CN116286426A - Bacillus and compound microbial agent and application thereof in refining high-concentration wastewater - Google Patents

Abstract

The invention relates to the technical field of biological wastewater treatment, in particular to bacillus and a compound microbial agent and application thereof in refining high-concentration wastewater, wherein the bacillus and the compound microbial agent are prepared according to the following steps: inoculating bacillus and campylobacter respectively into LB liquid culture medium for culture, respectively transferring to sterilized high-concentration wastewater for twice activation, transferring bacillus bacterial liquid and campylobacter bacterial liquid after twice activation to nutrient solution for continuous expansion culture, and finally mixing the obtained bacillus-containing fermentation agent and campylobacter-containing fermentation agent proportionally and adding the mixture into the high-concentration wastewater for hydrolysis and acidification treatment. The composite microbial agent has good high temperature resistance, impact resistance and adaptability, and can realize COD removal rate of more than 60% by adding the composite microbial agent into a hydrolysis acidification reactor containing refined high-concentration wastewater and filled with fibrous filler, thereby greatly reducing the cost of subsequent treatment of environmental pollution.

Description

Bacillus and compound microbial agent and application thereof in refining high-concentration wastewater

Technical Field

The invention relates to the technical field of biological wastewater treatment, in particular to bacillus and a compound microbial agent and application thereof in refining high-concentration wastewater.

Background

Along with the heavy and poor quality of crude oil, the pollution condition of the refining wastewater is also more and more complex, the existing 'old three sets' process, namely 'oil separation-air floatation-biochemistry' is difficult to treat the current complex sewage, and enterprises have to enhance the degradation of pollutants by superposing and prolonging the process flow. The hydrolysis acidification process has obvious advantages in treating high-concentration wastewater, and the hydrolysis acidification process breaks down macromolecular organic pollutants in the wastewater into micromolecular long-chain structures through anaerobic fermentation, so that micromolecular organic matters which are more easily broken down, such as acidic substances or alcohols, are further broken down. In the hydrolysis and acidification treatment process, the core key position is high-efficiency degradation bacteria, and secondly, the correct selection of the filler is the key for ensuring the good function of the functional bacteria.

Disclosure of Invention

The invention provides bacillus and a compound microbial agent and application thereof in refining high-concentration wastewater, overcomes the defects of the prior art, can treat the refining high-concentration wastewater with COD of 800mg/L to 2500mg/L and oil content not higher than 132mg/L, and realizes COD removal rate of more than 60 percent by the oil content of not higher than 50mg/L of effluent water quality after hydrolysis and acidification treatment of the compound microbial agent, thereby greatly reducing the cost of subsequent treatment of environmental pollution.

One of the technical schemes of the invention is realized by the following measures: bacillus sp.this strain was preserved in China general microbiological culture Collection center (CGMCC) under the preservation number of CGMCC No.21651 at 18/1/2021.

The second technical scheme of the invention is realized by the following measures: a compound microbial agent containing Bacillus.

The following is a further optimization and/or improvement of the second technical scheme of the invention:

the volume ratio of the composite microbial agent is 1:1 to 5 and a Bacillus containing starter culture comprising Bacillus and a nutrient solution, wherein the concentration of Bacillus in the Bacillus starter culture is 1×10 ⁸ The fermentation agent containing Campylobacter comprises Campylobacter and nutrient solution, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ CFU/mL or more.

The nutrient solution comprises 20g/L to 24g/L of glucose, 2g/L to 5g/L of peptone, 1g/L to 2g/L of yeast powder, 2g/L to 3g/L of ammonium nitrate, 4g/L to 5g/L of sodium chloride, 0.5g/L to 1g/L of monopotassium phosphate, 0.3g/L to 0.4g/L of manganese sulfate, 1g/L to 2g/L of magnesium sulfate, 0.02g/L to 0.05g/L of ferrous sulfate and 1g/L to 2g/L of calcium carbonate, and the pH value of the nutrient solution is 7.0 to 7.2.

The third technical scheme of the invention is realized by the following measures: an application of a compound microbial agent in refining high-concentration wastewater.

The following is a further optimization and/or improvement of the third aspect of the present invention:

the application of the composite microbial agent in refining high-concentration wastewater is carried out according to the following method:

in the first step, bacillus and campylobacter are separatedRespectively inoculating into LB liquid culture medium, shake culturing to obtain Bacillus fermentation seed liquid and Campylobacter fermentation seed liquid, 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 above 1X 108CFU/mL, the culture temperature is 30-35 ℃, and the rotation speed of a shaking table is 150-200 rpm;

the second step, inoculating the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into the sterilized low COD refining wastewater for activation, wherein the inoculum size of the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3 to 6 percent of the volume percentage of the sterilized low COD refining wastewater, the COD of the sterilized low COD refining wastewater is 500 to 800mg/L, the activation conditions comprise the culture temperature of 30 to 35 ℃, the pH of 8 to 10, the rotation speed of a shaking table of 150 to 200rpm and the activation time of 24 hours;

