Method for deeply degrading benzene series organic pollutants through cooperation of multiple strains

Abstract

The invention relates to the technical field of organic pollutant treatment, and discloses a method for deeply degrading benzene series organic pollutants through cooperation of multiple strains. Comprising the following steps: strain activation: respectively inoculating a pseudomonas strain A, a bacillus strain B, an acinetobacter strain C, a shewanella strain D and a pseudomonas strain E into an LB solid culture medium, culturing at 28 DEG C for 18 hours, activating, then transferring into an LB liquid culture medium, and carrying out shake culture to a stable phase to obtain each strain suspension; bacterial group compounding: mixing bacterial strains with the bacterial strain suspension according to a volume ratio of A: B: C: D: E of 3: 2: 2: 1: 2 to form a compound bacterial solution; and wastewater treatment: adding 0.1% (v / v) of the composite bacteria liquid into the wastewater containing the benzene series collected at the tail end of the aerobic tank, and carrying out oscillation aeration culture for 5-7 days under the conditions of 28 DEG C and 220rpm. The method for deeply degrading the benzene series organic pollutants through synergism of the multiple strains aims at solving the problems that in the prior art, benzene series treatment is low in efficiency, unstable, high in cost and prone to secondary pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic pollutant treatment, in particular to a method for deep degradation of benzene series organic pollutants by multiple strains in cooperation. BACKGROUND

[0002] Benzene series organic pollutants are widely derived from chemical synthesis, pharmaceutical production and other industries, which contain benzene, toluene, xylene, phenol, nitrobenzene and other substances. These pollutants have the characteristics of stable molecular structure, strong toxicity and difficult degradation. They can enter the ecological system through water and soil circulation, which not only inhibits the growth and reproduction of aquatic organisms and destroys the balance of soil microecology, but also enters the human body through respiratory tract and skin contact. Long-term exposure can cause damage to the nervous system, blood diseases and even cancer, posing a serious threat to human health and the ecological environment. In order to control pollution, physical, chemical and biological methods have been developed in the prior art: physical method separates pollutants by activated carbon adsorption, extraction and other methods, which is suitable for preliminary enrichment of low-concentration benzene series; chemical method such as Fenton oxidation and other advanced oxidation technologies degrades pollutants by strong oxidizing agents, which is suitable for rapid treatment of high-toxicity pollutants; biological method uses the metabolic action of microorganisms to mineralize benzene series into CO2 and water, which has the advantages of low cost, environmental friendliness and no secondary pollution risk, and becomes the mainstream research direction of benzene series pollution control, especially in large-scale wastewater treatment.

[0003] However, in the physical method, the adsorbent such as activated carbon is easy to saturate, and the regeneration process is complex and costly, which requires frequent replacement and leads to low treatment efficiency; in the extraction method, the recovery and harmless treatment of the extractant are difficult, which can easily cause secondary pollution; the chemical method can efficiently degrade benzene series, but a large amount of waste such as iron sludge is generated during the reaction process, and a large amount of chemical reagents and energy are consumed, which has high economic cost and environmental cost; in the biological method, conventional technology mainly relies on single strain or simple mixed bacteria, which has limited specific degradation ability of microorganisms to benzene series and poor adaptability to complex wastewater matrix, resulting in low degradation efficiency. For example, for pharmaceutical wastewater and chemical wastewater containing high-concentration benzene series, conventional microorganisms are difficult to break through the stable structure of benzene ring, and the removal rate of refractory COD is usually less than 30%, which cannot meet the discharge standard requirements; at the same time, single strain cannot metabolize complex intermediates in cooperation, which easily leads to accumulation of toxic intermediates, further limiting the stability of treatment effect. These defects make it difficult for the existing technology to achieve deep, efficient and low-cost treatment of benzene series pollutants, and it is urgent to develop high-efficiency bacteria groups with synergistic effect and optimize the process to break through the above bottlenecks. SUMMARY

[0004] The application aims at solving the problems of low efficiency, instability, high cost and secondary pollution in the prior art for treating benzene series, and provides a method for synergistically deep degradation of benzene series organic pollutants by multiple strains.

[0005] The technical scheme for solving the above technical problems is as follows:

[0006] The application provides a method for synergistically deep degradation of benzene series organic pollutants by multiple strains, comprising the following steps:

[0007] S10, strain activation: Pseudomonas strain A, Bacillus strain B, Acinetobacter strain C, Shewanella strain D and Pseudomonas strain E are inoculated into LB solid culture medium respectively, and are cultured at 28 DEG C for 18 hours for activation, and then are transferred into LB liquid culture medium for oscillation culture until the stable phase, so as to obtain a suspension of each strain;

[0008] S20, strain group compounding: the strains are mixed in a volume ratio of A:B:C:D:E = 3:2:2:1:2, and the mixed bacterial liquid is formed;

[0009] S30, wastewater treatment: 1 ‰ (v / v) of the mixed bacterial liquid is added into benzene series-containing wastewater collected at the end of an aerobic tank, and oscillation aeration culture is carried out at 28 DEG C and 220 rpm for 5-7 days;

[0010] S40, degradation monitoring: sampling is carried out every 24 hours, the COD concentration is determined by potassium dichromate method, and the COD degradation rate is calculated until the degradation effect is stable.

