dissolve it. Add NaOH solution dropwise to adjust the pH of the solution to 7.5±0.1 to obtain 1 L of sulfur autotrophic denitrification culture solution.

[0138] Example 3 Optimization of the cultivation of sulfur autotrophic denitrifying bacteria

[0139] The sludge in this embodiment was taken from the modified AAO anaerobic section activated sludge of Shenzhen Egongling Sewage Treatment Plant.

[0140] Step 1: Inoculate 80 ml of AAO anaerobic activated sludge with a suspended solids concentration of 10,000 mg / L into the sulfur autotrophic denitrification medium prepared in Example 1.

[0141] Step 2: Pass N2 for 20 minutes. After the ventilation is completed, quickly cover the rubber stopper. Place the culture medium in a water bath for constant temperature shaking culture at 30°C and a speed of 130 r / min. Connect the exhaust pipe of the rubber stopper to the water surface. The culture method is as follows: Figure 1 As shown;

[0142] Step 3: Cultivate under the conditions of step 2. When the culture medium changes from clear and transparent to yellow-brown turbid, and the pH does not drop within 2 hours, the first week of fermentation is over and the culture is stopped. At the end of the first cycle, the nitrate nitrogen concentration of the culture solution was tested, and the nitrate nitrogen removal rate was calculated to be 61.54%, and the total phosphorus removal rate was 18.90%. At the end of the period, take 200 ml of the liquid in the middle layer of the culture solution, centrifuge the liquid, and centrifuge it at 8000 rpm for 10 minutes. Take the lower layer of solid to obtain the concentrated strain;

[0143] Step 4: According to the nitrate nitrogen removal rate and total phosphorus removal rate calculated at the end of the first cycle, a new sulfur autotrophic denitrification medium is prepared. Since the nitrate nitrogen removal rate does not reach more than 70%, the medium is the same as the first cycle. The concentrated strain obtained in step 3 is inoculated into the new sulfur autotrophic denitrification medium for the next cycle of cultivation. The inoculation amount of the concentrated strain is 15% of the medium.

[0144] The second phase of cultivation was carried out in the same manner as step 2. At the end of the second period, the nitrate nitrogen concentration, total phosphorus concentration, dissolved oxygen concentration and ammonia nitrogen concentration of the culture solution were tested. The nitrate nitrogen removal rate was calculated to be 78.98%, and the total phosphorus removal rate was 17.91%. At the end of the period, 200 ml of the liquid in the middle layer of the culture solution was taken and centrifuged at 8000 rpm for 10 min. The solid layer at the bottom was taken to obtain the concentrated strain;

[0145] Step 5: According to the nitrate nitrogen removal rate and total phosphorus removal rate calculated at the end of the second cycle, the amount of KH2PO4 added to the culture medium is reduced to 0.5 g / L, a new sulfur autotrophic denitrification culture medium is prepared, and the concentrated strain obtained in step 4 is inoculated into the new sulfur autotrophic denitrification culture medium for the next cycle of cultivation. The inoculation amount of the concentrated strain is 15% of the culture medium;

[0146] The third period of cultivation was carried out in the same manner as step 2. At the end of the third period, the nitrate nitrogen concentration, total phosphorus concentration, dissolved oxygen concentration and ammonia nitrogen concentration of the culture solution were tested. The nitrate nitrogen removal rate was calculated to be 99.68%, and the total phosphorus removal rate was 58.11%. At the end of the period, 200 ml of the liquid in the middle layer of the culture solution was taken and centrifuged at 8000 rpm for 10 min. The solid layer at the bottom was taken to obtain the concentrated strain;

[0147] Step 6: According to the nitrate nitrogen removal rate and total phosphorus removal rate calculated at the end of the third cycle, the amount of KH2PO4 added to the culture medium is reduced to 0.3 g / L, a new sulfur autotrophic denitrification culture medium is prepared, and the concentrated strain obtained in step 5 is inoculated into the new sulfur autotrophic denitrification culture medium for the next cycle of cultivation. The inoculation amount of the concentrated strain is 15% of the culture medium;

[0148] The fourth period of cultivation was carried out in the same manner as step 2. At the end of the fourth period, the nitrate nitrogen concentration, total phosphorus concentration, dissolved oxygen concentration and ammonia nitrogen concentration of the culture solution were detected. The nitrate nitrogen removal rate was calculated to be 87.21%, the total phosphorus removal rate was 74.83%, and the ammonia nitrogen removal rate was 75.51%. The cultivation was terminated to obtain a high-concentration composite bacterial solution in which sulfur autotrophic denitrifying bacteria were the dominant species.