Transferring the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized high COD (chemical oxygen demand) refining wastewater for reactivation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3 to 6 percent of the volume percentage of the sterilized high COD refining wastewater, the COD of the sterilized high COD refining wastewater is 800 to 2500mg/L, the activation condition comprises that the culture temperature is 30 to 35 ℃, the pH is 8 to 10, the rotation speed of a shaking table is 150 to 200rpm, and the activation time is 24 hours;

transferring the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid to a fermentation tank containing a nutrient solution respectively for continuous fermentation and expansion culture to obtain a bacillus-containing fermentation agent and a campylobacter-containing fermentation agent respectively, wherein the inoculation amount of the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3-6% of the volume percentage of the nutrient solution respectively, and the concentration of bacillus in the bacillus-containing fermentation agent is 1 multiplied by 10 ⁸ CFU/ml or more, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ Over CFU/ml, the culture temperature is 28-38deg.C, pH is 7.5-9.5, and the rotation speed is high200rpm, the pressure of the fermentation tank is 0.03MPa to 0.06MPa, the aeration rate is 1.0 to 1.3:1, and the fermentation time is 16h to 20h;

fifthly, mixing a bacillus-containing starter and a campylobacter-containing starter to obtain a composite microbial agent, and adding the composite microbial agent into a hydrolysis acidification reactor containing refined high-concentration wastewater and filled with fiber fillers for hydrolysis acidification treatment, wherein the volume ratio of the bacillus-containing starter to the campylobacter-containing starter in the composite microbial agent is 1:1 to 5, the composite microbial agent accounts for 4 to 6 percent of the volume percentage of the refined high-concentration wastewater, the initial COD of the refined high-concentration wastewater is 800 to 2500mg/L, the oil content is not higher than 135mg/L, the temperature is 40 to 45 ℃ during hydrolytic acidification treatment, the time is not less than 55 hours, the fiber filler is made of polypropylene fiber, and the filling rate of the fiber filler in a hydrolytic acidification reactor is 20 to 70 percent.

Compared with the prior art, the invention has the beneficial effects that:

the bacillus and campylobacter are compounded to obtain the composite microbial agent, and the composite microbial agent is applied to the hydrolysis acidification treatment refining high-concentration wastewater, and can be suitable for refining high-concentration wastewater with the temperature of 40-45 ℃ for a long time, and has strong impact resistance when the upstream incoming water quality is greatly changed, so that the composite microbial agent has good high temperature resistance, impact resistance and adaptability.

The invention adds the composite microbial agent into the hydrolysis acidification reactor containing the refined high-concentration wastewater and filled with the fibrous filler, can treat the refined high-concentration wastewater with COD of 800mg/L to 2500mg/L and oil content of not higher than 132mg/L, has hydraulic retention time of more than 55h, and the oil content of the effluent water quality after the hydrolysis acidification treatment of the composite microbial agent of not higher than 50mg/L, realizes COD removal rate of more than 60 percent, greatly reduces the cost of subsequent treatment of environmental pollution, and simultaneously has organic load of 0.31 kg/(m) by adding the hydrolysis acidification reactor containing the composite microbial agent of two dominant degradation bacteria ³ D) lifting to 0.63 kg/(m) ³ D) and the bio-enhanced hydrolytic acidification reactor has good impact resistanceCapacity of load.

Drawings

FIG. 1 is an electron microscope image of Bacillus sp.

FIG. 2 is an electron microscope image of Campylobacter sp.

FIG. 3 shows COD removal rate of water quality after hydrolysis and acidification treatment of sterilized refined high-concentration wastewater by two dominant degrading bacteria with different proportions.

FIG. 4 shows COD and COD removal rate of effluent water quality within one week of treating refined high-concentration wastewater by adding dominant degrading bacteria with optimal proportion into a hydrolysis acidification reactor.

FIG. 5 shows COD and COD removal rate of effluent water quality when the hydrolysis acidification reactor is used for treating high-concentration COD inlet water after biological strengthening.

FIG. 6 shows the COD and COD removal rate of effluent water quality when the hydrolysis acidification reactor is used for treating high-concentration inlet water after biological strengthening, and the hydraulic retention time is reduced.

Detailed Description

The present invention is not limited by the following examples, and specific embodiments can be determined according to the technical scheme and practical situations of the present invention. The various chemical reagents and chemical supplies mentioned in the invention are all commonly known and used in the prior art unless specified otherwise; the percentages in the invention are mass percentages unless otherwise specified.

The invention is further described below with reference to examples:

example 1: the Bacillus sp has been preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.21651 at 1 month and 18 days of 2021.

Example 2: the volume ratio of the composite microbial agent is 1:1 to 5 and a Bacillus containing starter culture comprising Bacillus and a nutrient solution, wherein the concentration of Bacillus in the Bacillus starter culture is 1×10 ⁸ CFU/mL or more, the fermentation agent containing Campylobacter comprises Campylobacter and nutrient solution, and the fermentation agent contains CampylobacterThe concentration of Campylobacter was 1×10 ⁸ CFU/mL or more; the nutrient solution comprises 20g/L to 24g/L of glucose, 2g/L to 5g/L of peptone, 1g/L to 2g/L of yeast powder, 2g/L to 3g/L of ammonium nitrate, 4g/L to 5g/L of sodium chloride, 0.5g/L to 1g/L of monopotassium phosphate, 0.3g/L to 0.4g/L of manganese sulfate, 1g/L to 2g/L of magnesium sulfate, 0.02g/L to 0.05g/L of ferrous sulfate and 1g/L to 2g/L of calcium carbonate, and the pH value of the nutrient solution is 7.0 to 7.2.

Preservation description of Campylobacter:

strain name: campylobacter bacteria

Latin name: campylobacter sp.

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

Preservation number: CGMCC No.21652

Preservation address: china academy of sciences of microorganisms, no. 3, north Chen West Lu 1, chaoyang, beijing, china

Preservation date: 2021, 01, 18

Example 3: the application of the composite microbial agent in refining high-concentration wastewater.

