Bacillus and complex microbial agent and its application in refining high concentration wastewater

Abstract

The present application relates to the technical field of wastewater biological treatment, and is a kind of bacillus and composite microbial inoculant and its application in refining high-concentration wastewater, the latter is carried out according to the following method: bacillus and campylobacter are inoculated in LB liquid culture medium respectively, then are transferred to sterilized refining high-concentration wastewater respectively for twice activation, then, the bacillus and campylobacter liquid after twice activation are transferred to nutrient solution respectively for further culture, finally, the obtained fermentation agent containing bacillus and the fermentation agent containing campylobacter are mixed in proportion and added to refining high-concentration wastewater for hydrolysis acidification treatment.The composite microbial inoculant has good high-temperature resistance, impact resistance and adaptability, when it is added to the hydrolysis acidification reactor containing refining high-concentration wastewater and filled with fiber filler, the COD removal rate can reach more than 60%, which greatly reduces the cost of subsequent treatment of environmental pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of wastewater biological treatment, and is a bacillus and a compound microbial agent and application thereof in refining high-concentration wastewater. BACKGROUND

[0002] With the heavy and poor quality of crude oil, the pollution of refining wastewater is also more and more complex. The original "old three sets" process, i.e. "oil separation-air floatation-biochemical", is difficult to handle the current complex wastewater, and enterprises have to stack and extend the process flow to enhance the degradation of pollutants. The hydrolysis acidification process has obvious advantages in treating high-concentration wastewater. It can decompose large-molecule organic pollutants in water into small-molecule long-chain structures through anaerobic fermentation, and then decompose them into acidic substances or alcohol and other small-molecule organic matters which are easier to decompose. In the hydrolysis acidification process, the high-efficiency degrading bacteria are in the core and key position, and the correct selection of the filler is the key to ensure the good function of the functional bacteria. SUMMARY

[0003] The present application provides a bacillus and a compound microbial agent and application thereof in refining high-concentration wastewater, which overcomes the shortcomings of the prior art. The compound microbial agent can treat refining high-concentration wastewater with COD of 800 mg / L to 2500 mg / L and oil content of not higher than 132 mg / L, and the oil content of the effluent after hydrolysis acidification treatment by the compound microbial agent is not higher than 50 mg / L, realizing a COD removal rate of more than 60%, and greatly reducing the cost of subsequent treatment of environmental pollution.

[0004] One of the technical solutions of the present application is achieved by the following measures: a bacillus Bacillus sp. The strain has been preserved in the China General Microbiological Culture Collection Center on January 18, 2021, and the preservation number is CGMCC No. 21651.

[0005] The second technical solution of the present application is achieved by the following measures: a compound microbial agent containing bacillus.

[0006] The following is a further optimization or / and improvement of the second technical solution of the above application:

[0007] The above compound microbial agent is mixed by the bacillus-containing fermenting agent and the Campylobacter-containing fermenting agent in a volume ratio of 1:1 to 5. The bacillus-containing fermenting agent includes bacillus and nutrient solution, and the concentration of bacillus in the bacillus fermenting agent is 1x10 8 CFU / mL or more. The Campylobacter-containing fermenting agent includes Campylobacter and nutrient solution, and the concentration of Campylobacter in the Campylobacter-containing fermenting agent is 1x10 8CFU / mL or more.

[0008] The composition of the above-mentioned nutrient solution includes glucose 20g / L to 24g / L, proteose peptone 2g / L to 5g / L, yeast powder 1g / L to 2g / L, ammonium nitrate 2g / L to 3g / L, sodium chloride 4g / L to 5g / L, potassium dihydrogen phosphate 0.5g / L to 1g / L, manganese sulfate 0.3g / L to 0.4g / L, magnesium sulfate 1g / L to 2g / L, ferrous sulfate 0.02g / L to 0.05g / L, and calcium carbonate 1g / L to 2g / L, and the pH value of the nutrient solution is 7.0 to 7.2.

[0009] The third technical solution of the application is achieved by the following measures: application of a composite microbial agent in refining high-concentration wastewater.

[0010] The following is a further optimization or / and improvement of the third technical solution of the above-mentioned application:

[0011] The application of the above-mentioned composite microbial agent in refining high-concentration wastewater is carried out by the following method:

[0012] Firstly, Bacillus and Campylobacter are inoculated into LB liquid medium respectively, and after shaking culture, Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are obtained respectively, wherein the concentration of Bacillus in the Bacillus fermentation seed liquid is 1×10 8 CFU / mL or more, the concentration of Campylobacter in the Campylobacter fermentation seed liquid is 1×108CFU / mL or more, the culture temperature is 30℃ to 35℃, and the shaking speed is 150rpm to 200rpm;

[0013] Secondly, the Bacillus fermentation seed liquid and the Campylobacter fermentation seed liquid are inoculated into sterilized low-COD refining wastewater for activation, wherein the inoculation amount of the Bacillus fermentation seed liquid and the Campylobacter fermentation seed liquid is 3% to 6% of the volume percentage of the sterilized low-COD refining wastewater, the COD of the sterilized low-COD refining wastewater is 500mg / L to 800mg / L, the activation conditions include a culture temperature of 30℃ to 35℃, a pH of 8 to 10, a shaking speed of 150rpm to 200rpm, and an activation time of 24h;

[0014] Thirdly, the activated bacillus fermentation seed liquid and the activated campylobacter fermentation seed liquid are respectively transferred into the high COD refinery wastewater after sterilization for reactivation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the activated campylobacter fermentation seed liquid is 3% to 6% of the volume percentage of the high COD refinery wastewater after sterilization, the COD of the high COD refinery wastewater after sterilization is 800 mg / L to 2500 mg / L, the activation condition includes that the culture temperature is 30 DEG C to 35 DEG C, the pH is 8 to 10, the shaking table rotating speed is 150 rpm to 200 rpm, and the activation time is 24 h;

[0015] Fourthly, the reactivated bacillus fermentation seed liquid and the reactivated campylobacter fermentation seed liquid are respectively transferred into the fermentation tank containing the nutrient solution for continued fermentation and expansion culture, and the bacillus-containing fermentation agent and the campylobacter-containing fermentation agent are obtained, wherein the inoculation amount of the reactivated bacillus fermentation seed liquid and the reactivated campylobacter fermentation seed liquid is 3% to 6% of the volume percentage of the nutrient solution, the concentration of the bacillus in the bacillus-containing fermentation agent is 1*10 8 CFU / ml or above, the concentration of the campylobacter in the campylobacter-containing fermentation agent is 1*10 8 CFU / ml or above, the culture temperature is 28 DEG C to 38 DEG C, the pH is 7.5 to 9.5, the rotating speed is 200 rpm, the pressure of the fermentation tank is 0.03 MPa to 0.06 MPa, the aeration amount is 1.0 to 1.3:1, and the fermentation time is 16 h to 20 h;