[0011] On the basis of the above technical scheme, the application can also be improved as follows.

[0012] Further, the strain A in S20 is a gram-negative bacterium, and presents a milky white round colony on the LB culture medium; the strain B is a gram-positive bacterium, and presents a light yellow rough colony on the LB culture medium; the strain C is a gram-negative bacterium, and presents a transparent wrinkled colony on the LB culture medium; the strain D is a gram-negative bacterium, and presents a light pink irregular colony on the LB culture medium; the strain E is a gram-negative bacterium, and presents a grayish white dry colony on the LB culture medium; and each strain is confirmed by 16S rRNA gene sequence comparison, the similarity of the strain A and the strain E to Pseudomonas is greater than or equal to 99%, the similarity of the strain B to Bacillus is greater than or equal to 98%, the similarity of the strain C to Acinetobacter is greater than or equal to 97%, and the similarity of the strain D to Shewanella is greater than or equal to 98%.

[0013] Further, the compounding ratio of the step S20 is determined by the following flow:

[0014] S201. Construct 15 combinations from five strains of bacteria, the 15 combinations being ABC, ABD, ABE, ACD, ACE, ADE, BCD, BCE, BDE, CDE, ABCD, ABDE, ACDE, BCDE, and ABCDE, and mix them in equal volume ratios.

[0015] S202. Add each combination to simulated wastewater containing benzene series compounds at an inoculum rate of 1‰, with COD≥1000mg / L, and incubate at 28℃ for 5 days;

[0016] S203, Screening for the ABCDE combination with the highest COD degradation rate;

[0017] S204. Based on the combination of ABCDE, test 10 volume ratios, of which 10 volume ratios range from 1:1:1:1:1 to 3:1:1:3:2. Finally, determine that A:B:C:D:E = 3:2:2:1:2.

[0018] Furthermore, the wastewater of S30 includes pharmaceutical wastewater, coal gasification wastewater, coking wastewater, or chemical wastewater, and the wastewater needs to be pretreated by sterilization at 121℃ for 30 minutes to eliminate the original microbial interference; the benzene series compounds include at least three of phenol, aniline, benzene, o-xylene, m-xylene, p-xylene, nitrobenzene, o-p-chloronitrobenzene, and p-chloronitrobenzene, with an initial COD concentration of 220-360 mg / L.

[0019] Furthermore, the aeration and cultivation conditions for S30 are as follows: using microporous aeration heads (pore size ≤ 50 μm) to control the aeration rate at 0.8 m³ / s. 3 / (m 3 • h), initially cultured in a closed system for 24 hours, then stirred at 100 rpm for 5 minutes every 24 hours to promote bacterial contact.

[0020] Furthermore, the activation of the bacterial strain in S10 specifically includes:

[0021] The strain preserved in glycerol tubes at -80℃ was inoculated into LB solid medium containing 5 g / L yeast extract, 10 g / L NaCl, 10 g / L peptone, and pH 7.0-7.2.

[0022] Single colonies were picked and inoculated into LB liquid medium, and cultured with shaking at 220 rpm for 18 h to obtain primary seed culture.

[0023] Transfer to fresh LB liquid medium at a 1% inoculum rate and culture until OD600 = 0.6-0.8.

[0024] Furthermore, in the wastewater treatment of step S30, the composite microbial agent removes pollutants through an "adsorption-desorption-degradation" equilibrium mechanism:

[0025] a1, the strain metabolically secretes EPS degrading enzyme to decompose the surface layer of sludge floc, releasing the wrapped phenolic substances;

[0026] b1, the change of liquid phase phenolic concentration drives the adsorbed benzene series of sludge to convert into free state;

[0027] c1, the free state pollutants are mineralized into CO2 and water by the strain through the beta-ketoadipic acid pathway.