Example 4: the application of the composite microbial agent in refining high-concentration wastewater is carried out according to the following method:

the first step, respectively inoculating bacillus and campylobacter into LB liquid culture medium, and after shake cultivation, respectively obtaining bacillus fermentation seed liquid and campylobacter fermentation seed liquid, 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 30 ℃ above CFU/mL, and the rotation speed of the shaking table is 150rpm;

the second step, inoculating the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized low COD refining wastewater for activation, wherein the inoculum size of the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3 percent of the volume percentage of the sterilized low COD refining wastewater, the COD of the sterilized low COD refining wastewater is 500mg/L, the activation conditions comprise the culture temperature of 30 ℃, the pH value of 8, the rotation speed of a shaking table of 150rpm and the activation time of 24 hours;

transferring the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized high COD (chemical oxygen demand) refining wastewater for reactivation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3 percent of the volume percentage of the sterilized high COD refining wastewater, the COD of the sterilized high COD refining wastewater is 800mg/L, the activation condition comprises that the culture temperature is 30 ℃, the pH is 8, the rotation speed of a shaking table is 150rpm, and the activation time is 24 hours;

transferring the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid to a fermentation tank containing a nutrient solution respectively for continuous fermentation and expansion culture to obtain a bacillus-containing fermentation agent and a campylobacter-containing fermentation agent respectively, wherein the inoculation amount of the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3 percent of the volume percentage of the nutrient solution respectively, and the concentration of bacillus in the bacillus-containing fermentation agent is 1 multiplied by 10 ⁸ CFU/ml or more, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ The culture temperature is above CFU/ml, the pH is 7.5, the rotating speed is 200rpm, the pressure of a fermentation tank is 0.03MPa, the ventilation amount is 1.0:1, the fermentation time is 16h, the nutrient solution also comprises 20g/L glucose, 2g/L peptone, 1g/L yeast powder, 2g/L ammonium nitrate, 4g/L sodium chloride, 0.5g/L monopotassium phosphate, 0.3g/L manganese sulfate, 1g/L magnesium sulfate, 0.02g/L ferrous sulfate and 1g/L calcium carbonate, and the pH value of the nutrient solution is 7.0;

fifthly, mixing a bacillus-containing starter and a campylobacter-containing starter to obtain a composite microbial agent, and adding the composite microbial agent into a hydrolysis acidification reactor containing refined high-concentration wastewater and filled with fiber fillers for hydrolysis acidification treatment, wherein the volume ratio of the bacillus-containing starter to the campylobacter-containing starter in the composite microbial agent is 1:1, the composite microbial agent accounts for 4 percent of the volume percentage of the refined high-concentration wastewater, the initial COD of the refined high-concentration wastewater is 800mg/L, the oil content is 60mg/L, the temperature is 40 ℃ during hydrolysis and acidification treatment, the time is 55h, the fiber filler is made of polypropylene, and the filling rate of the fiber filler in a hydrolysis and acidification reactor is 20 percent.

Example 5: the application of the composite microbial agent in refining high-concentration wastewater is carried out according to the following method:

the first step, respectively inoculating bacillus and campylobacter into LB liquid culture medium, and after shake cultivation, respectively obtaining bacillus fermentation seed liquid and campylobacter fermentation seed liquid, 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 ℃ above CFU/mL, and the rotation speed of the shaking table is 200rpm;

the second step, inoculating the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized low COD refining wastewater for activation, wherein the inoculum size of the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 6 percent of the volume percentage of the sterilized low COD refining wastewater, the COD of the sterilized low COD refining wastewater is 800mg/L, the activation conditions comprise the culture temperature of 35 ℃, the pH value of 10, the rotation speed of a shaking table of 200rpm and the activation time of 24 hours;

transferring the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized high-COD refined wastewater for reactivation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 6% of the volume percentage of the sterilized high-COD refined wastewater, the COD of the sterilized high-COD refined wastewater is 2500mg/L, the activation condition comprises that the culture temperature is 35 ℃, the pH is 10, the rotation speed of a shaking table is 200rpm, and the activation time is 24 hours;

Transferring the re-activated bacillus fermentation seed liquid and campylobacter fermentation seed liquid to a fermentation tank containing a nutrient solution for continuous fermentation and expansion culture respectively to obtain a bacillus-containing fermentation agent and a campylobacter-containing fermentation agent, wherein the inoculation amount of the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 6% of the volume percentage of the nutrient solution respectively, and the bacillus-containing fermentation seed liquid and the campylobacter-containing fermentation seed liquid respectively containThe concentration of bacillus in the bacillus starter is 1×10 ⁸ CFU/ml or more, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ The culture temperature is above CFU/ml, the pH is 9.5, the rotating speed is 200rpm, the pressure of a fermentation tank is 0.06MPa, the ventilation amount is 1.3:1, the fermentation time is 20h, the nutrient solution also comprises 24g/L of glucose, 5g/L of peptone, 2g/L of yeast powder, 3g/L of ammonium nitrate, 5g/L of sodium chloride, 1g/L of monopotassium phosphate, 0.4g/L of manganese sulfate, 2g/L of magnesium sulfate, 0.05g/L of ferrous sulfate and 2g/L of calcium carbonate, and the pH value of the nutrient solution is 7.2;

fifthly, mixing a bacillus-containing starter and a campylobacter-containing starter to obtain a composite microbial agent, and adding the composite microbial agent into a hydrolysis acidification reactor containing refined high-concentration wastewater and filled with fiber fillers for hydrolysis acidification treatment, wherein the volume ratio of the bacillus-containing starter to the campylobacter-containing starter in the composite microbial agent is 1: and 5, the composite microbial agent accounts for 6% of the volume percentage of the refined high-concentration wastewater, the initial COD of the refined high-concentration wastewater is 2500mg/L, the oil content is 132mg/L, the temperature is 45 ℃ during hydrolysis and acidification treatment, the time is 65h, the fiber filler is made of polypropylene, and the filling rate of the fiber filler in the hydrolysis and acidification reactor is 70%.