[0016] Fifthly, the bacillus-containing fermentation agent and the campylobacter-containing fermentation agent are mixed to obtain the composite microbial inoculant, and the composite microbial inoculant is added into the hydrolysis acidification reactor containing the refinery high-concentration wastewater and filled with fiber filler for hydrolysis acidification treatment, wherein the volume ratio of the bacillus-containing fermentation agent and the campylobacter-containing fermentation agent in the composite microbial inoculant is 1:1 to 5, the composite microbial inoculant accounts for 4% to 6% of the volume percentage of the refinery high-concentration wastewater, the initial COD of the refinery high-concentration wastewater is 800 mg / L to 2500 mg / L, the oil content is not higher than 135 mg / L, during the hydrolysis acidification treatment, the temperature is 40 DEG C to 45 DEG C, the time is not less than 55 h, the fiber filler is made of polypropylene, and the filling rate of the fiber filler in the hydrolysis acidification reactor is 20% to 70%.

[0017] Compared with the prior art, the beneficial effects of the present application are that:

[0018] The present application is compound microbial inoculant prepared by compounding bacillus and campylobacter, and the compound microbial inoculant is applied to hydrolysis acidification treatment of high-concentration wastewater in refining and chemical industry, and the compound microbial inoculant can adapt to high-concentration wastewater in refining and chemical industry with temperature of 40-45 DEG C for a long time, and has strong impact resistance when upstream water quality changes greatly, so that the compound microbial inoculant has good high-temperature resistance, impact resistance and adaptability.

[0019] The present application is compound microbial inoculant prepared by compounding bacillus and campylobacter, and the compound microbial inoculant is applied to hydrolysis acidification treatment of high-concentration wastewater in refining and chemical industry, and the compound microbial inoculant can adapt to high-concentration wastewater in refining and chemical industry with temperature of 40-45 DEG C for a long time, and has strong impact resistance when upstream water quality changes greatly, so that the compound microbial inoculant has good high-temperature resistance, impact resistance and adaptability. 3 ·d) is promoted to 0.63 kg / (m 3 ·d), and the hydrolysis acidification reactor with biological strengthening has good impact load resistance. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is electron micrograph of bacillus sp.

[0021] Figure 2 It is electron micrograph of campylobacter sp.

[0022] Figure 3 It is COD removal rate of water quality after hydrolysis acidification treatment of sterilized high-concentration wastewater in refining and chemical industry by two kinds of dominant degrading bacteria with different proportions.

[0023] Figure 4 It is COD and COD removal rate of effluent water quality within one week after adding optimal proportion of dominant degrading bacteria to hydrolysis acidification reactor to treat high-concentration wastewater in refining and chemical industry.

[0024] Figure 5 It is COD and COD removal rate of effluent water quality when hydrolysis acidification reactor with biological strengthening treats high-concentration COD influent.

[0025] Figure 6 It is COD and COD removal rate of effluent water quality when hydrolysis acidification reactor with biological strengthening treats high-concentration influent by reducing hydraulic retention time. DETAILED DESCRIPTION

[0026] The present application is not limited by the following examples, and the specific implementation can be determined according to the technical solutions of the present application and the actual situation. The various chemical reagents and chemical supplies mentioned in the present application are commonly known and used in the prior art unless otherwise specified; the percentages in the present application are mass percentages unless otherwise specified.

[0027] The present application will be further described below in conjunction with examples:

[0028] Example 1: The Bacillus sp. has been preserved in the China General Microbiological Culture Collection Center on January 18, 2021, and the preservation number is CGMCC No. 21651.

[0029] Example 2: The composite microbial agent is mixed by the volume ratio of 1:1 to 5 of the fermentation agent containing Bacillus and the fermentation agent containing Campylobacter. The fermentation agent containing Bacillus includes Bacillus and nutrient solution, and the concentration of Bacillus in the Bacillus fermentation agent is 1x10 8 CFU / mL or more. The fermentation agent containing Campylobacter includes Campylobacter and nutrient solution, and the concentration of Campylobacter in the Campylobacter fermentation agent is 1x10 8 CFU / mL or more. The composition of the nutrient solution includes glucose 20g / L to 24g / L, peptone 2g / L to 5g / L, yeast powder 1g / L to 2g / L, ammonium nitrate 2g / L to 3g / L, sodium chloride 4g / L to 5g / L, potassium dihydrogen phosphate 0.5g / L to 1g / L, manganese sulfate 0.3g / L to 0.4g / L, magnesium sulfate 1g / L to 2g / L, ferrous sulfate 0.02g / L to 0.05g / L, and calcium carbonate 1g / L to 2g / L. The pH value of the nutrient solution is 7.0 to 7.2.

[0030] Preservation instructions of Campylobacter:

[0031] Strain name: Campylobacter

[0032] Latin name: Campylobacter sp.

[0033] Preservation agency: China General Microbiological Culture Collection Center

[0034] Preservation number: CGMCC No. 21652

[0035] Preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Yihuangyuan, Beijing, China

[0036] Preservation date: January 18, 2021

[0037] Example 3: Application of the complex microbial agent in high-concentration wastewater from refining and chemical industry.

[0038] Example 4: Application of the complex microbial agent in high-concentration wastewater from refining and chemical industry, which is carried out according to the following method:

[0039] Firstly, Bacillus and Campylobacter are inoculated into LB liquid medium respectively, and after shaking culture, Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are obtained respectively, wherein the concentration of Bacillus in the Bacillus fermentation seed liquid is above 1 x 10 8 CFU / mL, the concentration of Campylobacter in the Campylobacter fermentation seed liquid is above 1 x 10 8 CFU / mL, the culture temperature is 30°C, and the shaking speed is 150 rpm.

[0040] Secondly, the Bacillus fermentation seed liquid and the Campylobacter fermentation seed liquid are inoculated into sterilized low-COD wastewater from refining and chemical industry respectively for activation, wherein the inoculation amount of the Bacillus fermentation seed liquid and the Campylobacter fermentation seed liquid is 3% of the volume percentage of the sterilized low-COD wastewater from refining and chemical industry respectively, the COD of the sterilized low-COD wastewater from refining and chemical industry is 500 mg / L, and the activation conditions include a culture temperature of 30°C, a pH of 8, a shaking speed of 150 rpm, and an activation time of 24 h.

