Composite bacteria and its application in the treatment of high-salt and high-fat leather wastewater

Abstract

The present invention relates to the field of microbial technology, and in particular to a composite bacterial strain and its application in the treatment of high-salt and high-fat leather wastewater. The composite bacterial strain includes calcium-acetic acid acinetobacter with a preservation number of CGMCCNO.24322 and a subtilis with a preservation number of CGMCCNo.16167. Experiments show that when the total salt of the system is 100000 mg / L, the composite bacteria has a 24-hour degradation rate of 97.88% for oil with an initial concentration of 5000 mg / L, indicating that the composite bacteria and microbial agent of the present invention have a high efficiency in degrading animal oils in wastewater, can reduce the impact of high-salt and high-fat wastewater on the biochemical treatment system, improve the treatment efficiency of the wastewater, save pretreatment processes, and have significant economic and environmental benefits.

Description

Technical Field

[0001] The invention relates to the technical field of wastewater treatment, in particular to a composite bacterial strain and application thereof in the treatment of high-salt and high-fat leather wastewater. Background Art

[0002] Tanning wastewater refers to wastewater generated during the preparation and tanning stages of leather production. Tannery wastewater has a large discharge volume, a wide variety of pollutants, and complex components. Grease wastewater discharged from leather factories contains high concentrations of animal fats and a large amount of suspended solids, and has the characteristics of high salt content, high nutrients, easy to decay and stink. If it is discharged directly into natural water bodies without treatment, the formation of an oil film will hinder atmospheric dissolved oxygen and block the source of oxygen in the water, thereby reducing the dissolved oxygen content of the water, affecting the natural purification process of the water, endangering the water ecosystem, and seriously polluting the surrounding environment.

[0003] The main oil substance contained in leather wastewater is animal fat, with a total oil content of more than 10,000 mg / L and a total salt content of more than 100,000 mg / L. Animal fat is an organic matter that is difficult to biodegrade. If it is directly discharged into the wastewater treatment system without pretreatment, it will have an impact on the biochemical system and destroy the normal water treatment process. The commonly used treatment method for oil wastewater in leather factories is chemical flocculation. By adding ferrous sulfate, some of the oil in the wastewater is removed. The waste residue containing oil will be treated as hazardous waste, which not only increases the environmental burden, but also increases the cost of wastewater treatment.

[0004] In view of the high salt and fat characteristics of leather wastewater, there is an urgent need to provide an efficient salt-tolerant degreasing bacterial agent to achieve efficient degradation of animal fats in the wastewater. Summary of the invention

[0005] In view of this, the present invention provides a composite bacterial strain and its application in the treatment of high-salt and high-fat leather wastewater, wherein the composite bacterial strain has a highly efficient degradation effect on animal fats in the wastewater.

[0006] The composite bacterial strain of the invention comprises Acinetobacter calcoaceticus with a preservation number of CGMCC NO.24322 and Bacillus subtilis.

[0007] In the present invention, the Bacillus subtilis is preferably a strain with a preservation number of CGMCC No. 16167. Experiments show that the strains PG5 and PG8 are compounded in equal proportions to have a significant degradation effect on lard, with a 24h degradation rate of 97.88%. The strain PG5 is Bacillus subtilis with a preservation number of CGMCC No. 16167, and PG8 (YJY22-13) is Acinetobacter calcoaceticus with a preservation number of CGMCC No. 24322.

[0008] The Bacillus subtilis was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration in July 2018, with the deposit number CGMCC No. 16167, and the calcium-acetic acid Acinetobacter was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration in January 2022, with the deposit number CGMCC No. 24322.

[0009] The Bacillus subtilis is a Gram-positive bacterium without capsule and short rod shape. It is in a fast swimming state under a microscope and forms irregular protruding colonies on ordinary nutrient agar. The surface of the colonies is smooth, opaque and wrinkled. The calcium-acetic acid Acinetobacter is a Gram-negative bacterium, a cocci, which is usually arranged in pairs and can also exist alone, sometimes forming filaments and chains, with capsules, without flagella, and does not produce spores. It forms nearly circular medium-sized milky white colonies on ordinary nutrient agar. The colonies are flat and moist.

[0010] In some embodiments, the ratio of the effective bacterial count of the Acinetobacter calcoaceticus to that of Bacillus subtilis is 1:(0.8-3). In some specific embodiments, the ratio of the effective bacterial count of the Acinetobacter calcoaceticus to that of Bacillus subtilis is 1:1.

[0011] The present invention also provides a microbial agent, the raw materials of which include the composite bacteria species described in the present invention.