[0028] Compared with the prior art, the technical scheme of the present application has the following beneficial technical effects:

[0029] The present application is formed by compounding five different strains of Pseudomonas, Bacillus, Acinetobacter and Shewanella, and forming a complex bacterial liquid according to a specific ratio, and the metabolic characteristics of different strains are used to achieve synergistic effect - multiple strains can be used to degrade different substances such as benzene, toluene, xylene, phenol and nitrobenzene in benzene series, breaking through the degradation limit of single strain on the stable structure of benzene ring, and significantly improving the adaptability to complex substrates; at the same time, multiple strains can metabolize complex intermediates, avoiding the accumulation of toxic intermediates caused by single strain, and ensuring the stability of the treatment effect; through the addition amount of 1‰ (v / v) of the complex bacterial liquid and the optimization process of 28℃, 220rpm oscillation aeration, the COD degradation rate can reach ≥99% after 5-7 days of cultivation, meeting the discharge standard, and finally realizing the deep, efficient and low-cost treatment of benzene series pollutants. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a schematic diagram of the strain compounding of the present application;

[0031] Figure 2 It is the COD degradation rate of the simulated wastewater of different bacterial groups of the present application;

[0032] Figure 3 It is the COD degradation rate determination graph of the simulated wastewater of the bacterial group ratio optimization of the present application;

[0033] Figure 4 It is the COD degradation rate determination graph of the wastewater of a certain pharmaceutical in Zhejiang in the embodiment of the present application;

[0034] Figure 5 It is the COD degradation rate determination graph of the wastewater of a certain coal chemical industry in Xinjiang in the embodiment of the present application;

[0035] Figure 6 It is the COD degradation rate determination graph of the wastewater of a certain coking in the embodiment of the present application;

[0036] Figure 7 It is the COD degradation rate determination graph of the wastewater of a certain chemical industry in Hubei in the embodiment of the present application. DETAILED DESCRIPTION

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Benzene series organic pollutants are widely present in wastewater discharged from industries such as chemical, pharmaceutical, coal gasification, and coking. They are characterized by high toxicity, stable molecular structure, and difficulty in degradation, posing a serious threat to the ecological environment and human health. Existing physical and chemical methods for treating such wastewater suffer from high costs and the potential for secondary pollution, while conventional biological treatment technologies, due to the limited degradation capacity of microorganisms, are insufficient to meet wastewater discharge standards. This invention provides a method for the synergistic deep degradation of benzene series organic pollutants by multiple strains. Through the combination and optimization of specific strains, efficient and environmentally friendly treatment of benzene-containing wastewater is achieved.

[0039] Specific implementation methods

[0040] (I) Activation of microbial strains

[0041] This step aims to obtain suspensions of each strain with good activity and appropriate concentration, laying the foundation for subsequent bacterial group compounding.

[0042] Culture medium preparation

[0043] LB solid medium: Accurately weigh 5g yeast extract, 10g NaCl, and 10g peptone according to the formula, add 1L of distilled water, stir well, adjust the pH to 7.0-7.2, then add 15-20g agar powder and heat to dissolve. Dispense into Erlenmeyer flasks, autoclave at 121℃ for 30min, and when the temperature drops to 50-60℃, pour into plates in a laminar flow hood, cool and solidify before use.

[0044] LB liquid medium: The formula is the same as LB solid medium, but without agar powder. It is also autoclaved at 121°C for 30 minutes and then cooled to room temperature for use.

[0045] strain source and preservation

[0046] The strains A, B, C, D, and E used in this invention were all screened, isolated, purified, and stored in glycerol tubes at -80℃ by our research center in collaboration with Huazhong Agricultural University. Among them, strain A showed a 99% similarity to *Pseudomonas*; strain B showed a 98% similarity to *Bacillus*; strain C showed a 97% similarity to *Acinetobacter*; strain D showed a 98% similarity to *Shewanella*; and strain E was *Pseudomonas*.

[0047] Strain activation operation

[0048] (1) Take out each strain stored in a glycerol tube at -80℃, and in a clean bench, use a sterile inoculation loop to dip a small amount of bacterial solution into each strain and streak it onto an LB solid medium plate.

[0049] (2) Invert the inoculated plates and place them in a 28°C constant temperature incubator for 24 hours to complete the initial activation of the strains. At this time, the colony morphology of each strain can be observed: strain A is round, milky white, smooth and moist, with neat edges, and Gram staining is negative; strain B is oval, light yellow, rough, with irregular edges, and Gram staining is positive; strain C is round, transparent, wrinkled, with serrated edges, and Gram staining is negative; strain D is irregular in shape, light pink, moist, with blurred edges, and Gram staining is negative; strain E is round, grayish white, dry, with neat edges, and Gram staining is negative.

[0050] (3) Select single colonies of each strain from the activated LB solid medium plate using a sterile inoculation loop and inoculate them into test tubes containing 10 mL of LB liquid medium.

[0051] (4) Place the test tube in a shaker and culture it at 220 rpm and 28 ℃ for 18 h to obtain the primary seed solution.

[0052] (5) Transfer the primary seed culture to an Erlenmeyer flask containing 100 mL of fresh LB liquid medium at an inoculation rate of 1%. Continue to culture under the same shaker conditions until the bacteria grow to the logarithmic growth phase. At this time, the OD600 value of the bacterial suspension reaches 0.6-0.8. Then it is ready for use.

[0053] (II) Bacterial group compounding

[0054] By screening different combinations and ratios of bacterial strains, the optimal compound bacterial solution was determined to achieve efficient degradation of benzene series organic pollutants.