[0041] Thirdly, the activated Bacillus fermentation seed liquid and the activated Campylobacter fermentation seed liquid are transferred into sterilized high-COD wastewater from refining and chemical industry respectively for re-activation, wherein the inoculation amount of the activated Bacillus fermentation seed liquid and the activated Campylobacter fermentation seed liquid is 3% of the volume percentage of the sterilized high-COD wastewater from refining and chemical industry respectively, the COD of the sterilized high-COD wastewater from refining and chemical industry is 800 mg / L, and the activation conditions include a culture temperature of 30°C, a pH of 8, a shaking speed of 150 rpm, and an activation time of 24 h.

[0042] Fourthly, the re-activated Bacillus fermentation seed liquid and the re-activated Campylobacter fermentation seed liquid are transferred into fermentation tanks containing nutrient solution for further fermentation and expansion culture, and Bacillus-containing fermentation agent and Campylobacter-containing fermentation agent are obtained respectively, wherein the inoculation amount of the re-activated Bacillus fermentation seed liquid and the re-activated Campylobacter fermentation seed liquid is 3% of the volume percentage of the nutrient solution respectively, the concentration of Bacillus in the Bacillus-containing fermentation agent is above 1 x 10 8 CFU / mL, and the concentration of Campylobacter in the Campylobacter-containing fermentation agent is above 1 x 10 8CFU / ml or more, the culture temperature is 28℃, the pH is 7.5, the rotation speed is 200 rpm, the pressure of the fermenter is 0.03 MPa, the aeration rate is 1.0:1, the fermentation time is 16 h, and the composition of the nutrient solution further includes glucose 20 g / L, peptone 2 g / L, yeast powder 1 g / L, ammonium nitrate 2 g / L, sodium chloride 4 g / L, potassium dihydrogen phosphate 0.5 g / L, manganese sulfate 0.3 g / L, magnesium sulfate 1 g / L, ferrous sulfate 0.02 g / L, and calcium carbonate 1 g / L, and the pH of the nutrient solution is 7.0;

[0043] In the fifth step, the fermentation agent containing bacillus and the fermentation agent containing campylobacter are mixed to obtain a composite microbial agent, and the composite microbial agent is added to the hydrolysis acidification reactor containing the refining high-concentration wastewater and filled with fiber filler to perform hydrolysis acidification treatment, wherein the volume ratio of the fermentation agent containing bacillus and the fermentation agent containing campylobacter in the composite microbial agent is 1:1, the composite microbial agent accounts for 4% of the volume percentage of the refining high-concentration wastewater, the initial COD of the refining high-concentration wastewater is 800 mg / L, the oil content is 60 mg / L, the temperature during the hydrolysis acidification treatment is 40℃, the time is 55 h, the fiber filler is made of polypropylene, and the filling rate of the fiber filler in the hydrolysis acidification reactor is 20%.

[0044] In the fifth step, the composite microbial agent is applied in the refining high-concentration wastewater by the following method:

[0045] In the first step, bacillus and campylobacter are inoculated into LB liquid culture medium respectively, and after shaking culture, bacillus fermentation seed liquid and campylobacter fermentation seed liquid are obtained respectively, wherein the concentration of bacillus in the bacillus fermentation seed liquid is 1×10 8 The concentration of campylobacter in the campylobacter fermentation seed liquid is 1×10 8 The culture temperature is 35℃, and the rotation speed of the shaking table is 200 rpm.

[0046] In the second step, the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid are inoculated into sterilized low-COD refining wastewater respectively to activate, wherein the inoculation amount of the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 6% of the volume percentage of the sterilized low-COD refining wastewater respectively, the COD of the sterilized low-COD refining wastewater is 800 mg / L, and the activation conditions include a culture temperature of 35℃, a pH of 10, a rotation speed of the shaking table of 200 rpm, and an activation time of 24 h.

[0047] Third step, the activated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are respectively transferred to the high COD refinery wastewater after sterilization for reactivation, wherein the inoculation amount of the activated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid is respectively 6% of the volume percentage of the high COD refinery wastewater after sterilization, the COD of the high COD refinery wastewater after sterilization is 2500mg / L, the activation conditions include that the culture temperature is 35℃, the pH is 10, the shaking speed is 200rpm, and the activation time is 24h;

[0048] Fourth step, the reactivated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are respectively transferred to the fermentation tank containing nutrient solution for continued fermentation and expansion culture, and the fermentation agent containing Bacillus and the fermentation agent containing Campylobacter are respectively obtained, wherein the inoculation amount of the reactivated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid is respectively 6% of the volume percentage of the nutrient solution, the concentration of Bacillus in the fermentation agent containing Bacillus is 1×10 8 CFU / ml or above, the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 8 CFU / ml or above, the culture temperature is 38℃, the pH is 9.5, the rotation speed is 200rpm, the pressure of the fermentation tank is 0.06MPa, the aeration amount is 1.3:1, the fermentation time is 20h, the composition of the nutrient solution further includes glucose 24g / L, peptone 5g / L, yeast powder 2g / L, ammonium nitrate 3g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 1g / L, manganese sulfate 0.4g / L, magnesium sulfate 2g / L, ferrous sulfate 0.05g / L, and calcium carbonate 2g / L, and the pH value of the nutrient solution is 7.2;

[0049] Fifth step, the fermentation agent containing Bacillus and the fermentation agent containing Campylobacter are mixed to obtain a composite microbial inoculant, and the composite microbial inoculant is added to the hydrolysis acidification reactor containing refinery high-concentration wastewater and filled with fiber filler for hydrolysis acidification treatment, wherein the volume ratio of the fermentation agent containing Bacillus to the fermentation agent containing Campylobacter in the composite microbial inoculant is 1:5, the composite microbial inoculant accounts for 6% of the volume percentage of the refinery high-concentration wastewater, the initial COD of the refinery high-concentration wastewater is 2500mg / L, and the oil content is 132mg / L, during the hydrolysis acidification treatment, the temperature is 45℃, the time is 65h, the fiber filler is made of polypropylene, and the filling rate of the fiber filler in the hydrolysis acidification reactor is 70%.

