Synchronous oil removal and denitrification bacteria and screening, application, and preparation method of bacterial agent

Abstract

The invention discloses a synchronous de-oiling and denitrifying bacterium and a method for screening, applying and preparing a microbial agent. The strain is synchronous de-oiling and denitrifying bacterium CYCN‑1 (Pseudomonas koreensis CYCN‑1), which is deposited in China Center for Type Culture Collection, address: Wuhan University, China, deposit number: CCTCC NO:M 20221776; deposit date: November 14, 2022. The synchronous de-oiling and denitrifying bacterium of the invention is a denitrifying bacterium capable of producing lipase, which can be rapidly propagated in an aerobic pool of a sewage treatment device, become a dominant bacterium, continuously secrete lipase, degrade animal and plant oils and fats, and provide a carbon source necessary for denitrification, thereby improving the de-oiling and denitrification capacity of sewage treatment.

Description

Technical Field

[0001] The invention relates to a new strain of bacteria, namely, synchronous oil removal and denitrification bacteria CYCN-1 (Pseudomonas koreensis CYCN-1), and the use of the strain to improve the grease degradation and denitrification capabilities in the treatment of water environments with much kitchen grease. Background Art

[0002] In recent years, with the rapid development of the economy and the improvement of people's living standards, catering wastewater has been increasing, causing great trouble to our lives, seriously polluting the living environment and affecting human physical and mental health. Catering oily wastewater is a high-concentration organic wastewater with very complex components. It contains a variety of animal and plant oils, high turbidity, high salinity, high COD and ammonia nitrogen concentrations, and grease is easy to agglomerate, which can easily lead to pipe siltation and biochemical pool blockage, bringing difficulties to the treatment of oily wastewater. In order to solve this problem, many methods have been used to deal with this situation. Among them, although the physical and chemical methods have better treatment effects, they will produce secondary pollution and have high operation and maintenance costs. In contrast, biological methods will not cause secondary pollution to the environment and have received widespread attention.

[0003] At present, a large number of studies have been reported on the microbial degradation of oil in domestic sewage, mainly focusing on the screening and isolation of microbial strains and lipases with high oil degradation efficiency from the environment. Although many reports have been published on the isolation and identification of highly efficient oil-degrading microorganisms, most of them characterize the oil degradation performance of the strains under laboratory conditions, and the denitrification performance of the same strain has not been verified. According to the mechanism of biological denitrification, sufficient carbon source is a necessary condition to ensure that the total nitrogen meets the standard during sewage treatment. Waste cooking oil is decomposed into glycerol and fatty acids under the action of microorganisms, and finally decomposed into carbon dioxide and water. It is safe, non-toxic and does not produce secondary pollution, so it can be used as a carbon source for the normal growth of microorganisms. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art in treating oily catering wastewater by microbial methods, and to provide a new strain of bacteria - simultaneous oil removal and denitrification bacteria CYCN-1 (Pseudomonas koreensis CYCN-1), and a method for using the strain to improve the oil degradation efficiency and denitrification efficiency in wastewater treatment.

[0005] In order to achieve the above-mentioned purpose of the present invention, the present invention adopts the following technical solutions:

[0006] The present invention first provides a synchronous oil-removal and denitrification bacterium, the strain is synchronous oil-removal and denitrification bacterium CYCN-1 (Pseudomonas koreensis CYCN-1), which is preserved in the China Center for Type Culture Collection, address: Wuhan University, China, preservation number: CCTCC NO:M 20221776; preservation date: November 14, 2022.

[0007] As a preferred embodiment of the present invention, the colony morphology of the synchronous oil removal and denitrification bacteria is cultured on an LB plate at 30°C for 12 hours. The colonies are smooth and round, with a diameter of 2 to 3 mm, plump and raised, opaque, light yellow, with neat edges, smooth, moist and shiny, sticky and easy to pick up, and the colonies increase in size with time, and the diameter is ≥5 mm after a few days.