[0055] Strain combination screening

[0056] (1) Combination design: 15 combinations were constructed from 5 strains, namely ABC, ABD, ABE, ACD, ACE, ADE, BCD, BCE, BDE, CDE, ABCD, ABDE, ACDE, BCDE, and ABCDE.

[0057] The prepared 15 bacterial composite solutions were inoculated into Erlenmeyer flasks containing COD-simulated wastewater at an inoculation rate of 1‰ (v / v), with uninoculated simulated wastewater serving as a blank control. The flasks were incubated at 28℃ and 150 rpm with shaking for 5 days, and COD concentrations were measured every 24 hours. COD was determined using the potassium dichromate method, and the COD degradation rate of each bacterial composite solution was calculated. The bacterial combination with the highest COD degradation rate was selected as the optimal bacterial combination. Three biological replicates were performed for each experimental group to ensure data reliability.

[0058] The calculation formula is: COD degradation rate (%) = [(initial COD - final COD) / initial COD] × 100%.

[0059] (2) Preparation of compound bacterial solutions: Each strain was cultured in LB liquid medium at 28°C and 220 rpm until the logarithmic growth phase (OD600 = 0.6-0.8), and the above 15 initial compound bacterial solutions were prepared by mixing them in equal volume ratios. All operations were performed in a clean bench in strict accordance with aseptic techniques.

[0060] (3) Preparation of simulated wastewater: Prepare simulated wastewater containing benzene series compounds. Add benzene, phenol, aniline, o-xylene, m-xylene, p-xylene, nitrobenzene and other benzene series compounds to MM medium without carbon source, sonicate for 30 min, let stand overnight to obtain a homogeneous mixed solution containing a variety of typical benzene series compounds, so that the COD of the simulated wastewater is ≥1000mg / L. The MM medium formula is as follows: KH2PO4 0.5 g / L, K2HPO4 0.5 g / L, (NH4)2SO4 3 g / L, MgSO4·7H2O 0.3 g / L, trace element solution 1 mL / L, pH 7.2-7.4. The trace element solution consists of FeSO4 0.3 g / L, CuSO4·5H2O 0.038 g / L, ZnSO4·7H2O 0.115 g / L, MnSO4 0.169 g / L, H3BO3 0.116 g / L, CoCl2·6H2O 0.024 g / L, Na2Mo·2H2O 0.017 g / L, pH 7.2-7.4.

[0061] (4) Degradation experiment: 15 groups of compound bacterial solutions were added to simulated wastewater containing benzene compounds at an inoculation rate of 1%. Each combination was set up in 3 parallel groups, and simulated wastewater without bacterial inoculation was used as a blank control. The conical flasks were placed in a constant temperature shaker at 28℃ and shaken at 150rpm for 5 days. COD concentration was measured every 24 hours.

[0062] (5) Screening results: COD was determined using the potassium dichromate method (HJ828-2017), and the COD degradation rate of each composite bacterial solution was calculated. The results showed that the ABCDE combination had the best COD degradation effect, with the COD degradation rate increasing from 49.19% (day 1) to 91.84% (day 5) within 5 days, significantly higher than other combinations. Therefore, the ABCDE combination was determined to be the best bacterial combination.

[0063] Strains ratio optimization

[0064] (1) Proportion Design: Based on the ABCDE combination, the total inoculum amount of the strains was kept constant at 1‰ (v / v), and 10 volume ratios were designed, namely 1:1:1:1:1, 1:2:1:1:1, 2:2:1:1:1, 2:2:1:2:2, 3:1:2:2:1, 3:2:1:2:1, 3:1:1:2:2, 3:2:2:1:2, 3:1:1:1:3, and 3:1:1:3:2. The strain composition ratio was optimized based on the selected optimal strain combination. The total inoculum amount of the strains was kept constant at 1‰ (v / v), and different proportions of composite bacterial solutions were constructed by adjusting the volume ratio of each strain. Ten strain composition ratios were designed based on the performance of each strain. The composite bacterial solutions of each ratio were inoculated into simulated benzene series wastewater. COD concentration and degradation rate were measured every 24 hours, and the strain ratio combination with the best degradation effect was screened.

[0065] (2) Degradation experiment: The above 10 different proportions of compound bacterial solutions were added to simulated wastewater containing benzene compounds at an inoculation rate of 1‰, and the experimental conditions were the same as those for strain combination screening. COD concentration and degradation rate were measured every 24 hours. The experimental results are shown in the table below;

[0066] Different bacterial groups on COD degradation rate of simulated wastewater

[0067]

[0068]

[0069] As shown in the table, the combination of 5 bacterial strains resulted in the best degradation effect on benzene series compounds in simulated wastewater, with a COD degradation rate of 90.24% after 3 days. This degradation effect was significantly higher than that of the combinations of 4 and 3 bacterial strains, and the COD degradation rate of the bacterial-added groups was higher than that of the control group. The highest COD degradation rate in the control group was 17.34%. The results indicate that the combination of 5 bacterial strains has a good treatment effect on simulated wastewater containing benzene series compounds, and the control group mentioned above is the "uninoculated" group.