[0050] Example 6: The application of the composite microbial inoculant in refinery high-concentration wastewater is carried out by the following method:

[0051] The first step involved inoculating Bacillus and Campylobacter into LB liquid medium, respectively, and culturing them on a shaker to obtain Bacillus fermentation seed culture and Campylobacter fermentation seed culture, respectively. The concentration of Bacillus in the Bacillus fermentation seed culture was 1×10⁻⁶. 8 The concentration of Campylobacter in the Campylobacter fermentation seed culture is above 1×10⁻⁶ CFU / mL. 8 CFU / mL or higher, culture temperature 32℃, shaker speed 200 rpm;

[0052] The second step involves inoculating the Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid into the sterilized low-COD refining wastewater for activation. The inoculation amounts of the Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are 4% of the volume percentage of the sterilized low-COD refining wastewater, respectively. The COD of the sterilized low-COD refining wastewater is 600 mg / L. The activation conditions include a culture temperature of 32℃, a pH of 9, a shaking speed of 180 rpm, and an activation time of 24 h.

[0053] The third step involves transferring the activated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid to the sterilized high-COD refining wastewater for further activation. The inoculation amounts of the activated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are 4% of the volume percentage of the sterilized high-COD refining wastewater. The COD of the sterilized high-COD refining wastewater is 1500 mg / L. The activation conditions include a culture temperature of 32℃, a pH of 9, a shaking speed of 180 rpm, and an activation time of 24 h.

[0054] The fourth step involves transferring the reactivated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid to fermenters containing nutrient solution for further fermentation and scale-up culture, respectively, to obtain fermentation broths containing Bacillus and Campylobacter, respectively. The inoculation amounts of the reactivated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are 4% of the nutrient solution volume, respectively, and the concentration of Bacillus in the fermentation broth containing Bacillus is 1 × 10⁻⁶. 8 For starter cultures containing Campylobacter above CFU / ml, the concentration of Campylobacter is 1×10⁻⁶. 8 The fermentation conditions were as follows: CFU / ml above 30℃, pH 8.0, rotation speed 200 rpm, fermenter pressure 0.04 MPa, aeration rate 1.1:1, fermentation time 18 h, and nutrient solution composition including glucose 22 g / L, peptone 4 g / L, yeast powder 1.5 g / L, ammonium nitrate 2.5 g / L, sodium chloride 4.5 g / L, potassium dihydrogen phosphate 0.8 g / L, manganese sulfate 0.35 g / L, magnesium sulfate 1.5 g / L, ferrous sulfate 0.04 g / L, and calcium carbonate 1.5 g / L. The pH of the nutrient solution was 7.1.

[0055] Fifth step, the fermentation agent containing Bacillus and the fermentation agent containing Campylobacter are mixed to obtain a composite microbial inoculant, and the composite microbial inoculant is added to the hydrolysis acidification reactor containing the refining high-concentration wastewater and filled with fiber filler to perform hydrolysis acidification treatment, wherein the volume ratio of the fermentation agent containing Bacillus and the fermentation agent containing Campylobacter in the composite microbial inoculant is 1:3, the composite microbial inoculant accounts for 5% of the volume percentage of the refining high-concentration wastewater, the initial COD of the refining high-concentration wastewater is 1500 mg / L, the oil content is 100 mg / L, the temperature during the hydrolysis acidification treatment is 42℃, the time is 60 h, the fiber filler is made of polypropylene, and the filling rate of the fiber filler in the hydrolysis acidification reactor is 40%.

[0056] The fiber filler in the hydrolysis acidification reactor in the embodiments 4 to 6 of the present application is suspended, the fiber filler is made of polypropylene, and the filling rate of the filler in the hydrolysis acidification reactor is 40%. The Bacillus and the Campylobacter in the composite microbial inoculant are attached to the fiber filler, and the hydraulic retention time of the device is not less than 55 h.

[0057] The fiber filler adopted in the present application has a higher microbial capacity, which is 3 to 5 times of that of general fillers, can provide an optimal environment for the growth and reproduction of microorganisms, and can also play a good filtering and buffering role for continuous multi-stage filling of the filler.

[0058] Embodiment 7: Application of the composite microbial inoculant in refining high-concentration wastewater, which is performed according to the following method:

[0059] First step, Bacillus and Campylobacter are inoculated into LB liquid culture medium respectively, and after shaking culture, Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are obtained respectively, wherein the concentration of Bacillus in the Bacillus fermentation seed liquid is more than 1×10 8 CFU / mL, the concentration of Campylobacter in the Campylobacter fermentation seed liquid is more than 1×10 8 CFU / mL, the culture temperature is 35℃, and the shaking speed is 200 rpm;

[0060] Second step, the Bacillus fermentation seed liquid and the Campylobacter fermentation seed liquid are inoculated into sterilized low-COD refining wastewater respectively to activate, wherein the inoculation amount of the Bacillus fermentation seed liquid and the Campylobacter fermentation seed liquid is 4% of the volume percentage of the sterilized low-COD refining wastewater respectively, the COD of the sterilized low-COD refining wastewater is 800 mg / L, the activation conditions include that the culture temperature is 35℃, the pH is 8, the shaking speed is 200 rpm, and the activation time is 24 h;

[0061] Third step, the activated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are respectively transferred to the high COD refinery wastewater after sterilization for reactivation, wherein the inoculation amount of the activated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid is respectively 4% of the volume percentage of the high COD refinery wastewater after sterilization, the COD of the high COD refinery wastewater after sterilization is 2000mg / L, the activation conditions include that the culture temperature is 35℃, the pH is 8, the shaking speed is 200rpm, and the activation time is 24h;

[0062] Fourth step, the reactivated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are respectively transferred to the fermentation tank containing nutrient solution for continued fermentation and expansion culture, and the fermentation agent containing Bacillus and the fermentation agent containing Campylobacter are respectively obtained, wherein the inoculation amount of the reactivated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid is respectively 4% of the volume percentage of the nutrient solution, the concentration of Bacillus in the fermentation agent containing Bacillus is 1×10 8 CFU / ml or above, the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 8 CFU / ml or above, the culture temperature is 35℃, the pH is 8.0, the rotation speed is 200rpm, the pressure of the fermentation tank is 0.06MPa, the aeration amount is 1.1:1, the fermentation time is 20h, the composition of the nutrient solution further includes glucose 22g / L, proteose peptone 4g / L, yeast powder 1.5g / L, ammonium sulfate 2.5g / L, sodium chloride 4.5g / L, potassium dihydrogen phosphate 0.8g / L, manganese sulfate 0.35g / L, magnesium sulfate 1.5g / L, ferrous sulfate 0.04g / L and calcium carbonate 1.5g / L, and the pH value of the nutrient solution is 7.1;

[0063] Fifth step, the fermentation agent containing Bacillus and the fermentation agent containing Campylobacter are mixed to obtain a composite microbial inoculant, and the composite microbial inoculant is added to the hydrolysis acidification reactor containing refinery high-concentration wastewater and filled with fiber filler for hydrolysis acidification treatment, wherein the volume ratio of the fermentation agent containing Bacillus and the fermentation agent containing Campylobacter in the composite microbial inoculant is 1:2, the composite microbial inoculant accounts for 6% of the volume percentage of the refinery high-concentration wastewater, the initial COD of the refinery high-concentration wastewater is 2000mg / L, the oil content is 64mg / L, during the hydrolysis acidification treatment, the temperature is 45℃, the time is 60h, the fiber filler is polypropylene material, and the filling rate of the fiber filler in the hydrolysis acidification reactor is 40%.