[0149] At the end of each cycle, the cycle duration and cycle nitrate nitrogen removal rate were recorded. The data are shown in Table 1 below. A cycle removal curve was drawn based on Table 1. Figure 3 shown.

[0150] Table 1. Cycle nitrate nitrogen removal

[0151]

[0152] From Table 1 and Figure 3 It can be seen that the denitrification capacity of the bacterial community in period four is 25.67% higher than that in period one, indicating that after a certain period of directional intensive cultivation, the average daily denitrification capacity of microorganisms has been improved.

[0153] The high-concentration composite bacterial solution is sent to a third-party testing agency to determine its microbial composition, such as Figure 4 As shown, the analysis results showed that the total proportion of sulfur autotrophic denitrifying bacteria (Sulfurimonas and Thiobacillus) reached 76.80%.

[0154] Example 4 Optimization of the cultivation of sulfur autotrophic denitrifying bacteria

[0155] The sludge in this embodiment was taken from the bottom mud of the upper reaches of Xixi River in Dongguan.

[0156] Step 1: Inoculate 100 ml of river sediment with a suspended solid concentration of 8000 mg / L into the sulfur autotrophic denitrification culture medium prepared in Example 2.

[0157] Step 2: Pass N2 for 20 minutes. After the ventilation is completed, quickly cover the rubber stopper. Place the culture medium in a water bath for constant temperature shaking culture at 30°C and a speed of 130 r / min. Connect the exhaust pipe of the rubber stopper to the water surface. The culture method is as follows: Figure 1 As shown;

[0158] Step 3: Cultivate under the conditions of step 2. When the culture medium changes from clear and transparent to yellow-brown turbid, and the pH does not drop within 2 hours, the first week of fermentation is over and the culture is stopped. At the end of the first cycle, the nitrate nitrogen concentration of the culture solution was tested, and the nitrate nitrogen removal rate was calculated to be 59.18%, the total phosphorus removal rate was 54.73%, and the ammonia nitrogen removal rate was 18.85%. At the end of the period, take 200 ml of the liquid in the middle layer of the culture solution, centrifuge the liquid, and the centrifugation conditions are 8000 rpm and 10 min. Take the lower layer of solid to obtain the concentrated strain;

[0159] Step 4: According to the nitrate nitrogen removal rate and total phosphorus removal rate calculated at the end of the first cycle, a new sulfur autotrophic denitrification medium is prepared. Since the nitrate nitrogen removal rate does not reach more than 70%, the medium is the same as the first cycle. The concentrated strain obtained in step 3 is inoculated into the new sulfur autotrophic denitrification medium for the next cycle of cultivation. The inoculation amount of the concentrated strain is 15% of the medium.

[0160] The second phase of cultivation was carried out in the same manner as step 2. At the end of the second period, the nitrate nitrogen concentration, total phosphorus concentration, dissolved oxygen concentration and ammonia nitrogen concentration of the culture solution were detected. The nitrate nitrogen removal rate was calculated to be 84.45%, the total phosphorus removal rate was 57.21%, and the ammonia nitrogen removal rate was 57.21%. At the end of the period, 200 ml of the liquid in the middle layer of the culture solution was taken and centrifuged at 8000 rpm for 10 min. The solid layer at the bottom was taken to obtain the concentrated strain;

[0161] Step 5: According to the nitrate nitrogen removal rate and total phosphorus removal rate calculated at the end of the second cycle, the addition amount of KH2PO4 and NH4Cl in the culture medium is reduced to 0.3 g / L, a new sulfur autotrophic denitrification culture medium is prepared, and the concentrated strain obtained in step 4 is inoculated into the new sulfur autotrophic denitrification culture medium for the next cycle of cultivation. The inoculation amount of the concentrated strain is 15% of the culture medium;

[0162] The third period of cultivation was carried out in the same manner as step 2. At the end of the third period, the nitrate nitrogen concentration, total phosphorus concentration, dissolved oxygen concentration and ammonia nitrogen concentration of the culture solution were detected. The nitrate nitrogen removal rate was calculated to be 96.47%, the total phosphorus removal rate was 75.32%, and the ammonia nitrogen removal rate was 40.16%. At the end of the period, 200 ml of the liquid in the middle layer of the culture solution was taken and centrifuged at 8000 rpm for 10 minutes. The solid layer at the bottom was taken to obtain the concentrated strain;

[0163] Step 6: According to the nitrate nitrogen removal rate and total phosphorus removal rate calculated at the end of the third cycle, the amount of NH4Cl added to the culture medium is reduced to 0.1 g / L, a new sulfur autotrophic denitrification culture medium is prepared, and the concentrated strain obtained in step 5 is inoculated into the new sulfur autotrophic denitrification culture medium for the next cycle of cultivation. The inoculation amount of the concentrated strain is 15% of the culture medium;