[0012] In some embodiments, the microbial agent includes at least one of live bacteria of a composite bacterial species, or a culture thereof, or an extract thereof.

[0013] The present invention also provides a method for preparing the microbial agent, wherein the microbial agent is a liquid agent or a solid agent, and the preparation method thereof is as follows:

[0014] The Acinetobacter calcoaceticus of CGMCC No. 24322 and the Bacillus subtilis of CGMCC No. 16167 are activated and seed liquids are prepared respectively, and then they are inoculated into fermentation medium for fermentation, and the fermented bacteria are collected to obtain liquid bacterial agents;

[0015] The liquid bacterial agent is adsorbed, dried and crushed in sequence to obtain a solid bacterial agent.

[0016] In the present invention, the adsorption, drying and pulverization can be accomplished by conventional means in the art, and the present invention does not make any specific limitation.

[0017] In some embodiments, the activation is performed at room temperature for 3 to 8 hours. In some specific embodiments, the activation time may be 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours.

[0018] In some embodiments, the preparation of the seed solution comprises: inoculating the activated bacterial strain into a liquid culture medium, and culturing the culture medium under shaking conditions of 28-38° C. and a shaking speed of 100-200 r / min for 15-20 hours.

[0019] In some embodiments, the fermentation is as follows: the seed liquid of the bacterial species is inoculated into the fermentation medium at an inoculation rate of 1%-5% by volume for high-density fermentation; the conditions for the high-density fermentation are as follows: tank pressure 0.02-0.05MPa, temperature 28-38°C, rotation speed 200-400r / min, dissolved oxygen ≧20%, and time 15-36h.

[0020] The culture medium for the fermentation of Bacillus subtilis includes: 2%-3.5% soybean meal, 3%-4% corn flour, 1.8%-3.2% maltodextrin, 0.1%-0.16% urea, 0.1%-0.3% calcium carbonate, 0.2%-0.3% dimethyl hydrogen phosphate, 0.05%-0.15% magnesium sulfate, 0.01%-0.02% manganese sulfate, 0.1% polyether defoamer, and the balance is water;

[0021] The culture medium for fermentation of calcoacetic acinetobacter comprises: 0.3%-0.55% of maltodextrin, 0.35%-0.55% of corn flour, 1%-2% of yeast powder, 1%-3% of corn steep liquor powder, 0.1%-0.2% of potassium dihydrogen sulfate, 0.2%-0.4% of magnesium sulfate, 0.1% of polyether defoamer, and the balance is water.

[0022] The present invention also provides the composite bacteria, the microbial agent or the use of the composite bacteria in treating high-salt and high-fat leather wastewater.

[0023] The present invention also provides a method for treating high-salt and high-fat leather wastewater, which comprises: adding the composite bacteria species or the microbial agent of the present invention to the high-salt and high-fat leather wastewater.

[0024] In some embodiments, the effective live bacteria in the wastewater are ≥ 10 5 The composite bacteria or the microbial agent is added in an amount of cfu / mL.

[0025] Too high dissolved oxygen is not conducive to the division and reproduction of bacteria. During the treatment process, too high dissolved oxygen will also break up the activated sludge in the aerobic tank, which is not conducive to the attachment of bacterial agents. At the same time, it will also be accompanied by floating sludge and running sludge, causing the entire biochemical system to be paralyzed. In some embodiments, the treatment conditions of the high-salt and high-fat leather wastewater are: dissolved oxygen 1-6 mg / L, temperature 20-35°C, pH 6-8.

[0026] In the present invention, when a solid microbial agent is used to treat high-salt and high-fat leather wastewater, the solid agent is first activated, and the activation is specifically as follows: the microbial solid agent is uniformly mixed with water in a mass ratio of 1:3-10, and the activation is stirred in a stirring container for 16-24 hours; in some preferred embodiments, the water also includes 1-3% glucose, 0.02-0.05% ammonium sulfate and 0.02-0.05% dimethyl hydrogen phosphate.

[0027] The composite bacterial strain provided by the present invention comprises Acinetobacter calcoaceticus with a preservation number of CGMCC No. 24322 and Bacillus subtilis with a preservation number of CGMCC No. 16167. Experiments show that when the total salt of the system is 100000 mg / L, the composite bacteria has a 24-hour degradation rate of 97.88% for oil with an initial concentration of 5000 mg / L. When the bacterial agent is applied to the biochemical treatment of high-salt and high-fat leather wastewater, the total salt content of the system is about 110000 mg / L, the oil content is about 15000 mg / L, and the oil content of the effluent water is reduced to below 100 mg / L after adding one ten-thousandth of the bacterial agent for 4 days, and the effluent water is stabilized at below 5 mg / L after 6 days. It is shown that the composite bacteria and microbial agent of the present invention have a highly efficient degradation effect on animal fats in wastewater, can reduce the impact of high-salt and high-fat wastewater on the biochemical treatment system, improve the treatment efficiency of the wastewater, save pretreatment processes, reduce costs, and will not cause secondary pollution to the environment, with significant economic and environmental benefits.