Example 6: the application of the composite microbial agent in refining high-concentration wastewater is carried out according to the following method:

the first step, respectively inoculating bacillus and campylobacter into LB liquid culture medium, and after shake cultivation, respectively obtaining bacillus fermentation seed liquid and campylobacter fermentation seed liquid, 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 32 ℃ above CFU/mL, and the rotation speed of a shaking table is 200rpm;

the second step, inoculating the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized low COD refining wastewater for activation, wherein the inoculum size 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, the COD of the sterilized low COD refining wastewater is 600mg/L, the activation conditions comprise the culture temperature of 32 ℃, the pH value of 9, the rotation speed of a shaking table of 180rpm and the activation time of 24 hours;

transferring the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized high COD (chemical oxygen demand) refining wastewater for reactivation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 4% of the volume percentage of the sterilized high COD refining wastewater, the COD of the sterilized high COD refining wastewater is 1500mg/L, the activation condition comprises that the culture temperature is 32 ℃, the pH is 9, the rotation speed of a shaking table is 180rpm, and the activation time is 24 hours;

Transferring the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid to a fermentation tank containing a nutrient solution respectively for continuous fermentation and expansion culture to obtain a bacillus-containing fermentation agent and a campylobacter-containing fermentation agent respectively, wherein the inoculation amount of the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 4% of the volume percentage of the nutrient solution respectively, and the concentration of bacillus in the bacillus-containing fermentation agent is 1 multiplied by 10 ⁸ CFU/ml or more, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ The culture temperature is above CFU/ml, the pH is 8.0, the rotating speed is 200rpm, the pressure of a fermentation tank is 0.04MPa, the ventilation amount is 1.1:1, the fermentation time is 18h, the nutrient solution also comprises 22g/L glucose, 4g/L peptone, 1.5g/L yeast powder, 2.5g/L ammonium nitrate, 4.5g/L sodium chloride, 0.8g/L potassium dihydrogen phosphate, 0.35g/L manganese sulfate, 1.5g/L magnesium sulfate, 0.04g/L ferrous sulfate and 1.5g/L calcium carbonate, and the pH value of the nutrient solution is 7.1;

fifthly, mixing a bacillus-containing starter and a campylobacter-containing starter to obtain a composite microbial agent, and adding the composite microbial agent into a hydrolysis acidification reactor containing refined high-concentration wastewater and filled with fiber fillers for hydrolysis acidification treatment, wherein the volume ratio of the bacillus-containing starter to the campylobacter-containing starter in the composite microbial agent is 1:3, the composite microbial agent accounts for 5 percent of the volume percentage of the refined high-concentration wastewater, the initial COD of the refined high-concentration wastewater is 1500mg/L, the oil content is 100mg/L, the temperature is 42 ℃ during hydrolysis and acidification treatment, the time is 60 hours, the fiber filler is made of polypropylene, and the filling rate of the fiber filler in the hydrolysis and acidification reactor is 40 percent.

In the hydrolysis acidification reactors of the embodiments 4 to 6 of the present invention, fibrous filler is suspended, the fibrous filler is made of polypropylene, and the filling rate of the filler in the hydrolysis acidification reactor is 40%. Bacillus and campylobacter in the composite microbial agent are attached to the fiber filler, and the hydraulic retention time of the device is not less than 55 hours.

The fiber filler adopted by the invention has higher microorganism capacity which is 3 to 5 times that of common fillers, can provide an optimal environment for microorganism growth and propagation, and can play a good role in filtering and buffering impact by continuously filling the fillers in multiple sections.

Example 7: the application of the composite microbial agent in refining high-concentration wastewater is carried out according to the following method:

the first step, respectively inoculating bacillus and campylobacter into LB liquid culture medium, and after shake cultivation, respectively obtaining bacillus fermentation seed liquid and campylobacter fermentation seed liquid, 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 ℃ above CFU/mL, and the rotation speed of the shaking table is 200rpm;

the second step, inoculating the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized low COD refining wastewater for activation, wherein the inoculum size of the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 4 percent of the volume percentage of the sterilized low COD refining wastewater, the COD of the sterilized low COD refining wastewater is 800mg/L, the activation conditions comprise the culture temperature of 35 ℃, the pH value of 8, the rotation speed of a shaking table of 200rpm and the activation time of 24 hours;

Transferring the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized high COD (chemical oxygen demand) refining wastewater for reactivation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 4% of the volume percentage of the sterilized high COD refining wastewater, the COD of the sterilized high COD refining wastewater is 2000mg/L, the activation condition comprises that the culture temperature is 35 ℃, the pH is 8, the rotation speed of a shaking table is 200rpm, and the activation time is 24 hours;

transferring the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid to a fermentation tank containing a nutrient solution respectively for continuous fermentation and expansion culture to obtain a bacillus-containing fermentation agent and a campylobacter-containing fermentation agent respectively, wherein the inoculation amount of the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 4% of the volume percentage of the nutrient solution respectively, and the concentration of bacillus in the bacillus-containing fermentation agent is 1 multiplied by 10 ⁸ CFU/ml or more, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ The culture temperature is above 35 ℃ and the pH value is 8.0, the rotating speed is 200rpm, the pressure of a fermentation tank is 0.06MPa, the ventilation amount is 1.1:1, the fermentation time is 20h, the nutrient solution also comprises 22g/L glucose, 4g/L peptone, 1.5g/L yeast powder, 2.5g/L ammonium nitrate, 4.5g/L sodium chloride, 0.8g/L monopotassium phosphate, 0.35g/L manganese sulfate, 1.5g/L magnesium sulfate, 0.04g/L ferrous sulfate and 1.5g/L calcium carbonate, and the pH value of the nutrient solution is 7.1;

Fifthly, mixing a bacillus-containing starter and a campylobacter-containing starter to obtain a composite microbial agent, and adding the composite microbial agent into a hydrolysis acidification reactor containing refined high-concentration wastewater and filled with fiber fillers for hydrolysis acidification treatment, wherein the volume ratio of the bacillus-containing starter to the campylobacter-containing starter in the composite microbial agent is 1:2, the composite microbial agent accounts for 6 percent of the volume percentage of the refined high-concentration wastewater, the initial COD of the refined high-concentration wastewater is 2000mg/L, the oil content is 64mg/L, the temperature is 45 ℃ during hydrolysis and acidification treatment, the time is 60 hours, the fiber filler is made of polypropylene, and the filling rate of the fiber filler in a hydrolysis and acidification reactor is 40 percent.