[0064] In the embodiment 7 of the present application, the initial COD of the high-concentration wastewater from refinery and chemical industry is 2000 mg / L, the BOD is 24 mg / L, and the B / C is 0.012. The hydrolysis acidification reactor is provided with suspended fiber packing, and the loading amount is 40%. The hydrolysis acidification reactor uses the down-in and up-out type water inlet, the inoculation amount of the composite microbial agent accounts for 6% of the volume of the hydrolysis acidification reactor, the temperature of the reactor is maintained at 45°C, and the hydraulic retention time of the device is 60 h.

[0065] Embodiment 8: The application of the composite microbial agent in the high-concentration wastewater from refinery and chemical industry is carried out according to the following method.

[0066] Firstly, the bacillus and the campylobacter are respectively inoculated into LB liquid culture medium, and after shaking culture, the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid are obtained, wherein the concentration of the bacillus in the bacillus fermentation seed liquid is more than 1×10 8 CFU / mL, the concentration of the campylobacter in the campylobacter fermentation seed liquid is more than 1×10 8 CFU / mL, the culture temperature is 35°C, and the shaking speed is 200 rpm.

[0067] Secondly, the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid are respectively inoculated into the sterilized low-COD wastewater from refinery and chemical industry for activation, wherein the inoculation amount of the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is respectively 4% of the volume percentage of the sterilized low-COD wastewater from refinery and chemical industry, the COD of the sterilized low-COD wastewater from refinery and chemical industry is 800 mg / L, the activation conditions include that the culture temperature is 35°C, the pH is 8, the shaking speed is 200 rpm, and the activation time is 24 h.

[0068] Thirdly, the activated bacillus fermentation seed liquid and the activated campylobacter fermentation seed liquid are respectively transferred into the sterilized high-COD wastewater from refinery and chemical industry for re-activation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the activated campylobacter fermentation seed liquid is respectively 4% of the volume percentage of the sterilized high-COD wastewater from refinery and chemical industry, the COD of the sterilized high-COD wastewater from refinery and chemical industry is 2500 mg / L, and the re-activation conditions include that the culture temperature is 35°C, the pH is 8, the shaking speed is 200 rpm, and the re-activation time is 24 h.

[0069] Fourthly, the re-activated bacillus fermentation seed liquid and the re-activated campylobacter fermentation seed liquid are respectively transferred into the fermentation tank containing the nutrient solution for further fermentation and expansion culture, and the fermentation agent containing the bacillus and the fermentation agent containing the campylobacter are respectively obtained, wherein the inoculation amount of the re-activated bacillus fermentation seed liquid and the re-activated campylobacter fermentation seed liquid is respectively 4% of the volume percentage of the nutrient solution, and the concentration of the bacillus in the fermentation agent containing the bacillus is 1×10 8The concentration of the Campylobacter in the fermentation agent containing the Campylobacter is 1*10 8 The concentration of the Campylobacter in the fermentation agent containing the Campylobacter is 1*10

[0070] In the fifth step, the fermentation agent containing the Bacillus and the fermentation agent containing the Campylobacter are mixed to obtain a composite microbial agent, and the composite microbial agent is added into a hydrolysis acidification reactor containing the refining high-concentration wastewater and filled with fiber fillers to perform hydrolysis acidification treatment, wherein the volume ratio of the fermentation agent containing the Bacillus to the fermentation agent containing the Campylobacter in the composite microbial agent is 1:2, the composite microbial agent accounts for 6% of the volume percentage of the refining high-concentration wastewater, the initial COD of the refining high-concentration wastewater is 2500 mg / L, the oil content is 132 mg / L, the temperature during the hydrolysis acidification treatment is 45℃, the time is 60 h, the fiber fillers are made of polypropylene, and the filling rate of the fiber fillers in the hydrolysis acidification reactor is 40%.

[0071] In the eighth embodiment of the present application, the initial COD of the refining high-concentration wastewater is 2500 mg / L, the BOD is 32 mg / L, the B / C is 0.013, the fiber fillers are hung in the hydrolysis acidification reactor, the filling amount is 40%, the hydrolysis acidification reactor adopts the water inlet from the bottom and the water outlet from the top, the inoculation amount of the composite microbial agent accounts for 6% of the volume of the hydrolysis acidification reactor, the temperature of the reactor is maintained at 45℃, and the hydraulic retention time of the device is 65 h.

[0072] By using the treatment methods in the fourth embodiment to the eighth embodiment of the present application, the composite microbial agent is added into the hydrolysis acidification reactor containing the refining high-concentration wastewater and filled with fiber fillers, so that the refining high-concentration wastewater with a COD of 800 mg / L to 2500 mg / L and an oil content of not more than 132 mg / L can be treated, the oil content of the effluent after the hydrolysis acidification treatment of the composite microbial agent is not more than 50 mg / L, and the COD removal rate is more than 60%. The following is the evaluation of the strain screening, identification, and the treatment effect of the Campylobacter on the refining high-concentration wastewater in the seventh embodiment and the eighth embodiment of the present application.

[0073] Test 1: Strain screening and identification

[0074] Test method:

[0075] Step 1: Samples were taken from multiple high-concentration wastewater units in the petrochemical enterprise. This high-concentration wastewater included top wastewater from atmospheric and vacuum distillation towers in the distillation unit, wastewater from the sulfur-containing wastewater stripping unit, and electro-desalting wastewater from the crude oil coking unit. 50 ml of the high-concentration wastewater was placed in a 250 mL Erlenmeyer flask and incubated for 24 hours in a shaker at 30°C and 150 rpm. Then, the flask was serially diluted and spread onto LB agar plates. The LB agar plates were incubated at 30°C for 48 hours, and single colonies of different morphologies were selected.

[0076] Step 2: The obtained single colonies of different morphologies were inoculated into LB medium for enrichment and cultured for 24 hours to obtain bacterial solutions of different strains;

[0077] Step 3: High-concentration wastewater mixed from the industrial water unit of the refining and chemical enterprise is sterilized by high-pressure steam at a temperature of 120℃, a pressure of 0.1MPa, and a time of 30min.