[0008] As a preferred embodiment of the present invention, the cell morphology of the synchronous oil-removing and denitrifying bacteria is that the cells of the 24h culture have straight rods with round ends and a diameter of 0.5-1.2 μm; single, paired or short chain, no spores, no power; the physiological and biochemical characteristics are Gram-negative, facultative aerobic, chemoheterotrophic, and can degrade lipase substrates, forming transparent circles on soybean oil and short-chain oil substrate culture media, and the species identification is carried out using the bacterial 16S rDNA sequence sequencing method, and the sequence is as shown in EQ NO:1.

[0009] The present invention also provides the use of the above-mentioned simultaneous oil removal and denitrification bacteria in oil degradation.

[0010] The present invention also provides the use of the above-mentioned simultaneous oil-removing and denitrifying bacteria in denitrification treatment.

[0011] The present invention also provides the use of the above-mentioned synchronous oil-removing and denitrifying bacteria in the treatment of oily domestic sewage in farmhouses.

[0012] The present invention also provides a method for screening the above-mentioned simultaneous oil removal and denitrification bacteria, the screening method comprising the following steps:

[0013] a) Enrichment culture: Obtain a fat block, use the fat block as a carbon source, perform enrichment culture in an acclimation medium, and specify a shaking culture time, which is the first stage of culture; transfer the culture solution in the second acclimation cycle to the second stage of acclimation medium, specify a shaking culture time, which is the second stage of culture, and perform multiple acclimation;

[0014] b) Isolation and purification: The domesticated and enriched bacterial solution is diluted into different gradient bacterial suspensions by gradient dilution method, and streaked on LB solid plate culture medium to obtain single colonies;

[0015] c) Initial screening of oil removal performance: Use an inoculation loop to select a single colony point and inoculate it into the lipase plate culture medium for screening, place it in a constant temperature incubator and culture it for 2 days, select the strain that produces a clear transparent circle and inoculate it into a liquid culture medium, and place it in an oscillator for culture;

[0016] d) Initial screening of denitrification performance: When a single colony is selected by the inoculation loop and inoculated onto the lipase plate, the single colony is also selected and cultured on a 96-well plate. After the specified culture period, the bacterial solution is taken from each 96-well plate and placed on the NH 4+ with NO 2- In the verification plate, add ammonia nitrogen and nitrite color developing agent and observe the color change;

[0017] e) Selecting a strain that produces a larger transparent circle and obvious color development in a 96-well plate, culturing it in a medium with soybean oil as a carbon source and ammonium chloride as the only nitrogen source, taking samples regularly to detect the concentrations of ammonia nitrogen, total nitrogen and animal and vegetable oils in the medium; selecting the strain with the highest oil degradation rate and denitrification efficiency;

[0018] f) Rescreening: Select strains that produce larger transparent circles and obvious color in 96-well plates, culture them in a culture medium with soybean oil as the carbon source and ammonium chloride as the only nitrogen source, and take samples regularly to detect the concentrations of ammonia nitrogen, total nitrogen and animal and vegetable oils in the culture medium; select the strain with the highest oil degradation rate and denitrification efficiency and name it CYCN-1.

[0019] The present invention also provides a method for preparing the above-mentioned synchronous oil removal and denitrification bacteria agent, which comprises inoculating the synchronous oil removal and denitrification bacteria Pseudomonas koreensis CYCN-1 into a liquid LB culture medium and culturing it on a shaking table; then pouring the seed bacterial liquid into a fermentation tank for expansion culture; and mixing the cultured bacterial liquid and the culture medium, and sealing and refrigerating them.

[0020] As a preferred embodiment of the present invention, the culture medium for the fermentation tank expansion culture is configured according to the following proportions: 20L distilled water, 200g tryptone, 100g yeast powder, 200g sodium chloride, 40g soybean oil, pH is controlled at 7.0-7.2, and wet heat sterilization is performed at 121°C for 30min.