[0028] Biological Deposit Description

[0029] YJY22-13, classification name: Acinetobacter calcoaceticus, was deposited on January 17, 2022 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 24322. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph showing the results of the salt tolerance screening test of high-salt and high-fat leather wastewater degradation strains in Example 1;

[0031] Figure 2 This is the trend chart of the effluent of the blank group in the small-scale test of the high-salt and high-fat leather wastewater degradation bacterial agent in Example 4;

[0032] Figure 3 This is the effluent trend diagram of the bacteria group added in the small-scale test of the high-salt and high-fat leather wastewater degradation bacteria agent in Example 4;

[0033] Figure 4 This is a comparison chart of the biochemical pool for the application test of the high-salt and high-fat leather wastewater degradation bacterial agent in Example 5;

[0034] Figure 5 This is the effluent trend diagram of the test of applying the bacterial agent to degrade high-salt and high-fat leather wastewater in Example 5. DETAILED DESCRIPTION

[0035] The present invention discloses a composite bacterial strain and its application in the treatment of high-salt and high-fat leather wastewater. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] The present invention is further described below in conjunction with the embodiments, wherein Bacillus subtilis has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration in July 2018, with the deposit number of CGMCC No.16167.

[0037] Example 1 Screening of bacterial strains for degradation of high-salt and high-fat leather wastewater

[0038] 1) Screening of defatted strains

[0039] The activated sludge in the leather wastewater biochemical treatment device was used as the test matrix to separate and purify the strains. Finally, 7 strains were purified. Together with 5 strains with oil removal function preserved in the laboratory strain library, lard was used as the degradation object for effective strain screening. The fermentation liquid was inoculated into an inorganic salt culture medium containing 5000 mg / L lard at a 5% inoculation rate and cultured at 37°C and 180 rpm. The results are shown in Table 1. A total of 10 strains were screened that have lard degradation effects. Five strains had a 24h degradation rate of more than 50% for lard with an initial concentration of 5000 mg / L, of which PG5 and PG7 had a degradation rate of more than 90%.

[0040] Table 1 Degreasing strain screening test results

[0041]

[0042] 2) Screening of salt tolerance strains

[0043] The five strains with a degreasing rate of more than 50% were selected as test objects for salt tolerance screening. OD600 was used as the evaluation index. Single colonies were picked and inoculated into LB medium containing different salt concentrations. After overnight culture, the OD600 of the bacterial solution was tested. The results are shown in Figure 1 , strains PG5, PG8 and PG11 had the highest tolerance to total salt. When the total salt content in the system reached 90,000 mg / L, the growth of the strains was not affected, and they still reproduced when the total salt content reached 150,000 mg / L. The three strains were determined to be high-salt and high-fat leather wastewater degrading strains.

[0044] Example 2 Compounding of high-salt and high-fat leather wastewater degradation bacterial agent

[0045] The three screened high-salt and high-fat leather wastewater degradation strains PG5, PG8 and PG11 were used as test strains for compound optimization of the bacterial agent. The strains were compounded in equal proportions according to the principle of sequential order, and the effect of the bacterial agent was verified. The fermentation liquid was inoculated into an inorganic salt culture medium containing 5000 mg / L lard at a 5% inoculation amount, with a total salt content of 100000 mg / L, and cultured at 37°C and 180rpm. The results are shown in Table 2. The strain PG11 had a certain antagonistic effect on lard degradation after compounding. The strains PG5 / PG8 had the best degradation effect on lard after equal proportion compounding, with a degradation rate of 97.88% in 24 hours. The high-salt and high-fat leather wastewater degradation bacterial agent was determined to be the PG5 / PG8 compound bacterial agent. Both strains were laboratory preserved strains, PG5 was Bacillus subtilis, and PG8 was Acinetobacter calcoaceticus YJY22-13.