[0164] The fourth period of cultivation was carried out in the same manner as step 2. At the end of the fourth period, the nitrate nitrogen concentration, total phosphorus concentration, dissolved oxygen concentration and ammonia nitrogen concentration of the culture solution were detected. The nitrate nitrogen removal rate was calculated to be 86.74%, the total phosphorus removal rate was 76.01%, and the ammonia nitrogen removal rate was 82.69%. The cultivation was terminated to obtain a high-concentration composite bacterial solution in which sulfur autotrophic denitrifying bacteria were the dominant species.

[0165] The cycle duration and cycle nitrate nitrogen removal rate were recorded. The data are shown in Table 2 below. A cycle removal curve was drawn based on Table 2. Figure 5 shown.

[0166] Table 2. Cycle nitrate nitrogen removal

[0167]

[0168] From Table 2 and Figure 5 It can be seen that the denitrification capacity of the bacterial community in period four was 27.56% higher than that in period one, and the denitrification capacity of the bacterial community was enhanced.

[0169] The high-concentration composite bacterial solution is sent to a third-party testing agency to determine its microbial composition, such as Figure 6 As shown, the analysis results showed that the total proportion of sulfur autotrophic denitrifying bacteria (Sulfurimonas and Thiobacillus) reached 86.60%.

[0170] Example 5 Sulfur autotrophic denitrifying bacteria biofilm experiment

[0171] Two experimental columns were built in the field, named column A and column B, with a height of 1800 mm and a diameter of 144 mm. The matrix of the experimental columns was the same, which was: 100 mm gravel, 1200 mm light sulfur-based filler, and 50 mm zeolite.

[0172] Column A was added with 5L of high-concentration composite bacterial solution obtained in Example 4 and allowed to form biofilm for one week; column B was allowed to form biofilm naturally. The experimental column used a peristaltic pump to evenly inlet water, and the inlet water quality was the first-level A effluent from a municipal sewage treatment plant. The hydraulic retention time was 6h. After the effluent was stable, samples were taken twice a week and the operation was stable for 2 months. After sampling every week, the nitrate nitrogen concentration (NO3 - -N), ammonia nitrogen concentration (NH4 +-N), and calculate the nitrate nitrogen removal rate and total nitrogen removal rate. Operation data is shown below Figure 7 and Figure 8 .

[0173] Depend on Figure 7 and Figure 8 It can be seen that during operation,

[0174] The total nitrogen removal efficiency of column A was 49.57% to 94.87%, and the nitrate nitrogen removal efficiency of column A was 59.21% to 98.97%;

[0175] The total nitrogen removal efficiency of column B was 21.09% to 51.71%, and the nitrate nitrogen removal efficiency of column B was 20.53% to 50.25%;

[0176] The total nitrogen removal rate and nitrate nitrogen removal rate of column A were significantly better than those of column B.

[0177] The experimental results show that the use of sulfur autotrophic bacteria to form biofilm can accelerate the domestication process of sulfur autotrophic denitrification reactors / process sections and greatly shorten the domestication cycle. The use of improved culture medium formula reduces the risk of nitrogen and phosphorus pollution of the water environment during the domestication process. At the same time, the addition of sulfur autotrophic bacteria can significantly increase the removal rate of total nitrogen under low carbon-nitrogen ratios, which can be used in the field of deep sewage purification and low carbon-nitrogen ratio sewage denitrification and phosphorus removal.

