A production process for producing autotrophic denitrifying bacteria by fermentation

Abstract

The present invention belongs to the field of wastewater treatment and relates to a production process for fermenting and producing an autotrophic denitrifying bacterial agent, comprising: inoculating sludge containing denitrifying autotrophic bacteria into an ascending anaerobic reactor, using bluestone, siderite and elemental sulfur as fillers of the reactor, and sequentially loading the fillers and sludge in batches during the filling process to mix the mud and water evenly; after the ascending anaerobic reactor is filled, biofilm acclimation is carried out to obtain autotrophic denitrifying bacterial cells; the autotrophic denitrifying bacterial cells are produced on a large scale using a fermentation method. The present invention constructs a bacterial cell that uses elemental sulfide and iron compounds as electron donors for autotrophic denitrification; and uses a fermentation method to produce an autotrophic denitrifying bacterial agent on a large scale. The bacterial agent has a stronger denitrification ability and is more suitable for denitrification treatment of industrial and municipal wastewater.

Description

Technical Field

[0001] The invention belongs to the field of wastewater treatment, and particularly relates to a production process for producing an autotrophic bacterial agent by a fermentation method. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] The total nitrogen content in wastewater is increasing, with nitrate being the primary pollutant. Directly discharging nitrate-containing wastewater into the environment can cause eutrophication. Therefore, removing nitrate from water has become an urgent issue.

[0004] Currently, in the field of denitrification, biological denitrification technology is the most economical and effective method for removing nitrates. Traditional nitrate removal processes generally use heterotrophic denitrifying bacteria for denitrification. This strain uses organic carbon sources such as methanol and sodium acetate as electron donors. In an anaerobic environment, heterotrophic denitrifying bacteria convert nitrates into nitrogen gas, thereby achieving the purpose of denitrification. Although this strain can effectively remove total nitrogen from sewage, it consumes a large amount of carbon sources, has high operating costs, and produces a large amount of sludge, which brings great cost pressure to enterprises.

[0005] However, in traditional sewage treatment plants, the proportion of autotrophic denitrifying bacteria in the activated sludge is relatively low, and only a small amount survives in the anaerobic section. Due to the lack of reaction substrates, it is almost impossible to achieve the denitrification effect. The acclimation cycle is long when the original activated sludge is used for acclimation, the process system starts slowly, and the debugging and operation are too complicated. Summary of the Invention

[0006] In view of the above defects, the purpose of the present invention is to produce a highly efficient autotrophic denitrifying bacterial agent that can be directly added into an anoxic tank to increase the abundance of denitrifying bacteria.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a production process for producing an autotrophic denitrifying bacterial agent by fermentation, comprising:

[0009] The sludge containing denitrifying autotrophic bacteria is inoculated into the ascending anaerobic reactor. Bluestone, siderite and elemental sulfur are used as the fillers of the reactor. The fillers and sludge are loaded in batches during the filling process to ensure that the mud and water are evenly mixed.

[0010] After the ascending anaerobic reactor is filled, biofilm formation and acclimation are carried out to obtain autotrophic denitrification bacteria;

[0011] The autotrophic denitrification bacteria are produced on a large scale by a fermentation method to obtain the product.

[0012] Compared with traditional denitrification, the present invention provides a chemoautotrophic bacterium, the main strain of which, Thiobacillus denitrificans, can use elemental sulfur and its sulfide as electron donors to directly convert nitrate into nitrogen gas under anaerobic conditions. Elemental sulfur and sulfide are abundant and inexpensive. Using such reducing substances as electron donors for denitrification can fundamentally reduce the amount of organic carbon source added and reduce the cost of sewage operation.

[0013] At the same time, batch fermentation produces efficient autotrophic denitrifying bacteria and adds them into the reactor, which can quickly start the reactor, greatly shorten the commissioning period, and improve system stability.

[0014] The second aspect of the present invention provides an autotrophic denitrifying bacterial agent produced by the above process.

[0015] The third aspect of the present invention provides the use of the above-mentioned autotrophic denitrifying bacteria agent in the denitrification treatment of industrial wastewater or municipal wastewater.

[0016] The beneficial effects of the present invention are:

[0017] (1) The present invention adopts the method of sludge acclimation to screen strains. Although the purity of the bacterial agent is not as high as that of a single denitrifying Thiobacillus, the bacterial community is rich and contains a variety of autotrophic denitrifying bacteria. The various denitrifying bacteria grow synergistically during the fermentation process. The acid-producing autotrophic bacteria and the alkali-producing autotrophic bacteria work together to achieve their respective good growth rates. Therefore, the denitrification ability is stronger and it is more suitable for the denitrification treatment of industrial wastewater and municipal wastewater.