[0046] Table 2 Compounding of degradation bacteria

[0047]

[0048] Example 3 Preparation of high-salt and high-fat leather wastewater degradation bacterial agent

[0049] (1) Activation of bacterial strains: Take out the Bacillus subtilis and Acinetobacter calcoaceticus strains stored at 4°C and activate them at room temperature for 3 hours. Since the strains are stored on slant nutrient agar medium in test tubes, no nutrients need to be added during the activation process.

[0050] (2) Liquid seed preparation: In a sterile operating table, use a pipette to take 10 ml of sterile water and add it to the culture storage tube, and use the pipette to repeatedly blow and beat to prepare a bacterial suspension, and then inoculate the bacterial suspension into 200 ml of liquid culture medium and shake and culture for 20 h. The culture conditions are: temperature 35°C, shaking speed 170 r / min;

[0051] (3) High-density fermentation: Bacillus subtilis or Acinetobacter calcoaceticus liquid seeds were inoculated at a volume ratio of 1.5% into the corresponding fermentation medium for liquid submerged fermentation; the fermentation conditions were: temperature 35° C., rotation speed 200 r / min, and culture time 20 h;

[0052] Formula of high-density culture medium for Bacillus subtilis: soybean meal 2%-3.5%, corn flour 3%-4%, maltodextrin 1.8%-3.2%, urea 0.1%-0.16%, calcium carbonate 0.1%-0.3%, dimethyl hydrogen phosphate 0.2%-0.3%, magnesium sulfate 0.05%-0.15%, manganese sulfate 0.01%-0.02%, polyether defoamer 0.1%, and the balance is water;

[0053] The high-density fermentation conditions of Bacillus subtilis were as follows: tank pressure 0.02 MPa, temperature 35°C, rotation speed 200 r / min, dissolved oxygen 30%, and culture time 36 h;

[0054] Formula of high-density culture medium for Acinetobacter calcoaceticus: 0.3%-0.55% maltodextrin, 0.35%-0.55% corn flour, 1%-2% yeast powder, 1%-3% corn steep liquor powder, 0.1%-0.2% potassium dihydrogen sulfate, 0.2%-0.4% magnesium sulfate, 0.1% polyether defoamer, and the balance is water;

[0055] High-density fermentation conditions of Acinetobacter calcoaceticus: tank pressure 0.02MPa, temperature 35℃, speed 200r / min, dissolved oxygen 30%, culture time 12h;

[0056] (4) Drying and crushing: adsorbing, drying and crushing the bacterial cells obtained by the above fermentation to obtain a solid high-salt and high-fat leather wastewater degradation bacterial agent;

[0057] The plate counting method was used to detect that the effective live bacterial count of the high-salt and high-fat leather wastewater degradation agent was 13.652 billion cfu / g.

[0058] Example 4 Small-scale test of high-salt and high-fat leather wastewater degradation agent

[0059] Wastewater and biochemical sludge from a leather factory in Zhanhua, Shandong were used for small-scale microbial agent testing. The total oil content of high-salt and high-fat leather wastewater was 12800 mg / L, and the total salt content was 95300 mg / L. A 5L beaker was taken and 4L of biochemical sludge was added. The water inlet of high-salt and high-fat leather wastewater was 50%. Water was added and discharged once a day. The preserved Bacillus subtilis and Acinetobacter calcoaceticus were respectively inoculated into sterile LB liquid culture medium in an ultra-clean workbench, and cultured at 37°C and 180rpm for 16-20h. The cultured bacterial solution was inoculated into the small-scale test system with an inoculation volume ratio of 5%, and cultured under aeration at room temperature. A blank control system was set up at the same time. The total oil content of the effluent was tracked and detected every day, and the detection was continuously tracked for 20 days. The results are shown in the figure. Figure 2 and Figure 3The total oil content in the effluent of the blank group showed an upward trend, and after 7 days, the total oil content was maintained above 10000 mg / L. The total oil content in the effluent of the bacteria group showed a linear downward trend in the first 7 days, from 535.18 mg / L to 7.23 mg / L. From the 8th day, the total oil in the effluent of the system was maintained below 5 mg / L. Compared with the blank group, the oil degradation rate of the bacteria group was maintained above 99%.