[0078] Step 4: After cooling the sterilized high-concentration refining wastewater to room temperature, take 5 ml of bacterial suspension from different strains and inoculate them into 100 mL of sterilized high-concentration refining wastewater for hydrolysis and acidification treatment. After inoculation, place them in a shaker at 45℃ and 120 rpm for evaluation. The optimal strain is selected by the COD value of the bacterial suspension from different strains in the sterilized high-concentration refining wastewater after 120 h of hydrolysis and acidification treatment. At the same time, the optimal strain is morphologically identified.

[0079] Experimental results: As shown in Table 1, Table 1 shows the COD values ​​of high-concentration refining wastewater after sterilization treated by different strains. As shown in Table 1, strain 5 (Bacillus) and strain 7 (Campylobacter) have the best treatment effect on COD values ​​of high-concentration refining wastewater after sterilization. Strains 5 and 7 have high degradation capacity and are the best dominant degrading bacteria.

[0080] Morphological identification results of strain 5: as follows Figure 1 As shown, the bacterial characteristics are: colonies appear as small, opaque, milky-white dots. After morphological, physiological, biochemical, and molecular biological identification, strain No. 5 was identified as Bacillus sp., which was deposited on January 18, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21651, located at Building 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0081] Morphological identification results of strain 7: as follows Figure 2As shown, the strain characteristics are colony morphology protruding wet, irregular shape, rough surface, and milky white. After morphological, physiological and biochemical properties and molecular biology identification, strain 7 is Campylobacter sp. and has been preserved in the China General Microbiological Culture Collection Center on January 18, 2021, with the preservation number CGMCC No. 21652 and the preservation address No. 3, Beichen West Road, Chaoyang District, Beijing.

[0082] The nucleic acid sequences of strains 5 and 7 are shown in the sequence table.

[0083] Strains 5 and 7 are both conventional facultative anaerobes after puncture culture.

[0084] Test 2: Investigation of the COD removal effect of Bacillus and Campylobacter in the compound microbial agent on mixed sewage under different proportions.

[0085] Test method: The orthogonal test is used to determine the optimal compounding ratio and strain dosage. The total dosage of the two kinds of dominant degrading bacteria (i.e. the dosage of the compound microbial agent) is factor A, and the dosage ratio of the two kinds of dominant degrading bacteria Bacillus and Campylobacter is factor B. The orthogonal test design scheme is shown in Table 2. The enriched Bacillus (strain 5) and Campylobacter (strain 7) are added into the sterilized refining high-concentration wastewater according to the orthogonal design scheme for shaking bed evaluation. The COD removal rates after hydrolysis acidification treatment for 48h, 72h and 5d are investigated respectively.

[0086] Test results: Table 2 is the orthogonal test design table, and Table 3 is the COD value and COD removal rate in the water quality after hydrolysis acidification treatment of the sterilized refining high-concentration wastewater under different test schemes, Figure 3 as shown in Table 3 and Figure 3 Scheme 5 has the fastest COD removal rate and better degradation rate on the sterilized refining high-concentration wastewater. The COD removal rate of scheme 5 reaches 63.86% after hydrolysis acidification treatment for 48h, and the COD removal rate of scheme 5 reaches 77.72% after hydrolysis acidification treatment for 5d.

[0087] It is shown that the optimal volume ratio of the two kinds of dominant degrading bacteria Bacillus (strain 5) and Campylobacter (strain 7) is 1:2, and the total dosage of the two kinds of dominant degrading bacteria is 6% (i.e. the optimal dosage of the compound microbial agent is 6%).

[0088] Test 3: Evaluation of the effect of the compound microbial agent on the hydrolysis acidification treatment of refining high-concentration wastewater

[0089] Test method: according to the treatment method of Example 7 and Example 8 of the present application, the composite microbial agent is used for hydrolysis acidification treatment of the refining high-concentration wastewater, and the water quality after the hydrolysis acidification treatment is detected, the water quality indexes include chemical oxygen demand COD, ammonia nitrogen, sulfide, total phosphorus, oil content and effluent water sludge concentration, the chemical oxygen demand COD is determined according to the method specified in GB / T 11914-2015 'Determination of Chemical Oxygen Demand in Water Potassium Dichromate Method', the ammonia nitrogen is determined according to the method specified in HJ536-2015 'Determination of Ammonia Nitrogen in Water Nessler's Reagent Spectrophotometric Method', the sulfide is determined according to the method specified in GB / T16489-2015 'Determination of Sulfide in Water Methylene Blue Spectrophotometric Method', the total phosphorus is determined according to the method specified in GB / T11893-2015 'Determination of Total Phosphorus in Water Ammonium Molybdate Spectrophotometric Method', and the oil content is determined according to the method specified in HJ637-2015 'Determination of Oil and Animal Oil in Water Infrared Spectrophotometric Method'.

[0090] Test results: the indexes of the refining high-concentration wastewater before and after the hydrolysis acidification treatment of Example 7 of the present application are shown in Table 4, and it can be known from Table 4 that the indexes of the refining high-concentration wastewater before the hydrolysis acidification treatment in Example 7 of the present application are as follows: COD is 2000 mg / L, oil content is 64 mg / L, sulfide content is 25 mg / L, and ammonia nitrogen content is 16 mg / L, and the indexes of the refining high-concentration wastewater after the hydrolysis acidification treatment by the composite microbial agent containing bacillus and campylobacter are as follows: COD value is 1000 mg / L, oil content is 21 mg / L, sulfide content is 14 mg / L, and ammonia nitrogen content is 6 mg / L.

[0091] The indexes of the refining high-concentration wastewater before and after the hydrolysis acidification treatment of Example 8 of the present application are shown in Table 5, and it can be known from Table 5 that the indexes of the refining high-concentration wastewater before the hydrolysis acidification treatment in Example 8 of the present application are as follows: COD is 2500 mg / L, oil content is 132 mg / L, sulfide content is 31 mg / L, and ammonia nitrogen content is 21 g / L, and the indexes of the refining high-concentration wastewater after the hydrolysis acidification treatment by the composite microbial agent containing bacillus and campylobacter are as follows: COD value is 1100 mg / L, oil content is 43 mg / L, sulfide content is 17 mg / L, and ammonia nitrogen content is 10 mg / L.

[0092] It is shown that the composite microbial agent of the present application can treat the refining high-concentration wastewater with COD of 800 mg / L to 2500 mg / L and oil content of not more than 132 mg / L, and after the hydrolysis acidification treatment by the composite microbial agent, the oil content in the effluent wastewater is not more than 50 mg / L, and the COD removal rate reaches more than 60%.