[0018] (2) Construct a bacterial cell that uses elemental sulfide and its compounds as electron donors for autotrophic denitrification; and use fermentation to produce autotrophic denitrification bacteria on a large scale.

[0019] (3) The main bacterial species of this agent is Denitrifying Bacillus, a Gram-negative bacterium with a short rod-shaped cell structure, an internal folded structure, a flagellum at the end, and strong motility. It is an obligate autotrophic and facultative anaerobic bacterium.

[0020] (4) The production process is characterized by: the inoculation amount of denitrifying bacteria is 4%-8%, the culture temperature is, the fermentation time is 5 hours, and the reaction substrate is elemental sulfur.

[0021] (5) The operation method of the present application is simple, low-cost, universal, and easy to scale up for production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 This is a process flow diagram of an ascending reactor;

[0024] Among them, the reactor body is a plexiglass column with a height of 45 cm and a bottom diameter of 10 cm. 1 is a water distribution bucket, 2 is a water inlet pump, 3 is a gas collecting bottle, 4 is a lower sampling port, 5 is a middle sampling port, 6 is an upper sampling port, 7 is a filler, and 8 is a secondary sedimentation tank.

[0025] Figure 2 This is a graph showing the changes in the water quality of the reactor effluent. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] A production process for producing an autotrophic denitrifying bacterial agent by fermentation, comprising:

[0028] Obtaining denitrifying autotrophic bacteria:

[0029] 300 ml of flocculent sludge in the anaerobic tower of the municipal sewage treatment plant and activated sludge in the anoxic tank were taken and inoculated into the ascending anaerobic reactor. Bluestone, siderite and elemental sulfur were used as fillers of the reactor. The particle size was 2-3 mm and the filling height was 40 cm. The fillers and sludge should be loaded in batches in the filling process to ensure uniform mixing of mud and water.

[0030] The inlet water is artificially distributed, and the test water component is: NO3 - -N is prepared with NaNO3, the concentration of NaNO3 is 200ml / L, and the other culture medium components are: potassium dihydrogen phosphate 10mg / L, calcium chloride dihydrate 240mg / L, calcium chloride heptahydrate 320mg / L, sodium bicarbonate 800mg / L, trace element I 1mg / L, trace element II 1.25mg / L. See Table 1

[0031] Table 1 Trace element ratio

[0032]

[0033] After the reactor was filled, biofilm formation was carried out under the following conditions. Since denitrifying autotrophic bacteria formed biofilms slowly, an appropriate amount of sodium acetate was added as a carbon source in the early stage of acclimation, with COD / N = 5:1. The sodium acetate content was then gradually reduced, and the reactor finally entered the complete autotrophic stage. On the 50th day of operation, the biofilms in the upper, middle and lower parts of the reactor were taken for scanning electron microscopy observation.

[0034] The microorganisms observed in the present invention were all short rod-shaped. High-throughput sequencing technology was used to detect and analyze 16srRNA biodiversity. The results showed that the abundance of Sulfurimanas was 21%-24%, and the abundance of Methylophilales was 51%-57%.

[0035] The strains screened out by the present invention have multiple sulfur oxidation and denitrification capabilities. Although their purity is not as high as that of a single strain of bacterial agent, they have higher biological abundance, stronger shock resistance and denitrification capabilities, and are more suitable for denitrification treatment of industrial wastewater and municipal sewage treatment plants.

[0036] The present invention provides a method for preparing an autotrophic denitrifying bacterial agent, which mainly comprises the following steps:

[0037] Step 1: Prepare seed solution, prepare the configured culture medium, inoculate 4% of the selected autotrophic denitrifying bacteria into the culture medium, and after culturing for 2 hours, detect the OD value of the culture medium to monitor the growth of the strain, and detect the OD value of the culture medium every 1 hour, while gradually changing the inoculation amount. After 12 hours of cultivation, determine the culture medium with the largest OD value as the seed solution, and record the inoculation amount and culture time as the optimal inoculation amount and inoculation time.

[0038] Step 2: Fermentation: Prepare a fermentation broth. Inoculate the seed solution prepared in Step 1 into the fermenter. Set the pH to 7-7.5 and the temperature to 30°C. The entire fermentation process is anaerobic, but nitrogen will be produced during the fermentation process. Ensure that the air pressure in the tank does not fluctuate significantly during the fermentation process and that the breathing valve operates normally. After 2 hours of fermentation, test the OD value and sodium nitrate concentration in the tank. Based on the test results, draw a bacterial growth curve to determine the optimal fermentation end point. The fermentation time is 20 hours, and the OD concentration reaches approximately 2.3 at the end. 16srRNA biodiversity analysis shows that the abundance of denitrifying Thiobacillus is approximately 21%.