[0060] Example 5 Application of bacterial agent for degradation of high-salt and high-fat leather wastewater

[0061] The biochemical system of a leather factory in Zhanhua, Shandong Province was impacted by high-concentration oil and fat wastewater, causing foam to overflow the pool, dissolved oxygen to decrease, and water effluent indicators to remain high. After testing and analysis, the oil content in the biochemical system reached about 15,000 mg / L, and the total salt content of the system was about 110,000 mg / L. In view of this situation, a high-salt and high-fat leather wastewater degradation agent was added. The specific usage method is as follows:

[0062] (1) Dosing of bacterial agents: Calculate the effective pool capacity (or water retention) of the aeration tank of the sewage treatment system and add bacterial agents at a dosage of one ten-thousandth;

[0063] (2) Debugging and testing: After the addition of the bacterial agent is completed, the aeration tank parameters are debugged: dissolved oxygen 4 mg / L, pH 7.0, and the effluent of the aeration tank is sampled every 24 hours to test the pollutant indicators;

[0064] The tracking test was carried out for 14 days, and the results were as follows Figure 4 When the system shown in the figure was impacted, foam was all over the surface of the aerobic pool. After 7 days of adjustment with the addition of bacterial agents, the foam on the pool surface dissipated, revealing the aerobic sludge. Figure 5 The change of oil content in the effluent water showed a linear downward trend. After 6 days of adding the bacterial agent, the oil content in the effluent water dropped to about 5 mg / L, and remained stable below 5 mg / L in the later period.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite bacterial strain, characterized in that: Including the calcoacetic acid Acinetobacter calcoaceticus (CGMCC NO. 24322) Acinetobacter calcoaceticus ) and Bacillus subtilis with a deposit number of CGMCC No.16167 ( Bacillus subtilis ).

2. The composite strain according to claim 1, characterized in that: The ratio of the effective bacterial counts of the calcoacetic acid Acinetobacter and the Bacillus subtilis is 1:(0.8-3).

3. A microbial agent, characterized in that: The raw materials include the composite bacteria described in claim 1 or 2.

4. The method for preparing the microbial agent according to claim 3, characterized in that: The microbial agent is a liquid agent or a solid agent, and its preparation method is as follows: The Acinetobacter calcoaceticus with a preservation number of CGMCC No. 24322 and the Bacillus subtilis with a preservation number of CGMCC No. 16167 are activated to prepare seed liquids, and then inoculated into fermentation medium for fermentation, and the fermented bacteria are collected to obtain liquid bacterial agents; The liquid bacterial agent is adsorbed, dried and crushed in sequence to obtain a solid bacterial agent.

5. The preparation method according to claim 4, characterized in that: The activation is performed at room temperature for 3h to 8h; The preparation of the seed solution comprises: inoculating the activated bacterial strain into a liquid culture medium, and culturing the culture medium under the conditions of a temperature of 28-38° C. and a shaking speed of 100-200 r / min for 16-20 hours; The fermentation is as follows: the seed liquid of the bacterial species is inoculated into the fermentation medium at an inoculation rate of 1%-5% by volume for high-density fermentation; the conditions of the high-density fermentation are: tank pressure 0.02-0.05 MPa, temperature 28-38°C, rotation speed 200-400r / min, dissolved oxygen ≧20%, and time 15-36h.

6. The preparation method according to claim 4, characterized in that: The culture medium for the Bacillus subtilis fermentation comprises: 2%-3.5% soybean meal, 3%-4% corn flour, 1.8%-3.2% maltodextrin, 0.1%-0.16% urea, 0.1%-0.3% calcium carbonate, 0.2%-0.3% dimethyl hydrogen phosphate, 0.05%-0.15% magnesium sulfate, 0.01%-0.02% manganese sulfate, 0.1% polyether defoamer, and the balance is water; The culture medium for fermentation of calcoacetic acinetobacter comprises: 0.3%-0.55% maltodextrin, 0.35%-0.55% corn flour, 1%-2% yeast powder, 1%-3% corn steep liquor powder, 0.1%-0.2% potassium dihydrogen sulfate, 0.2%-0.4% magnesium sulfate, 0.1% polyether defoamer, and the balance is water.

7. Use of the composite strain described in claim 1 or 2, the microbial agent described in claim 3, or the microbial agent prepared by the preparation method described in any one of claims 4 to 6 in treating high-salt and high-fat leather wastewater.

8. A method for treating high-salt and high-fat leather wastewater, characterized in that: The composite strain described in claim 1 or 2, the microbial agent described in claim 3, or the microbial agent prepared by the preparation method described in any one of claims 4 to 6 is added to high-salt and high-fat leather wastewater.

9. The processing method according to claim 8, characterized in that: According to the effective living bacteria in the wastewater ≥ 10 5 The composite bacteria or the microbial agent is added in an amount of cfu / mL.

10. The processing method according to claim 8, characterized in that: The treatment conditions of the high-salt and high-fat leather wastewater are: dissolved oxygen 1-6 mg / L, temperature 20-35° C., and pH 6-8.

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