In the embodiment 7 of the invention, the initial COD of the refined high-concentration wastewater is 2000mg/L, BOD is 24mg/L, and B/C is 0.012. The fiber filler is suspended in the hydrolysis acidification reactor, the filling amount is 40%, the hydrolysis acidification reactor adopts water inlet with lower inlet and upper outlet, 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 60h.

Example 8: the application of the composite microbial agent in refining high-concentration wastewater is carried out according to the following method:

The first step, respectively inoculating bacillus and campylobacter into LB liquid culture medium, and after shake cultivation, respectively obtaining bacillus fermentation seed liquid and campylobacter fermentation seed liquid, 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 ℃ above CFU/mL, and the rotation speed of the shaking table is 200rpm;

the second step, inoculating the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized low COD refining wastewater for activation, wherein the inoculum size of the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 4 percent of the volume percentage of the sterilized low COD refining wastewater, the COD of the sterilized low COD refining wastewater is 800mg/L, the activation conditions comprise the culture temperature of 35 ℃, the pH value of 8, the rotation speed of a shaking table of 200rpm and the activation time of 24 hours;

transferring the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized high-COD refined wastewater for reactivation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 4% of the volume percentage of the sterilized high-COD refined wastewater, the COD of the sterilized high-COD refined wastewater is 2500mg/L, the activation condition comprises that the culture temperature is 35 ℃, the pH is 8, the rotation speed of a shaking table is 200rpm, and the activation time is 24 hours;

Fourth, transferring the re-activated bacillus fermentation seed liquid and campylobacter fermentation seed liquid into a fermentation tank containing nutrient solution respectively for continuous fermentation and expansion culture to obtain respectivelyThe bacillus-containing starter and the campylobacter-containing starter, wherein the seed liquid of the bacillus fermentation and the seed liquid of the campylobacter fermentation after reactivation are respectively 4 percent of the volume percentage of the nutrient solution, and the concentration of the bacillus in the starter containing the bacillus is 1 multiplied by 10 ⁸ CFU/ml or more, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ The culture temperature is above 35 ℃ and the pH value is 8.0, the rotating speed is 200rpm, the pressure of a fermentation tank is 0.06MPa, the ventilation amount is 1.1:1, the fermentation time is 20h, the nutrient solution also comprises 22g/L glucose, 4g/L peptone, 1.5g/L yeast powder, 2.5g/L ammonium nitrate, 4.5g/L sodium chloride, 0.8g/L monopotassium phosphate, 0.35g/L manganese sulfate, 1.5g/L magnesium sulfate, 0.04g/L ferrous sulfate and 1.5g/L calcium carbonate, and the pH value of the nutrient solution is 7.1;

fifthly, mixing a bacillus-containing starter and a campylobacter-containing starter to obtain a composite microbial agent, and adding the composite microbial agent into a hydrolysis acidification reactor containing refined high-concentration wastewater and filled with fiber fillers for hydrolysis acidification treatment, wherein the volume ratio of the bacillus-containing starter to the campylobacter-containing starter in the composite microbial agent is 1:2, the composite microbial agent accounts for 6 percent of the volume percentage of the refined high-concentration wastewater, the initial COD of the refined high-concentration wastewater is 2500mg/L, the oil content is 132mg/L, the temperature is 45 ℃ during hydrolysis and acidification treatment, the time is 60 hours, the fiber filler is polypropylene, and the filling rate of the fiber filler in the hydrolysis and acidification reactor is 40 percent.

In the embodiment 8 of the invention, the initial COD of the refined high-concentration wastewater is 2500mg/L, the BOD is 32mg/L, and the B/C is 0.013. The fiber filler is suspended in the hydrolysis acidification reactor, the filling amount is 40%, the hydrolysis acidification reactor adopts water inlet with lower inlet and upper outlet, 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 65h.

By adopting the treatment methods of the embodiments 4 to 8, the composite microbial agent is added into the hydrolysis acidification reactor containing the refined high-concentration wastewater and filled with the fiber filler, the refined high-concentration wastewater with COD of 800mg/L to 2500mg/L and oil content not higher than 132mg/L can be treated, and the oil content of the effluent water quality after the hydrolysis acidification treatment of the composite microbial agent is not higher than 50mg/L, so that the COD removal rate is over 60 percent. The following are screening and identification of strains and evaluation of reduction treatment effect of the refined high-concentration wastewater by using Campylobacter bacteria in examples 7 and 8 of the present invention.