[0039] The components of the seed liquid culture medium described in step 1 are: sodium nitrate 100 ml / L, peptone 50 mg / L, potassium dihydrogen phosphate 10 mg / L, calcium chloride dihydrate 240 mg / L, calcium chloride heptahydrate 32 mg / L, sodium bicarbonate 800 mg / L, trace element I 1 mg / L, trace element II 1.25 mg / L, and the trace element ratio is shown in Table 1.

[0040] The components of the fermentation medium in step 2 are: 100 mg of sulfur powder, 50 mg of iron powder, 50 ml / L of potassium dihydrogen phosphate, 32 ml / L of calcium chloride, 300 ml / L of magnesium sulfate, 800 mg / L of sodium bicarbonate, 1 ml / L each of trace element I and trace element II. The trace element ratio is shown in Table 1.

[0041] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0042] A composite bacterial agent for denitrification in a sewage treatment plant comprises Thiobacillus denitrificans and other autotrophic denitrifying bacteria, wherein the abundance of Thiobacillus denitrificans is 21%-24%.

[0043] Example 1:

[0044] Obtaining autotrophic denitrifying bacteria:

[0045] Test water components: NO3 - -N is prepared from Na NO3, and the concentration of Na NO3 is 200 ml / L. The other components of the culture medium are: potassium dihydrogen phosphate 10 mg / L, calcium chloride dihydrate 240 mg / L, calcium chloride heptahydrate 320 mg / L, sodium bicarbonate 800 mg / L, trace element I 1 mg / L, trace element II 1.25 mg / L. The trace element ratio is shown in Table 1.

[0046] 300ml of flocculent sludge from the anaerobic tower of a municipal sewage treatment plant and activated sludge from the anoxic tank were inoculated into an ascending anaerobic reactor. Bluestone, siderite, and elemental sulfur were used as fillers, all with a particle size of 2-3mm. First, a 10cm layer of bluestone was added as a backing layer, which also served as an alkaline substance to replenish the alkalinity consumed by sulfur autotrophic denitrification. Siderite and elemental sulfur were then added as substrates for the sulfur autotrophic reaction at a ratio of 2:1. The reaction substrates were filled to a height of 30cm, for a total height of 40cm. The reactor was then filled with municipal sewage treatment plant sludge at a concentration of 5g / ml, reaching 80% of its capacity. The filler and sludge were added in batches to ensure uniform mixing of the sludge and water.

[0047] After the reactor is filled, biofilm formation is carried out under the following conditions. Since the biofilm formation of denitrifying autotrophic bacteria is slow, an appropriate amount of sodium acetate is added as a carbon source in the early stage of acclimation, COD / N=5:1, and then the sodium acetate content is gradually reduced. Finally, the reactor enters the complete autotrophic stage.

[0048] In the early stage of reactor startup, the nitrogen removal rate was very low, only 2 mgN / (L·h). On the 20th day of reactor operation, the nitrogen removal rate increased significantly, reaching 80%. By controlling the influent sodium nitrate concentration and shortening the HRT to 4h, the reactor effluent sodium nitrate concentration was below 20 mg / L. On the 40th day, the effluent sodium nitrate concentration was below 5 mg / L, and the removal rate reached 0.95 kg / (m 3 ·d).

[0049] On the 50th day of operation, the biofilms in the upper, middle and lower parts of the reactor were observed under a scanning electron microscope.

[0050] The microorganisms observed in the present invention were all short rod-shaped. High-throughput sequencing technology was used to detect and analyze 16srRNA biodiversity. The results showed that the abundance of Sulfurimanas was 21%-24%, and the abundance of Methylophilales was 51%-57%.

[0051] Example 2:

[0052] Fermentation seed liquid preparation:

[0053] The components of the seed liquid culture medium are: sodium nitrate 100 ml / L, peptone 50 mg / L, potassium dihydrogen phosphate 10 mg / L, calcium chloride dihydrate 240 mg / L, calcium chloride heptahydrate 32 mg / L, sodium bicarbonate 800 mg / L, trace element I 1 mg / L, trace element II 1.25 mg / L.

[0054] Prepared culture medium was inoculated with 5% of the autotrophic denitrifying bacteria selected in Example 1. After 2 hours of incubation, the OD value of the culture medium was measured to monitor the growth of the strain. The OD value of the culture medium was also measured every 4 hours. After 12 hours of incubation, the removal efficiency of the composite bacterial agent seed solution was observed. The data are shown in Table 2. A gradient inoculum size experiment was also conducted, with the inoculum size ranging from 3% to 8%. The data are shown in Table 3.