[0021] The present invention also provides an application of the bacterial agent obtained by the above-mentioned preparation method, taking the influent of the sewage treatment station of the decentralized sewage station of the farmhouse in the village scenic area, controlling the concentrations of ammonia nitrogen and total nitrogen to 50mg / L-60mg / L, and the oil content to 2g / L; the synchronous oil removal and denitrification bacterial agent obtained by the preparation method described above is added to the prepared oily sewage at an amount of 1%.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The simultaneous oil removal and denitrification bacteria of the present invention are denitrification bacteria that can produce lipase, can rapidly multiply in the aerobic pool of the sewage treatment equipment, become dominant bacteria, continuously secrete lipase, degrade animal and plant oils and fats, and provide the necessary carbon source for denitrification, thereby improving the oil removal and denitrification capacity of the sewage treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the transparent circle produced on the lipase verification culture medium of the synchronous oil removal and denitrification bacteria Pseudomonas koreensis CYCN-1 in Example 1.

[0025] Figure 2 It is a growth curve diagram of the synchronous oil removal and denitrification bacteria Pseudomonas koreensis CYCN-1 in Example 3;

[0026] Figure 3 This is a diagram showing the oil degradation effect of the synchronous oil removal and denitrification bacteria Pseudomonas koreensis CYCN-1 of Example 5 when soybean oil is the only carbon source;

[0027] Figure 4 This is a diagram showing the removal effect of ammonia nitrogen and total nitrogen by the synchronous oil-removing and denitrifying bacteria Pseudomonas koreensis CYCN-1 of Example 5 when ammonia nitrogen is the only nitrogen source. Specific implementation methods

[0028] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0029] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0030] The simultaneous oil-removal and denitrification bacteria CYCN-1 (Pseudomonas koreensis CYCN-1) provided in the present invention is deposited in the China Center for Type Culture Collection, address: Wuhan University, China, deposit number: CCTCC NO:M 20221776; deposit date: November 14, 2022.

[0031] Furthermore, the colony morphology was determined by culture on LB plates at 30°C for 12 h. The colonies were smooth and round, 2 to 3 mm in diameter, plump and raised, opaque, light yellow in color, with neat edges, smooth, moist and shiny, sticky and easy to pick up. The colonies increased in size over time, and after a few days, the diameter could reach more than 5 mm.

[0032] Example 1

[0033] Isolation, screening and performance identification of simultaneous oil removal and denitrification bacteria.

[0034] 1. Enrichment culture: salvage the grease block from the farmhouse grease trap, use the grease block as the carbon source, carry out enrichment culture in a specific acclimation medium, set the shaking culture time, which is the first stage of culture; transfer the culture liquid in the second acclimation cycle to the second stage of acclimation medium, set the shaking culture time, which is the second stage of culture, and carry out multiple acclimation;

[0035] 2. Isolation and purification: Dilute the domesticated and enriched bacterial suspension into different gradient bacterial suspensions according to the gradient dilution method, and perform streak culture on LB solid plate culture medium to obtain single colonies;

[0036] 3. Initial screening of oil removal performance: Use an inoculation loop to select a single colony point and inoculate it into the lipase plate culture medium for screening, place it in a constant temperature incubator and culture it for 2 days, select the strain that produces a clear transparent circle and inoculate it into the liquid culture medium, and place it in an oscillator for culture;

[0037] 4. Initial screening of denitrification performance: When a single colony is selected by the inoculation loop and inoculated onto the lipase plate, the single colony is also selected to be cultured on a 96-well plate. After a specified period of culture, bacterial liquid is taken from the 96-well plate to each NH4+ and NO2- verification plate, and ammonia nitrogen and nitrite color developer are added to observe the color change;

[0038] 5. Select the strain that produces a large transparent circle and obvious color in the 96-well plate, and culture it in a medium with soybean oil as the carbon source and ammonium chloride as the only nitrogen source. Take samples regularly to detect the concentration of ammonia nitrogen, total nitrogen and animal and vegetable oil in the medium. Select the strain with the highest oil degradation rate and denitrification efficiency and name it CYCN-1.

[0039] 6. Rescreening: Select strains that produce larger transparent circles and obvious color in 96-well plates, and culture them in a medium with soybean oil as the carbon source and ammonium chloride as the only nitrogen source. Take samples regularly to detect the concentrations of ammonia nitrogen, total nitrogen, and animal and vegetable oils in the medium. Select the strain with the highest oil degradation rate and denitrification efficiency, named CYCN-1, and transfer it to LB slant and refrigerate at 2-8℃.