Test 1: screening and identification of strains

The test method comprises the following steps:

step 1: sampling from a plurality of high-concentration sewage devices of petrochemical enterprises, wherein the high-concentration sewage comprises the sewage at the top of a distillation tower of a distillation device under normal pressure and reduced pressure, the sewage generated by a sulfur-containing sewage stripping device and the electric desalting sewage of a crude oil coking device. 50mL of high-concentration sewage is put into a 250mL triangular flask, the triangular flask is placed into a shaking table for culturing for 24 hours under the conditions that the temperature is 30 ℃ and the rotating speed is 150rpm, and then the high-concentration sewage is gradually diluted in a gradient way and is coated on an LB plate. Culturing the coated LB plate for 48 hours at the temperature of 30 ℃ and selecting single colonies with different forms;

Step 2: inoculating the obtained single colonies with different forms into LB culture medium respectively for enrichment, and culturing for 24 hours to obtain bacterial solutions of different strains;

step 3: high-concentration sewage after being mixed by industrial water devices of refining enterprises is subjected to high-pressure steam sterilization, wherein the sterilization temperature is 120 ℃, the sterilization pressure is 0.1MPa, and the sterilization time is 30min;

step 4: and (3) cooling the sterilized refined high-concentration wastewater to room temperature, respectively inoculating 5mL of bacterial solutions of different strains into 100mL of sterilized refined high-concentration wastewater for hydrolysis and acidification treatment, placing the inoculated bacterial solutions into a shaking table with the temperature of 45 ℃ and the rotating speed of 120rpm for evaluation, screening the optimal bacterial strains according to COD values of the sterilized refined high-concentration wastewater after 120h of hydrolysis and acidification treatment of the bacterial solutions of the different bacterial strains, and carrying out morphological identification on the optimal bacterial strains.

Test results: as shown in Table 1, table 1 shows COD values in the sterilized high-concentration wastewater treated by different strains, and the strains No. 5 (bacillus) and No. 7 (Campylobacter) have the best treatment effect on the COD values in the sterilized high-concentration wastewater, and the strains No. 5 and No. 7 have higher degradation capacity and are the best dominant degradation bacteria.

Morphological identification of strain No. 5: as shown in fig. 1, the strain characteristics: the colony forms as milky opaque dots. Through morphological, physiological and biochemical properties and molecular biological identification, the No. 5 strain is Bacillus sp, which is preserved in China general microbiological culture Collection center (CGMCC) No.21651 in 2021, 1 month and 18 days, and the preservation address is building No. 3 of North Chenxi Lu No. 1 in the Chaoyang area of Beijing city.

Morphological identification of strain No. 7: as shown in FIG. 2, the strain is characterized by moist colony-form protrusions, irregular shapes, rough surfaces and milky white surfaces. Through morphological, physiological and biochemical properties and molecular biological identification, the No. 7 strain is Campylobacter sp, which is preserved in China general microbiological culture Collection center (CGMCC) for 1 month 18 in 2021, and the preservation number is CGMCC No.21652, and the preservation address is building No. 3 of North Xielu No. 1, a region of Chaoyang in Beijing city.

The nucleic acid sequences of the strain No. 5 and the strain No. 7 are shown in the sequence table in detail.

The strain 5 and the strain 7 are conventional facultative anaerobes after puncture culture.

Test 2: and (3) examining the COD removal effect of bacillus and campylobacter in the composite microbial agent on the mixed sewage under different proportions.

The test method comprises the following steps: the optimal compounding proportion and the strain adding amount are determined by adopting an orthogonal test, the total adding amount of two dominant degradation bacteria (namely, the adding amount of a composite microbial agent) is taken as a factor A, the adding proportion of two dominant degradation bacteria bacillus and campylobacter is taken as a factor B, and the design scheme of the orthogonal test is shown in a table 2. Putting the enriched bacillus (strain No. 5) and campylobacter (strain No. 7) into sterilized refined high-concentration wastewater according to an orthogonal design scheme for carrying out shaking table evaluation, and respectively examining COD removal rates in 48h, 72h and 5d of hydrolysis acidification treatment.

Test results: table 2 is an orthogonal test design table, table 3 is the COD value and COD removal rate in water quality after hydrolysis acidification treatment of sterilized high-concentration wastewater by different test schemes, FIG. 3 is a bar graph of Table 3, the results are shown in Table 3 and FIG. 3, scheme 5 has the fastest COD removal rate and better degradation rate for sterilized high-concentration wastewater, scheme 5 can reach 63.86% COD removal rate after hydrolysis acidification treatment for 48 hours, and scheme 5 reaches 77.72% COD removal rate of water quality after hydrolysis acidification treatment for 5 days.

The optimal volume ratio of two dominant degrading bacteria bacillus (strain 5) to campylobacter (strain 7) is shown to be 1:2, the total adding amount of the two dominant degradation bacteria is 6 percent (namely, the optimal adding amount of the composite microbial agent is 6 percent).

Test 3: evaluating the effect of the composite microbial agent on hydrolysis and acidification treatment of the refined high-concentration wastewater

The test method comprises the following steps: according to the treatment methods of examples 7 and 8 of the present invention, the composite microbial inoculum is subjected to hydrolysis acidification treatment, the water quality after the hydrolysis acidification treatment is detected, the water quality indexes including chemical oxygen demand COD, ammonia nitrogen, sulfide, total phosphorus, oil content and external drainage sludge concentration are measured, the chemical oxygen demand COD is measured by referring to a method specified in a potassium dichromate method for measuring chemical oxygen demand of water quality, ammonia nitrogen is measured by referring to a method specified in a Nashi reagent spectrophotometry for measuring ammonia nitrogen of water quality, the sulfide is measured by referring to a method specified in a methylene blue spectrophotometry for measuring sulfide of water quality, the total phosphorus is measured by referring to a method specified in an ammonium molybdate spectrophotometry for measuring total phosphorus of water quality, and the oil content is measured by referring to a method specified in an infrared spectrophotometry for measuring water quality oil and animal oil, which is measured by referring to a method specified in a method for measuring water quality oil and animal oil, which is called GB/T11893-2015.