[0055] Table 2 Growth of seed solution at different culture times

[0056]

[0057] Table 3 Growth of different inoculum amounts of seed solution

[0058]

[0059] Example 3

[0060] Microbial fermentation culture:

[0061] The components of the fermentation medium are: sodium nitrate 200 ml / L, sulfur powder 100 mg, iron powder 50 mg, potassium dihydrogen phosphate 50 ml / L, calcium chloride 32 ml / L, magnesium sulfate 300 ml / L, sodium bicarbonate 800 mg / L, trace element I and trace element II 1 ml / L each. The trace element ratio is shown in Table 1.

[0062] The seed liquid prepared in Example 2 was inoculated into the fermentation tank, and the pH was set to 7-7.5 and the temperature was set to 30°C. The entire fermentation process was anaerobic fermentation, but nitrogen gas was produced during the fermentation process. During the fermentation process, it was necessary to ensure that the air pressure in the tank did not change significantly and to ensure the normal operation of the breathing valve. After 2 hours of fermentation, the OD value and sodium nitrate concentration in the tank were detected. The bacterial growth curve was drawn according to the test results to determine the optimal fermentation end point. The fermentation time was 20 hours, and the OD concentration reached about 2.1 at the end. According to the 16srRNA biodiversity detection and analysis, the abundance of denitrifying Thiobacillus was about 21%. The experimental data are shown in Table 4

[0063] Table 4 Fermentation tank bacterial concentration monitoring data

[0064]

[0065] Example 4

[0066] Actual wastewater treatment:

[0067] Table 5 Actual wastewater quality parameters

[0068]

[0069] The reactor is an ascending reactor, using bluestone, siderite and elemental sulfur as the reactor filler, the particle size is 2-3mm, the filling height is 40cm, the filling process is loaded in batches and the composite bacterial agent prepared in Example 3 is loaded to ensure that the mud and water are mixed evenly. The reactor is operated at room temperature (21-31℃) for 17 weeks, and the water quality changes are shown in Figure 2. Figure 2 .

[0070] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or to replace portions thereof with equivalents. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A production process for producing an autotrophic denitrifying bacterial agent by fermentation, characterized in that: include: The sludge containing denitrifying autotrophic bacteria is inoculated into the ascending anaerobic reactor. Bluestone, siderite and elemental sulfur are used as the fillers of the reactor. The fillers and sludge are loaded in batches during the filling process to ensure that the mud and water are evenly mixed. After the ascending anaerobic reactor is filled, biofilm formation and acclimation are carried out to obtain autotrophic denitrifying bacteria; The autotrophic denitrification bacteria are produced on a large scale using a fermentation method to obtain; The inlet water is artificially distributed, with a concentration of 200 ml / L of NaNO3, 10 mg / L of potassium dihydrogen phosphate, 240 mg / L of calcium chloride dihydrate, 320 mg / L of calcium chloride heptahydrate, 800 mg / L of sodium bicarbonate, 1 mg / L of trace elements I, and 1.25 mg / L of trace elements II. The trace elements I include: EDTA 5000 mg / L, ferrous sulfate 5000 mg / L; The trace elements II include: EDTA 1000 mg / L, boric acid 14 mg / L, manganese chloride tetrahydrate 990 mg / L, copper sulfate pentahydrate 250 mg / L, zinc sulfate heptahydrate 430 mg / L, nickel chloride hexahydrate 190 mg / L, sodium selenate decahydrate 210 mg / L, sodium molybdate dihydrate 220 mg / L; In the early acclimation stage, sodium acetate was added as a carbon source, COD / N=4~5:1, and then the sodium acetate content was gradually reduced, and finally the reactor entered the complete autotrophic stage.

2. The process for producing an autotrophic denitrifying agent by fermentation according to claim 1, wherein: The particle size of the fillers is 2-3 mm, and the filling height is 40-42 cm.

3. The process for producing an autotrophic denitrifying agent by fermentation according to claim 1, wherein: The components of the seed liquid culture medium are: sodium nitrate 100 ml / L, peptone 50 mg / L, potassium dihydrogen phosphate 10 mg / L, calcium chloride dihydrate 240 mg / L, calcium chloride heptahydrate 32 mg / L, sodium bicarbonate 800 mg / L, trace element I 1 mg / L, trace element II 1.25 mg / L.

4. The process for producing an autotrophic denitrifying bacterial agent by fermentation according to claim 1, wherein: The ingredients of the fermentation medium are: 100 mg of sulfur powder, 50 mg of iron powder, 50 ml / L of potassium dihydrogen phosphate, 32 ml / L of calcium chloride, 300 ml / L of magnesium sulfate, 800 mg / L of sodium bicarbonate, and 1 ml / L each of trace element I and trace element II.

5. The autotrophic denitrifying bacterial agent produced by the process according to any one of claims 1 to 4.

6. Use of the autotrophic denitrifying bacterial agent according to claim 5 in denitrification treatment of industrial wastewater or municipal wastewater.

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