[0093] Experiment 4: Investigating the shock load resistance of the hydrolysis acidification reactor after initial addition of compound microbial inoculant.

[0094] Experimental Methods: Following the ratio and total dosage of the two dominant degrading bacteria, Bacillus and Campylobacter, determined in Experiment 2, a bio-enhanced hydrolysis acidification reactor was constructed using bacterial broth. The bacterial strains were added in stages. Each time the COD concentration of the high-concentration refining wastewater influent was increased, the compound bacterial broth was promptly replenished in the reactor to minimize the impact of load shocks on the overall stability of the microbial community within the reactor. Specifically, taking the first addition with an influent COD of 1800 mg / L as an example, with a total hydraulic retention time of 55 h as the cycle, influent of the same reactor volume was prepared and replenished in three batches, approximately every 18 h. Simultaneously, strains 5 and 7 were enriched to a concentration of 1 × 10⁻⁶ using inorganic salt culture medium. 8 CFU / ml, according to the optimal addition ratio, Bacillus (strain 5): Campylobacter (strain 7) = 1:2, and the total bacterial solution volume is 6% of the influent volume each time, so that the bacterial solution enters the hydrolysis acidification reactor together with the influent. The COD of the influent and effluent of the hydrolysis acidification reactor is continuously recorded, and the COD and COD removal rate of the effluent quality of the hydrolysis acidification reactor within one week after the addition of the compound bacterial solution are investigated.

[0095] Test results: such as Figure 4 As shown, by Figure 4 It can be seen that within one week of adding the compound bacterial solution, the COD treatment efficiency of the hydrolysis acidification reactor rapidly increased, and the COD removal rate stabilized at over 50%. Simultaneously, the organic load of the hydrolysis acidification reactor increased from 0.31 kg / (m³) before bacterial addition. 3 ·d) Increased to 0.56 kg / (m 3 ·d), which meets the requirements for the full operation of the hydrolysis acidification reactor.

[0096] Experiment 5: Investigating the resistance to shock loads of the bio-enhanced hydrolysis acidification reactor.

[0097] Experimental method: After the hydrolysis acidification reactor was fully started and operated stably in Experiment 4, the addition of compound bacterial solution to the hydrolysis acidification reactor was stopped, and the COD concentration of the influent was gradually increased to above 2400 mg / L. The COD of the influent and effluent of the hydrolysis acidification reactor was continuously recorded to examine the resistance of the hydrolysis acidification reactor to load shock after biological enhancement.

[0098] Test results: such as Figure 5 As shown, by Figure 5 It can be seen that even after increasing the influent COD concentration to above 2400 mg / L, the hydrolysis acidification reactor still exhibits good resistance to load shocks, and the organic loading rate of the hydrolysis acidification reactor is increased to 0.63 kg / (m³).3 Approximately d). This indicates that the added compound bacterial solution achieved good and stable growth in the hydrolysis acidification reactor, gradually settling on the fiber packing material within the reactor. The bacterial concentration gradually stabilized, the overall activity was high, and the hydrolysis acidification reactor was operating stably.

[0099] Experiment 6: Investigating the hydraulic retention time of the bio-enhanced hydrolysis acidification reactor in treating high-concentration refinery wastewater.

[0100] Experimental method: In order to determine the stability of the microbial community in the reactor and further shorten the hydraulic retention time of the reactor to increase the overall treatment efficiency of the reactor, the hydraulic retention time of the reactor was reduced from 56h to 48h, and the COD of the reactor influent and effluent were continuously recorded.

[0101] Experimental results: Figure 6 As shown, by Figure 6 It can be seen that the overall organic load of the hydrolysis acidification reactor is reduced to 0.51 kg / (m³). 3 Around d), the treatment effect decreased slightly, but the organic load of the hydrolysis acidification reactor was still 0.31 kg / (m³) higher than the organic load before the addition of the compound bacterial solution. 3 •d) indicates that the bio-enhanced treatment of the reactor by adding two dominant degrading bacteria significantly improved the degradation effect of the hydrolysis acidification reactor, confirming that adding two dominant degrading bacteria to improve the degradation effect of the reactor is effective.

[0102] In summary, this invention, by adding a compound microbial agent to a hydrolysis acidification reactor containing high-concentration refining wastewater and packed with fiber packing, can treat high-concentration refining wastewater with a COD of 800 mg / L to 2500 mg / L and an oil content not exceeding 132 mg / L. The hydraulic retention time is over 55 hours, and the oil content of the effluent after hydrolysis acidification treatment with the compound microbial agent is no higher than 50 mg / L, achieving a COD removal rate of over 60%. This significantly reduces the cost of subsequent environmental pollution treatment. Furthermore, the organic load of the hydrolysis acidification reactor, after the addition of two dominant degrading bacteria, is reduced from 0.31 kg / (m³). 3 ·d) Increased to 0.63 kg / (m 3 ·d), and the bio-enhanced hydrolysis acidification reactor has a good ability to resist shock loads.

[0103] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

[0104] Table 1

[0105]

[0106] Table 2

[0107]

[0108] Table 3

[0109]

[0110] Table 4

[0111] Property Influent Effluent COD, mg / L 2000 1000 Ammonia nitrogen, mg / L 16 6 Sulfide, mg / L 25 14 Total phosphorus, mg / L 2.2 1.3 Oil content, mg / L 64 21