[0040] The acclimation medium is prepared according to the following proportions: 0.36 g NH4Cl, 0.5 g K2HPO4, 0.5 g KH2PO4, 0.4 g MgSO4·7H2O, 2 ml trace salt solution in 1000 mL distilled water, pH 7.0-7.2, 120°C, wet heat sterilization for 30 min; the trace salt solution includes: 50 g ethylenediaminetetraacetic acid, 2.2 g ZnSO4, 5.5 g CaCl2, 1.57 g CuSO4·5H2O, 5.06 g MnCl2·4H2O, 5 g FeSO4·7H2O, and 1.61 g CoCl2·6H2O; 1000 mL distilled water; pH 7.0-7.2.

[0041] The LB solid medium was prepared according to the following proportions: 10.0 g of tryptone, 5 g of yeast powder, 10.0 g of NaCl, pH 7.0-7.2, and 20 g of agar powder, and sterilized with wet heat at 121° C. for 30 min.

[0042] The lipase plate culture medium is prepared by adding 2% agar and 1% soybean oil emulsion to the acclimated culture medium.

[0043] The soybean oil emulsion was prepared in the following ratio: 4% polyvinyl alcohol: soybean oil = 3:1, and processed with a high-speed homogenizer for 6 minutes.

[0044] The oil concentration is determined according to HJ 637-2012 “Water quality - Determination of petroleum, animal and vegetable oils - Infrared spectrophotometry”, and the instrument is Huaxia Technology OIL 460 infrared spectrophotometer.

[0045] Schematic diagram of the transparent circle produced on the lipase verification medium of the strain Pseudomonas koreensis CYCN-1 is shown in Figure 1 .

[0046] Example 2

[0047] Molecular Biological Identification of Simultaneous Oil Removal and Denitrification Bacteria

[0048] The simultaneous oil removal and denitrification bacteria were identified as Pseudomonas by 16Sr DNA analysis; its scientific name is Pseudomonaskoreensis.

[0049] Sequencing results SEQ ID NO.1:

[0050]

[0051] Example 3

[0052] Growth curve

[0053] Keeping the initial ammonia nitrogen concentration in the restaurant oily wastewater at 50-60 mg / L, using soybean oil 2 g / L as the only carbon source, the synchronous oil removal and denitrification bacteria were activated and cultured in LB medium for 24 h, and the bacterial solution was inoculated into the restaurant oily wastewater at a 1% inoculation rate, and placed in a constant temperature shaking incubator at 30 ° C and 150 r / min for culture, and the OD was measured every 12 h. 600 , draw a growth curve.

[0054] See also Figure 2 From the growth curve of synchronous oil removal and denitrification bacteria within 72 hours, we can see that OD 600 The value reached the highest value of 1.684 after 48h of cultivation. The exponential growth phase of strain CYCN-1 was 0-48, and the growth entered the stable phase at 48h-72h.

[0055] Example 4

[0056] Preparation of inoculants

[0057] The synchronous de-oiling and denitrifying bacteria Pseudomonas koreensis CYCN-1 was inoculated into the liquid LB culture medium and cultured at 30°C and 200r / min on a shaking table for 24 hours. The seed bacterial liquid was then poured into the fermentation tank for expansion culture; the inoculation amount was 5%. The cultured bacterial liquid and the culture medium were mixed in a ratio of 2:1, and then bucketed and sealed for refrigeration.

[0058] The culture medium for the fermentation tank expansion culture is configured according to the following proportions: 20L distilled water, 200g tryptone, 100g yeast powder, 200g sodium chloride, 40g soybean oil, pH is controlled at 7.0-7.2, and wet heat sterilization is performed at 121°C for 30min.