Claims

1. A method for optimizing the cultivation of sulfur autotrophic denitrifying bacteria, characterized in that: Anaerobic activated sludge is used as the original bacterial strain, and sulfur autotrophic denitrification culture medium is used for constant temperature continuous periodic directional culture. At the end of each period, the fermentation liquid is concentrated and inoculated into the next period culture medium; the nitrate nitrogen concentration, total phosphorus concentration and / or ammonia nitrogen concentration of the culture liquid at the end of each period are detected, and the ammonia nitrogen concentration and / or total phosphorus concentration in the next period culture medium are adjusted according to the detection results, and the optimization culture of the sulfur autotrophic denitrifying bacteria group is completed after the nitrate nitrogen removal rate, total phosphorus removal rate and ammonia nitrogen removal rate of the culture liquid meet the standards; The sulfur autotrophic denitrification culture medium contains per liter: KNO3 1~ 2g, NaHCO3 1~ 2g, KH2PO4 0.3~ 2g, MgCl2·6H2O 0.2~0.80g, NH4Cl 0.1~0.5g, FeSO4·7H2O 0.01~0.03g, Na2S2O3·5H2O 3~6.00g; The solvent of the sulfur autotrophic denitrification culture medium is sterile water; The specific steps of the culture method are: (1) Inoculate the anaerobic activated sludge into a conical flask containing sulfur autotrophic denitrification culture medium, introduce N2 into the conical flask, stop aeration after 20 min, seal the conical flask, and place it in a water bath for constant temperature culture; (2) The culture cycle is 3 to 5 days. During the fermentation process, when the sulfur autotrophic denitrification culture medium changes from clear to yellow-brown turbid, and the pH does not drop within 2 hours, the first stage of fermentation ends. At the end of the cycle, the nitrate nitrogen (NO3 - -N) concentration, total phosphorus (TP) concentration, dissolved oxygen (DO) concentration, ammonia nitrogen (NH4 + -N) concentration and chemical oxygen demand (COD) concentration, centrifuge the liquid at the middle height of the culture solution to obtain concentrated strains; (3) Re-prepare a new sulfur autotrophic denitrification medium, inoculate the concentrated bacteria into the new sulfur autotrophic denitrification medium for the next cycle of cultivation, and detect the nitrate nitrogen (NO3 - -N) concentration, total phosphorus (TP) concentration, dissolved oxygen (DO) concentration, ammonia nitrogen (NH4 + -N) concentration and chemical oxygen demand (COD) concentration, centrifuge the upper liquid in the culture solution to obtain concentrated strains; (4) The same method as step (3) is used to carry out cultivation in each cycle. When the nitrate nitrogen removal rate of at least one cycle reaches more than 70%, a new sulfur autotrophic denitrification medium is re-prepared, and the total phosphorus unit concentration of the sulfur autotrophic denitrification medium is reduced by 20-85%. The concentrated strain obtained in the previous cycle is inoculated into the new sulfur autotrophic denitrification medium for cultivation in the next cycle; (5) Continue to cultivate each cycle in the same manner as step (3). When the nitrate nitrogen removal rate of at least one cycle reaches more than 70% and the total phosphorus removal rate reaches 55% to 90%, re-prepare a new sulfur autotrophic denitrification culture medium, adjust the unit concentration of ammonia nitrogen in the culture medium to 20% to 80%, and adjust the unit concentration of total phosphorus in the culture medium to the total phosphorus unit concentration optimized in the previous cycle. Inoculate the concentrated strain obtained in the previous cycle into the new sulfur autotrophic denitrification culture medium for the next cycle of cultivation. (6) Continue to cultivate each cycle in the same manner as in step (3). Stop cultivation when the nitrate nitrogen removal rate of the culture medium reaches more than 80%, the total phosphorus removal rate reaches 75% to 90%, and the ammonia nitrogen removal rate reaches 75% to 90%.

2. The method for optimizing the cultivation of a sulfur autotrophic denitrifying bacteria community according to claim 1, characterized in that: The method for preparing the sulfur autotrophic denitrification culture medium comprises the following steps: (1) Weigh KNO3, NaHCO3, MgCl2·6H2O, NH4Cl and Na2S2O3·5H2O, fully dissolve them, and sterilize them to obtain sterile solution A; (2) Weigh KH2PO4, fully dissolve it, and sterilize it to obtain sterile solution B; (3) Sterile solution A and sterile solution B are mixed, FeSO4·7H2O is added, and a pH adjuster is added dropwise to adjust the pH of the solution to 7.5±0.1 to obtain a sulfur autotrophic denitrification medium.

3. The method for optimizing the cultivation of a sulfur autotrophic denitrifying bacteria community according to claim 1, characterized in that: The anaerobic activated sludge is taken from one or more of the anaerobic process section of the domestic sewage treatment system, river bottom mud, filter tank, and UASB reactor.

4. The method for optimizing the cultivation of a sulfur autotrophic denitrifying bacteria group according to claim 1 or 3, characterized in that: The anaerobic activated sludge is inoculated in the sulfur autotrophic denitrification medium for the first cycle of cultivation. The suspended solid concentration of the anaerobic activated sludge is 8000-12000 mg / L, the dissolved oxygen concentration is 0.5 mg / L, and the inoculation ratio of the anaerobic activated sludge is 5%-20% of the sulfur autotrophic denitrification medium.

5. The method for optimizing the cultivation of a sulfur autotrophic denitrifying bacteria community according to claim 1, characterized in that: The inoculation ratio of the concentrated bacteria is 10% to 20% of the sulfur autotrophic denitrification culture medium.

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