[0112] Table 5

[0113] Property Influent Effluent COD, mg / L 2500 1100 Ammonia nitrogen, mg / L 21 10 Sulfide, mg / L 31 17 Total phosphorus, mg / L 2.1 1.0 Oil content, mg / L 132 43 SEQUENCE LISTING <110> PetroChina Karamay Petrochemical Co., Ltd., China National Petroleum Corporation <120> Bacillus and composite microbial agent and its application in refining high-concentration wastewater <130> 2021 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1000 <212> DNA <213> Bacillus sp. <400> 1 ggaagtggcg ggtgctatac tgcaagtcga gcggacagat gggagcttgc tccctgatgt 60 tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120 gaaaccgggg ctaataccgg atggttgttt gaaccgcatg gttcaaacat aaaaggtggc 180 ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240 accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300 cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360 cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420 gaacaagtac cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct 480 aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540 cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg 600 agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag 660 cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720 actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780 gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840 cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900 ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960 gtcttgacat cctctgacaa tcctagagat aggacgtccc 1000 <210> 2 <211> 1000 <212> DNA <213> Campylobacter sp. <400> 2 aagtgatccc cttcgacggc tccttccaca agggttaggc caccggcttc gggtgttacc 60 gactttcatg acttgacggg cggtgtgtac aaggcccggg aacgtattca ccgcagcgtt 120 gctgatctgc gattactagc gactccgact tcatgaggtc gagttgcaga cctcaatccg 180 aactgagacc ggctttttgg gattcgctcc accttacggt atcgcagccc tttgtaccgg 240 ccattgtagc atgcgtgaag cccaagacat aaggggcatg atgatttgac gtcatcccca 300 ccttcctccg agttgacccc ggcagtctcc tatgagtccc cggcataacc cgctggcaac 360 atagaacgag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 420 gacaaccatg caccacctgt acaccgacca caagggggcg accatctctg gccgtttccg 480 gtgtatgtca agccttggta aggttcttcg cgttgcatcg aattaatccg catgctccgc 540 cgcttgtgcg ggcccccgtc aattcctttg agttttagcc ttgcggccgt actccccagg 600 cggggcgctt aatgcgttag ctacgacaca gaaaccgtgg aaaggtccct acatctagcg 660 cccaacgttt acggcatgga ctaccagggt atctaatcct gttcgctccc catgctttcg 720 ctcctcagcg tcagttacgg cccagagatc tgccttcgcc atcggtgttc ctcctgatat 780 ctgcgcattc caccgctaca ccaggaattc caatctcccc taccgcactc tagtctgccc 840 gtacccactg caagcccgag gttgagcctc gggatttcac agcagacgcg acaaaccgcc 900 tacgagctct ttacgcccaa taattccgga caacgcttgc accctacgta ttaccgcggc 960 tgctggcacg tagttagccg gtgcttttct gcaggtaccg 1000

Claims

1. A compound microbial agent, characterized in that... The compound microbial inoculant is composed of a Bacillus-containing fermentation agent and a Campylobacter-containing fermentation agent mixed at a volume ratio of 1:1 to 1:

5. The Bacillus-containing fermentation agent includes Bacillus and nutrient solution, and the concentration of Bacillus in the Bacillus-containing fermentation agent is 1×10⁻⁶. 8 Fermentation broth containing Campylobacter above CFU / mL includes both Campylobacter and nutrient solution, with a Campylobacter concentration of 1×10⁻⁶ in the fermentation broth. 8 CFU / mL or higher, including Bacillus ( Bacillus sp. The strain was deposited on January 18, 2021, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 21651. Campylobacter (… Campylobacter sp. The strain was deposited at the China General Microbiological Culture Collection Center on January 18, 2021, with accession number CGMCC No. 21652.

2. The compound microbial agent according to claim 1, characterized in that... The nutrient solution consists of 20 g / L to 24 g / L glucose, 2 g / L to 5 g / L peptone, 1 g / L to 2 g / L yeast extract, 2 g / L to 3 g / L ammonium nitrate, 4 g / L to 5 g / L sodium chloride, 0.5 g / L to 1 g / L potassium dihydrogen phosphate, 0.3 g / L to 0.4 g / L manganese sulfate, 1 g / L to 2 g / L magnesium sulfate, 0.02 g / L to 0.05 g / L ferrous sulfate, and 1 g / L to 2 g / L calcium carbonate. The pH of the nutrient solution is 7.0 to 7.

2.

3. The application of a composite microbial agent according to claim 1 or 2 in high-concentration refining wastewater.

4. The application of the composite microbial agent according to claim 3 in high-concentration refining wastewater, characterized in that... Perform it as follows: The first step involved inoculating Bacillus and Campylobacter into LB liquid medium, respectively, and culturing them on a shaker to obtain Bacillus fermentation seed culture and Campylobacter fermentation seed culture, respectively. The concentration of Bacillus in the Bacillus fermentation seed culture was 1×10⁻⁶. 8 The concentration of Campylobacter in the Campylobacter fermentation seed culture is above 1×10⁻⁶ CFU / mL. 8 CFU / mL or higher, culture temperature 30℃ to 35℃, shaker speed 150 rpm to 200 rpm; The second step involves inoculating the Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid into the sterilized low-COD refining wastewater for activation. The inoculation amounts of the Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are 3% to 6% of the volume percentage of the sterilized low-COD refining wastewater, respectively. The COD of the sterilized low-COD refining wastewater is 500 mg / L to 800 mg / L. The activation conditions include a culture temperature of 30℃ to 35℃, a pH of 8 to 10, a shaking speed of 150 rpm to 200 rpm, and an activation time of 24 h. The third step involves transferring the activated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid to the sterilized high-COD refining wastewater for further activation. The inoculation amounts of the activated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are 3% to 6% of the volume percentage of the sterilized high-COD refining wastewater, respectively. The COD of the sterilized high-COD refining wastewater is 800 mg / L to 2500 mg / L. The activation conditions include a culture temperature of 30℃ to 35℃, a pH of 8 to 10, a shaking speed of 150 rpm to 200 rpm, and an activation time of 24 h. The fourth step involves transferring the reactivated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid to fermenters containing nutrient solution for further fermentation and scale-up culture, respectively, to obtain fermentation broths containing Bacillus and Campylobacter, respectively. The inoculation amounts of the reactivated Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid are 3% to 6% of the nutrient solution volume, respectively, and the concentration of Bacillus in the fermentation broth containing Bacillus is 1 × 10⁻⁶. 8 For starter cultures containing Campylobacter above CFU / ml, the concentration of Campylobacter is 1×10⁻⁶. 8 The fermentation conditions are as follows: CFU / ml or higher, culture temperature is 28℃ to 38℃, pH is 7.5 to 9.5, rotation speed is 200 rpm, fermenter pressure is 0.03 MPa to 0.06 MPa, aeration rate is 1:1 to 1.3:1, and fermentation time is 16h to 20h. The fifth step involves mixing a fermentation agent containing Bacillus and a fermentation agent containing Campylobacter to obtain a compound microbial agent. This compound microbial agent is then added to a hydrolysis acidification reactor containing high-concentration refining wastewater and filled with fiber packing material for hydrolysis acidification treatment. The volume ratio of the Bacillus-containing fermentation agent to the Campylobacter-containing fermentation agent in the compound microbial agent is 1:1 to 1:

5. The compound microbial agent accounts for 4% to 6% of the volume of the high-concentration refining wastewater. The initial COD of the high-concentration refining wastewater is 800 mg / L to 2500 mg / L, and the oil content is not higher than 135 mg / L. During the hydrolysis acidification treatment, the temperature is 40℃ to 45℃, and the time is not less than 55 hours. The fiber packing material is polypropylene, and the filling rate of the fiber packing material in the hydrolysis acidification reactor is 20% to 70%.

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