Test results: as shown in Table 4, the index of the water quality of the high-concentration wastewater after and before the hydrolysis and acidification treatment in example 7 of the present invention is shown in Table 4, and the index of the high-concentration wastewater after the hydrolysis and acidification treatment in example 7 of the present invention is shown in Table 4: COD is 2000mg/L, oil content is 64mg/L, sulfide content is 25mg/L, ammonia nitrogen content is 16mg/L, and the indexes of the water quality of the refined high-concentration wastewater after hydrolysis and acidification treatment by the composite microbial agent containing bacillus and campylobacter are as follows: COD value is 1000mg/L, oil content is 21mg/L, sulfide content is 14mg/L, and ammonia nitrogen content is 6mg/L.

As shown in Table 5, the index of the water quality of the high-concentration wastewater after and before the hydrolysis and acidification treatment in example 8 of the present invention is shown in Table 5, and the index of the high-concentration wastewater after the hydrolysis and acidification treatment in example 8 of the present invention is as follows: COD is 2500mg/L, oil content is 132mg/L, sulfide content is 31mg/L, ammonia nitrogen content is 21g/L, and indexes of water quality of refined high-concentration wastewater after hydrolysis and acidification treatment of composite microbial agents containing bacillus and campylobacter are as follows: COD value is 1100mg/L, oil content is 43mg/L, sulfide content is 17mg/L, and ammonia nitrogen content is 10mg/L.

The composite microbial agent can treat refined high-concentration wastewater with COD of 800mg/L to 2500mg/L and oil content not higher than 132mg/L, and after hydrolysis and acidification treatment of the composite microbial agent, the oil content in the discharged wastewater is not higher than 50mg/L, so that the COD removal rate is over 60 percent.

Test 4: investigating the impact load resistance of hydrolysis acidification reactor after preliminary adding compound microbial agent

The test method comprises the following steps: and (3) performing bacterial liquid feeding biological strengthening on the hydrolysis acidification reactor according to the compounding proportion and the total feeding amount of the two dominant degradation bacteria bacillus and campylobacter determined in the test 2. The bacterial strain addition is carried out in stages, and when the COD concentration of the inflow water of the high-concentration wastewater is raised each time, the compound bacterial liquid is supplemented into the reactor in time, so that the influence of load impact on the stability of the whole microbial community in the reactor is reduced as much as possible. The method comprises the following steps: taking the first feeding when the COD of the inflow water is 1800mg/L as an example, taking the total hydraulic retention time of the reactor as a period of 55h, proportioning the inflow water with the same volume of the reactor, and carrying out inflow water updating once every 18h for three times. Meanwhile, the strain No. 5 and the strain No. 7 are respectively enriched to the strain concentration of 1 multiplied by 10 by using an inorganic salt culture medium ⁸ CFU/ml, bacillus (strain No. 5) according to the optimal addition ratio: campylobacter (strain No. 7) =1: 2, and the total bacterial liquid amount is 6% of the water inflow each timeIn water, bacterial liquid enters the hydrolysis acidification reactor along with the water inlet, and COD of the water inlet and outlet of the hydrolysis acidification reactor is continuously recorded, and COD removal rate of the water quality of the water outlet of the hydrolysis acidification reactor in a week after the bacterial liquid is added are inspected.

Test results: as shown in FIG. 4, it is clear from FIG. 4 that the COD treatment efficiency of the hydrolysis acidification reactor is rapidly improved within one week of adding the compound bacterial liquid, the COD removal rate is stabilized to be more than 50%, and the organic load of the hydrolysis acidification reactor is 0.31 kg/(m) before bacterial addition ³ D) lifting to 0.56 kg/(m) ³ D) meeting the need for complete operation of the hydrolytic acidification reactor.

Test 5: investigating the impact load resistance of the hydrolysis acidification reactor after biological strengthening

The test method comprises the following steps: after the hydrolytic acidification reactor is completely started and stably operated in the test 4, the addition of the compound bacterial liquid into the hydrolytic acidification reactor is suspended, the COD concentration of inflow water is gradually increased to be more than 2400mg/L, the COD of inflow water and outflow water of the hydrolytic acidification reactor is continuously recorded, and the load impact resistance of the hydrolytic acidification reactor after biological reinforcement is examined.

Test results: as shown in FIG. 5, it can be seen from FIG. 5 that after increasing the COD concentration of the influent water to above 2400mg/L, the hydrolysis acidification reactor still has better load impact resistance, and the organic load of the hydrolysis acidification reactor is increased to 0.63 kg/(m) ³ D) left and right. The method shows that the added compound bacterial liquid obtains better stable growth in the hydrolysis acidification reactor, gradually inhabits on the fiber filler in the hydrolysis acidification reactor, gradually stabilizes the concentration of the bacterial strain, has higher overall activity, and stably operates the hydrolysis acidification reactor.

Test 6: the hydraulic retention time of the hydrolysis acidification reactor after biological reinforcement in the treatment of the refined high-concentration wastewater is examined

The test method comprises the following steps: in order to judge the stability of the microbial community in the reactor, further shorten the hydraulic retention time of the reactor, increase the overall treatment efficiency of the reactor, reduce the hydraulic retention time of the reactor from 56 hours to 48 hours, and continuously record the COD of the inlet water and the outlet water of the reactor.

Test knotThe method comprises the following steps: as can be seen from FIG. 6, the overall organic load of the hydrolysis acidification reactor was reduced to 0.51 kg/(m) as can be seen from FIG. 6 ³ About d), the treatment effect was slightly reduced, but the organic load of the hydrolytic acidification reactor was still higher than that of the case where no complex bacterial liquid was added before by 0.31 kg/(m) ³ D), showing that after the reactor is subjected to biological strengthening treatment by adding the two dominant degradation bacteria, the degradation effect of the hydrolysis acidification reactor is obviously improved, and proving that the degradation effect of the reactor is effectively improved by adding the two dominant degradation bacteria.