[0070] (3) Optimization results: The results show that when the volume ratio of strain A:B:C:D:E is 3:2:2:1:2, the COD degradation effect of simulated wastewater is the best, with the highest degradation rate reaching 99.34%. Therefore, this ratio is determined to be the optimal compound ratio.

[0071] Ten bacterial strain combinations were designed based on their characteristics, and applied to simulated wastewater at an inoculation rate of 1‰. The optimal combination ratio was selected based on the COD degradation rate. The results showed that the optimal COD degradation effect was achieved when the volume ratio of strains A:B:C:D:E was 3:2:2:1:2, with a maximum degradation rate of 99.34%, indicating that the benzene series compounds in the wastewater were basically completely degraded.

[0072] Validation of the effect of optimized bacterial community ratio on COD degradation in simulated wastewater

[0073]

[0074]

[0075] All compound bacterial suspensions were prepared under strict aseptic conditions, with mixing completed in a laminar flow hood. Three replicates were created for each compound bacterial suspension, with LB broth without the bacterial strain serving as a blank control to verify the reliability of the experimental results. The groups are as follows: Figure 1 As shown.

[0076] (III) Wastewater Treatment

[0077] The prepared compound bacterial solution was applied to actual wastewater treatment, and the deep degradation of benzene series organic pollutants was achieved through specific process conditions.

[0078] Wastewater Sources and Pretreatment

[0079] (1) Wastewater source: The wastewater treated by this invention includes pharmaceutical wastewater, coal gasification wastewater, coking wastewater or chemical wastewater, which is collected from the end of the aerobic tank. At this time, some of the easily degradable organic matter in the wastewater has been initially degraded by the original microorganisms in the aerobic tank, and the residual difficult-to-degrade organic matter affects the COD of the effluent.

[0080] The table below shows the verification effect of recalcitrant COD degradation in wastewater from different sources.

[0081]

[0082] Determination of the degradation rate of recalcitrant COD in wastewater from different sources

[0083]

[0084]

[0085] Combination Figures 4 to 7 As shown, the optimal combination of bacteria, under the best compounding ratio, can further degrade recalcitrant COD components in wastewater from different sources and of different types by more than 50% on the basis of the original degradation level. It can deeply degrade COD components in a variety of wastewaters, proving that the bacteria group has broad applicability to industries containing benzene series wastewater and has good application potential in actual wastewater treatment.

[0086] (2) Pretreatment: The collected wastewater was sterilized at 121℃ for 30 minutes to eliminate interference from existing microorganisms. The benzene series compounds in the wastewater included at least three of phenol, aniline, benzene, o-xylene, m-xylene, p-xylene, nitrobenzene, o-p-chloronitrobenzene, and p-chloronitrobenzene, with an initial COD concentration of 220-360 mg / L.

[0087] Experimental setup and conditions

[0088] (1) Experimental setup: A conical flask was used as the reaction vessel. For pilot-scale testing, a simmering aeration experimental setup was constructed, including an air compressor and a gas flow meter (range 0-2m). 3 It is equipped with a per-h aeration head (pore size ≤ 50 μm), a pH meter (accuracy ± 0.01), and a dissolved oxygen meter (accuracy ± 0.1 mg / L).

[0089] (2) Inoculation and Cultivation: Add 1% (v / v) of compound bacterial solution (mixed at a volume ratio of A:B:C:D:E = 3:2:2:1:2) to the pretreated benzene-containing wastewater collected at the end of the aerobic tank. In the laboratory setting, place the conical flasks at 28℃ and 220rpm for shaking cultivation for 5-7 days. In the pilot-scale setting, divide the wastewater into equal volumes into ton containers, filling each container with 180L, leaving 20L space for aeration, mixing, and sampling. Add the compound bacterial agent at a 1‰ inoculation rate, and stir thoroughly for 5 minutes using a stirring rod to ensure uniform mixing of the agent and wastewater. Place the aeration heads at the four corners and center of the bottom of the ton container to ensure uniform aeration. Connect the air compressor and gas flow meter, and adjust the aeration rate to 0.8m³. 3 / (m 3 •h). For the first 24 hours of aeration, keep the container completely sealed to avoid external interference; thereafter, open the container every 24 hours and use a stirrer to stir rapidly at 100 rpm for 5 minutes to promote full contact between the bacterial solution and the wastewater. After stirring, immediately seal the container and continue aeration. The total aeration time is set to 7 days.