[0059] Example 5

[0060] Verification of the oil degradation and denitrification performance of the strain

[0061] Prepare oily kitchen wastewater, control the ammonia nitrogen concentration in the wastewater to 50-60mg / L, and control the soybean oil content to 2g / L. Activate and culture the synchronous oil removal and denitrification bacteria in LB liquid culture medium for 24 hours, and inoculate the wastewater with a 1% inoculation amount. Culture in a constant temperature shaking box at 30℃ and 150r / min. Take samples every 12 hours, and measure the total nitrogen, ammonia nitrogen and animal and vegetable oil concentrations in the supernatant after centrifugation. The results are as follows Figure 3As shown. It can be seen that with the increase of culture time, the oil concentration gradually decreases. After 36 hours of culture, the oil degradation rate can reach 100%, and soybean oil can no longer be detected. Therefore, the new strain of the present invention has a good effect on oil degradation. Figure 4 The degradation trends of total nitrogen and ammonia nitrogen are consistent. After 48 hours of cultivation, the maximum degradation rate of ammonia nitrogen can reach 99.5%, and the maximum degradation rate of total nitrogen can reach 90.0%. Therefore, the new strain of the present invention has a good effect on sewage denitrification.

[0062] Example 6

[0063] Application of bacterial activation and oil removal and nitrogen removal in sewage treatment pools

[0064] In order to better exert the performance of simultaneous oil removal and denitrification bacteria in the sewage treatment pool, the low-temperature preserved bacterial agent needs to be simply activated before use. On-site, add the influent water in the sewage to the plastic barrel and mix the bacterial agent in a 1:1 ratio. Stir and aerate in the expansion device for 24 hours. Add this mixed liquid according to 1% of the daily processing volume of the aerobic tank of the sewage treatment plant. Samples are taken every 24 hours to test the total nitrogen, ammonia nitrogen and animal and vegetable oil concentrations in the effluent. After on-site sampling and testing, 48 hours after the addition of the bacterial agent, the effluent water quality was significantly improved, and basically no oil was detected at the outlet. The total nitrogen and ammonia nitrogen met the effluent discharge indicators.

[0065] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. The application of synchronous oil removal and denitrification bacteria in the treatment of oily domestic sewage in farmhouses is characterized by: The simultaneous oil-removal and denitrification bacteria is the simultaneous oil-removal and denitrification bacteria CYCN-1 (Pseudomonas koreensis CYCN-1), which is deposited in the China Center for Type Culture Collection, address: Wuhan University, China, collection number: CCTCC NO:M 20221776; collection date: November 14, 2022; the 16S rDNA sequence of the simultaneous oil-removal and denitrification bacteria CYCN-1 is shown in SEQ ID NO:

1.

2. A method for preparing a simultaneous oil removal and denitrification bacterial agent, characterized in that: The synchronous oil-removal and denitrification bacteria CYCN-1 are inoculated into liquid LB culture medium and cultured on a shaking table; the seed bacterial liquid is then poured into a fermentation tank for expansion culture; the cultured bacterial liquid and culture medium are mixed, sealed and refrigerated; wherein the synchronous oil-removal and denitrification bacteria CYCN-1 is preserved in the China Center for Type Culture Collection, address: Wuhan University, China, preservation number: CCTCC NO:M 20221776; preservation date: November 14, 2022; the 16S rDNA sequence of the synchronous oil-removal and denitrification bacteria CYCN-1 is shown in SEQ ID NO:

1.

3. The method for preparing the simultaneous oil removal and denitrification bacteria agent according to claim 2, characterized in that: The culture medium for the fermentation tank expansion culture is configured according to the following proportions: 20L distilled water, 200g tryptone, 100g yeast powder, 200g sodium chloride, 40g soybean oil, pH is controlled at 7.0-7.2, and wet heat sterilization is performed at 121°C for 30min.

4. An application of the bacterial agent obtained by the preparation method according to claim 2, characterized in that: Take the influent of the decentralized sewage station of the farmhouse in the village scenic area and the sewage treatment station, control the concentration of ammonia nitrogen and total nitrogen to 50 mg / L-60 mg / L, and the oil content to 2 g / L; add 1% of the synchronous oil removal and denitrification bacterial agent obtained by the preparation method described in claim 2 into the prepared oily sewage.

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