In summary, the composite microbial agent is added into the hydrolysis acidification reactor which contains the refined high-concentration wastewater and is filled with the fiber filler, the refined high-concentration wastewater with COD of 800mg/L to 2500mg/L and oil content of not more than 132mg/L can be treated, the hydraulic retention time is more than 55h, the oil content of the effluent water quality after the hydrolysis acidification treatment of the composite microbial agent is not more than 50mg/L, the COD removal rate is more than 60%, the cost of subsequent treatment of environmental pollution is greatly reduced, and meanwhile, the organic load of the hydrolysis acidification reactor through adding two dominant degradation bacteria is 0.31 kg/(m) ³ D) lifting to 0.63 kg/(m) ³ D) and the bio-enhanced hydrolytic acidification reactor has good capability of resisting impact load.

The technical characteristics form the embodiment of the invention, have stronger adaptability and implementation effect, and can increase or decrease unnecessary technical characteristics according to actual needs so as to meet the requirements of different situations.

TABLE 1

TABLE 2

TABLE 3 Table 3

TABLE 4 Table 4

Properties of (C)	Inflow of water	Effluent water
			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

TABLE 5

Properties of (C)	Inflow of water	Effluent water
			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> Petroleum and Clamah petrochemical Co., ltd., china Petroleum and Natural gas Co., ltd

<120> bacillus and composite microbial agent and application thereof 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 (6)

1. The Bacillus sp is characterized in that the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 21651 in the year 1 and 18 of 2021.

2. A composite microbial agent comprising the bacillus of claim 1.

3. The composite microbial agent according to claim 2, which is characterized by comprising the following components in volume ratio of 1:1 to 5 and a Bacillus containing starter comprising Bacillus and a nutrient solution, wherein the concentration of Bacillus in the Bacillus containing starter is 1×10 ⁸ The fermentation agent containing Campylobacter comprises Campylobacter and nutrient solution, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ CFU/mL or more.

4. The compound microbial agent according to claim 3, wherein the composition of the nutrient solution comprises 20g/L to 24g/L of glucose, 2g/L to 5g/L of peptone, 1g/L to 2g/L of yeast powder, 2g/L to 3g/L of ammonium nitrate, 4g/L to 5g/L of sodium chloride, 0.5g/L to 1g/L of potassium dihydrogen phosphate, 0.3g/L to 0.4g/L of manganese sulfate, 1g/L to 2g/L of magnesium sulfate, 0.02g/L to 0.05g/L of ferrous sulfate and 1g/L to 2g/L of calcium carbonate, and the pH value of the nutrient solution is 7.0 to 7.2.

5. Use of the composite microbial agent according to claim 2 or 3 or 4 for refining high-concentration wastewater.

6. The application of the composite microbial agent in refining high-concentration wastewater according to claim 5, which is characterized by comprising the following steps:

the first step, respectively inoculating bacillus and campylobacter into LB liquid culture medium, and after shake cultivation, respectively obtaining bacillus fermentation seed liquid and campylobacter fermentation seed liquid, 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 CFU/mL, culture temperature of 30-35deg.C, shaking table rotation speed of 150rpm to 200rpm;

the second step, inoculating the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into the sterilized low COD refining wastewater for activation, wherein the inoculum size of the bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3 to 6 percent of the volume percentage of the sterilized low COD refining wastewater, the COD of the sterilized low COD refining wastewater is 500 to 800mg/L, the activation conditions comprise the culture temperature of 30 to 35 ℃, the pH of 8 to 10, the rotation speed of a shaking table of 150 to 200rpm and the activation time of 24 hours;

transferring the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid into sterilized high COD (chemical oxygen demand) refining wastewater for reactivation, wherein the inoculation amount of the activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3 to 6 percent of the volume percentage of the sterilized high COD refining wastewater, the COD of the sterilized high COD refining wastewater is 800 to 2500mg/L, the activation condition comprises that the culture temperature is 30 to 35 ℃, the pH is 8 to 10, the rotation speed of a shaking table is 150 to 200rpm, and the activation time is 24 hours;

Transferring the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid to a fermentation tank containing a nutrient solution respectively for continuous fermentation and expansion culture to obtain a bacillus-containing fermentation agent and a campylobacter-containing fermentation agent respectively, wherein the inoculation amount of the re-activated bacillus fermentation seed liquid and the campylobacter fermentation seed liquid is 3-6% of the volume percentage of the nutrient solution respectively, and the concentration of bacillus in the bacillus-containing fermentation agent is 1 multiplied by 10 ⁸ CFU/ml or more, and the concentration of Campylobacter in the fermentation agent containing Campylobacter is 1×10 ⁸ The culture temperature is above CFU/ml, the temperature is 28-38 ℃, the pH is 7.5-9.5, the rotating speed is 200rpm, the pressure of a fermentation tank is 0.03-0.06 MPa, the ventilation rate is 1.0-1.3:1, and the fermentation time is 16-20 h;

fifthly, mixing a bacillus-containing starter and a campylobacter-containing starter to obtain a composite microbial agent, and adding the composite microbial agent into a hydrolysis acidification reactor containing refined high-concentration wastewater and filled with fiber fillers for hydrolysis acidification treatment, wherein the volume ratio of the bacillus-containing starter to the campylobacter-containing starter in the composite microbial agent is 1:1 to 5, the composite microbial agent accounts for 4 to 6 percent of the volume percentage of the refined high-concentration wastewater, the initial COD of the refined high-concentration wastewater is 800 to 2500mg/L, the oil content is not higher than 135mg/L, the temperature is 40 to 45 ℃ during hydrolytic acidification treatment, the time is not less than 55 hours, the fiber filler is made of polypropylene fiber, and the filling rate of the fiber filler in a hydrolytic acidification reactor is 20 to 70 percent.

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