[0090] Degradation mechanism

[0091] The compound microbial agent removes pollutants through an "adsorption-desorption-degradation" equilibrium mechanism, the specific process of which is as follows:

[0092] (1) The strain metabolizes and secretes EPS degrading enzymes to decompose the surface layer of sludge flocs and release encapsulated phenolic substances.

[0093] (2) Changes in the concentration of phenols in the liquid phase drive the conversion of benzene compounds adsorbed by sludge into a free state.

[0094] (3) Free pollutants are mineralized into CO2 and water by the strain via the β-ketoadipic acid pathway. Under aerobic conditions, monooxygenases and dioxygenases in the microorganism mediate the hydroxylation of the benzene ring, converting it into intermediates such as catechol or protocatechuic acid. Subsequently, ring-cleaving dioxygenases mediate the cleavage of the benzene ring intermediates. Catechol 1,2-dioxygenase and protocatechuic acid 3,4-dioxygenase work together with molecular oxygen to cleave the benzene rings of catechol and protocatechuic acid, respectively, generating cis, cis-mucocarboxylic acid and β-carboxyl derivatives. Finally, these products are further metabolized into intermediates of the tricarboxylic acid (TCA) cycle via the β-ketoadipic acid pathway and are further bio-utilized and mineralized into CO2 and water.

[0095] (iv) Degradation monitoring

[0096] The effectiveness of wastewater treatment is evaluated by monitoring indicators such as COD concentration and phenol concentration.

[0097] COD concentration determination

[0098] Samples were taken every 24 hours and centrifuged. The supernatant was collected, and the COD concentration was determined using the potassium dichromate method (HJ828-2017). The COD degradation rate was calculated until the degradation effect stabilized. Microbial growth was characterized by measuring the OD600 value of the bacterial suspension and by gradient dilution plating. All experimental data were averaged from parallel experiments. Data processing and plotting were performed using Origin software, and significance analysis was conducted using SPSS software (p<0.05 indicates significant difference). For actual wastewater, the degradation effect of this method was as follows: COD of pharmaceutical wastewater decreased from 360 mg / L to 65 mg / L (degradation rate 82%), COD of coal gasification wastewater decreased from 280 mg / L to 96 mg / L (degradation rate 66%), COD of coking wastewater decreased from 220 mg / L to 130 mg / L (degradation rate 41%), and COD of chemical wastewater decreased from 320 mg / L to 101 mg / L (degradation rate 68%).

[0099] Phenolic substance concentration monitoring

[0100] The concentration of phenolic substances was monitored simultaneously during the degradation process to verify the "detoxification" effect of the bacterial agent. After 164 hours of treatment, the phenol concentration in the compound bacterial agent group decreased from 23.9 mg / L to 11.7 mg / L, while the concentration in the commercially available bacterial agent group and the uninoculated group did not decrease significantly.

[0101] Assessment of the ability to degrade recalcitrant COD

[0102] The compound bacterial solution's ability to deeply degrade recalcitrant COD is demonstrated by the fact that, based on the existing degradation of wastewater at the end of the aerobic tank, it further degrades 40-80% of the residual COD, and the degradation rate is significantly higher than that of commercially available bacterial agents (the COD of the control bacterial agent B / C group was ≥235 mg / L after 164 h, while the compound bacterial agent group was reduced to 92 mg / L).

[0103] Phenol concentration measurement results

[0104] The table below shows the biodegradation of phenolic substances in aerobic wastewater.

[0105]

[0106] Note: A is the compound bacterial agent, B is the COD bacterial agent ① from an environmental protection company in Qingdao, C is the COD degradation bacterial agent ② from our company, and D is the control group (uninoculated group). The data in the table are in mg / L.

[0107] Data observation over the first 116 hours showed that the bacterial-treated groups (groups A, B, and C) did not exhibit an advantage in phenol degradation compared to the control group (group D), and the phenol concentration in the bacterial-treated groups even increased to some extent. However, group A initially showed an increase, then began to decrease after 92 hours, and continued to decline thereafter. After 140 hours of reaction, the phenol concentration in group A decreased to 17.5 mg / L, and by 164 hours, it had reached a minimum of 11.7 mg / L. After 164 hours of reaction, groups B and C did not show an advantage in phenol degradation compared to the untreated group (group D).

[0108] After the microbial agent is added to the reaction system, during the process of the bacterial strains combining with the activated sludge, some of the phenolic substances originally adsorbed in the sludge will be released. However, this release is a degradation-driven positive process, not a simple desorption. This release is a necessary step in the degradation process, not a negative effect. The released phenols are further acted upon by the bacterial strains, simultaneously improving the removal efficiency of both free and adsorbed pollutants. This process involves two mechanisms: First, according to the adsorption-desorption equilibrium principle, changes in the concentration of phenols in the liquid phase will cause the phenols adsorbed by the sludge to be released into the liquid phase (desorption), forming a dynamic equilibrium of "adsorbed state → free state → degradation". Second, during metabolism, phenol-degrading bacteria secrete EPS-degrading enzymes (such as glycanase), which moderately decompose the EPS layer on the surface of the sludge flocs, exposing and releasing the phenolic substances encapsulated in the EPS into the liquid phase for further degradation by the bacterial community; this release is controllable and will not lead to large-scale disintegration of the sludge flocs. The release and further degradation of phenolic substances in the sludge demonstrates that the microbial agent can assist in the "detoxification" of sludge.

[0109] COD concentration measurement results

[0110] The table below shows the biodegradation of COD in aerobic pond wastewater.

[0111]

[0112] Note: A is a compound bacterial agent, B is a COD bacterial agent ① from an environmental protection company in Qingdao, C is a COD-degrading bacterial agent ② from our company and a commercially available COD-degrading bacterial agent, and D is the control group (uninoculated group). The data in the table are in mg / L.

[0113] Group A, with added bacteria, showed the best COD degradation. The COD concentration decreased from an initial level of about 280 mg / L to below 183 mg / L after 92 hours of reaction, and further to about 92 mg / L after 164 hours. Group B did not show any significant degradation effect at present. Group C showed some degradation effect, but compared with Group D (control), the final COD concentration was higher and there was no advantage.

[0114] III. Examples

[0115] Example 1: Simulated Wastewater Treatment

[0116] Preparation of experimental materials

[0117] (1) Strains: Bacterial suspensions of strains A, B, C, D and E were prepared according to the above-mentioned bacterial activation steps, and the OD600 values ​​were all between 0.6 and 0.8.

[0118] (2) Simulated wastewater: Prepare simulated wastewater containing benzene compounds according to the above formula, wherein the COD concentration of phenol is 1000 mg / L, and other benzene compounds are added in proportion to make the total COD of the simulated wastewater ≥ 1000 mg / L.

[0119] (3) Compound bacterial solution: Mix the suspensions of each strain according to the volume ratio of A:B:C:D:E = 3:2:2:1:2 to prepare the compound bacterial solution.

[0120] Experimental steps (1) Take a 250mL conical flask, add 100mL of prepared benzene-containing simulated wastewater, and inoculate the compound bacterial solution at a rate of 1‰. Set up 3 parallel experiments for each group.

[0121] (2) Place the conical flask in a constant temperature shaker at 28℃ and shake it at 220rpm for 5 days.

[0122] (3) During the culture process, samples were taken every 24 hours. The samples were placed in a centrifuge and centrifuged at 4000 rpm for 10 minutes. The supernatant was then used for COD determination.

[0123] Experimental results

[0124] After 5 days of cultivation, the COD degradation rate of the simulated wastewater reached 99.34%, indicating that the composite bacterial solution has an excellent degradation effect on benzene series organic pollutants in the simulated wastewater.

[0125] Example 2: Actual Pharmaceutical Wastewater Treatment

[0126] Preparation of experimental materials

[0127] (1) Strains: Same as in Example 1, prepare the compound bacterial solution.

[0128] (2) Actual wastewater: Pharmaceutical wastewater from the end of the aerobic tank of a pharmaceutical factory in Zhejiang was collected, pretreated by sterilization at 121℃ for 30 min, and its initial COD concentration was measured to be 360 ​​mg / L.

[0129] (3) Control group setup: Three control groups were set up, namely the sterilized treatment group without inoculation and the non-sterilized treatment group without inoculation, with 3 replicates in each group.

[0130] Experimental steps (1) Take a 250mL conical flask, add 100mL of sterilized pretreated pharmaceutical wastewater to each flask, and inoculate the compound bacterial solution at a rate of 1‰ to form the experimental group; at the same time, set up a control group.

[0131] (2) Place the conical flask in a constant temperature shaker at 28℃ and 220rpm for 5 days.

[0132] (3) Samples were taken every 24 hours, and the supernatant was taken after centrifugation to determine the COD concentration.

[0133] Experimental results

[0134] After 5 days of cultivation, the COD concentration of the pharmaceutical wastewater in the experimental group decreased from 360 mg / L to 65 mg / L, with a degradation rate of 82%. The COD degradation rate of the sterilized but uninoculated group was only 4%, and the COD degradation rate of the unsterilized but uninoculated group was 11%. The results show that the composite bacterial solution has a significant degradation effect on recalcitrant COD in actual pharmaceutical wastewater, which is far superior to the control group.

[0135] In summary, the method for the synergistic deep degradation of benzene-series organic pollutants provided by this invention achieves highly efficient treatment of benzene-containing wastewater through scientific strain activation, rational bacterial composition, optimized wastewater treatment conditions, and precise degradation monitoring, and has broad application prospects. This method is simple to operate, low in cost, and environmentally friendly, providing a practical and feasible new biological treatment approach for solving the pollution problems of benzene-containing wastewater and pharmaceutical and chemical synthesis wastewater.

[0136] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0137] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the synergistic deep degradation of benzene series organic pollutants by multiple bacterial strains, characterized in that, Includes the following steps: S10. Activation of strains: Pseudomonas strain A, Bacillus strain B, Acinetobacter strain C, Shewanella strain D and Pseudomonas strain E were inoculated into LB solid medium and cultured at 28°C for 24 hours to activate them. Then they were transferred to LB liquid medium and cultured with shaking until the stationary phase to obtain suspensions of each strain. S20. Bacterial group compounding: Mix the bacterial strains in a volume ratio of A:B:C:D:E = 3:2:2:1:2 to form a compound bacterial solution; S30. Wastewater treatment: Add 1‰ (v / v) of compound bacterial solution to the benzene-containing wastewater collected at the end of the aerobic tank, and culture it under shaking and aeration at 28℃ and 220rpm for 5-7 days. S40. Degradation monitoring: Samples are taken every 24 hours, and the COD concentration is determined by the potassium dichromate method. The COD degradation rate is calculated until the degradation effect reaches a stable level.

2. The method for the synergistic deep degradation of benzene series organic pollutants by multiple strains according to claim 1, characterized in that, Strain A in S20 is a Gram-negative bacterium that forms milky white, round colonies on LB medium; strain B is a Gram-positive bacterium that forms pale yellow, rough colonies on LB medium; and strain C is a Gram-negative bacterium that forms transparent, wrinkled colonies on LB medium. Strain D is a Gram-negative bacterium, exhibiting pale pink irregular colonies on LB medium; strain E is a Gram-negative bacterium, exhibiting grayish-white dried colonies on LB medium; and each strain was confirmed by 16S rRNA gene sequence comparison, with strains A and E showing ≥99% similarity to Pseudomonas spp., strain B showing ≥98% similarity to Bacillus spp., strain C showing ≥97% similarity to Acinetobacter spp., and strain D showing ≥98% similarity to Shewanella spp.

3. The method for the synergistic deep degradation of benzene series organic pollutants by multiple strains according to claim 1, characterized in that, The compounding ratio in step S20 is determined through the following process: S201. Construct 15 combinations from five strains of bacteria, the 15 combinations being ABC, ABD, ABE, ACD, ACE, ADE, BCD, BCE, BDE, CDE, ABCD, ABDE, ACDE, BCDE, and ABCDE, and mix them in equal volume ratios. S202. Add each combination to simulated wastewater containing benzene series compounds at an inoculum rate of 1‰, with COD≥1000mg / L, and incubate at 28℃ for 5 days; S203, Screening for the ABCDE combination with the highest COD degradation rate; S204. Based on the combination of ABCDE, test 10 volume ratios, of which 10 volume ratios range from 1:1:1:1:1 to 3:1:1:3:

2. Finally, determine that A:B:C:D:E = 3:2:2:1:

2.

4. The method for the synergistic deep degradation of benzene series organic pollutants by multiple strains according to claim 1, characterized in that, The wastewater in S30 includes pharmaceutical wastewater, coal gasification wastewater, coking wastewater, or chemical wastewater, and the wastewater needs to be pretreated by sterilization at 121℃ for 30 minutes to eliminate the interference of the original microorganisms; the benzene series compounds in the wastewater include at least three of phenol, aniline, benzene, o-xylene, m-xylene, p-xylene, nitrobenzene, o-p-chloronitrobenzene, and p-chloronitrobenzene, and the initial COD concentration is 220-360 mg / L.

5. The method for the synergistic deep degradation of benzene series organic pollutants by multiple strains according to claim 1, characterized in that, The aeration and cultivation conditions for S30 are as follows: Aeration rate of 0.8 m³ / s is controlled using microporous aeration heads. 3 / (m 3 • h), initially cultured in a closed system for 24 hours, then stirred at 100 rpm for 5 minutes every 24 hours to promote bacterial contact.

6. The method for the synergistic deep degradation of benzene series organic pollutants by multiple strains according to claim 1, characterized in that, The specific activation of the bacterial strain in S10 includes: The strain preserved in glycerol tubes at -80℃ was inoculated into LB solid medium containing 5 g / L yeast extract, 10 g / L NaCl, 10 g / L peptone, and pH 7.0-7.

2. Single colonies were picked and inoculated into LB liquid medium, and cultured with shaking at 220 rpm for 18 h to obtain primary seed culture. Transfer to fresh LB liquid medium at a 1% inoculum rate and culture until OD600 = 0.6-0.

8.

7. The method for the synergistic deep degradation of benzene series organic pollutants by multiple strains according to claim 1, characterized in that, In the wastewater treatment of step S30, the composite microbial agent removes pollutants through an "adsorption-desorption-degradation" equilibrium mechanism. a1. The strain metabolizes and secretes EPS-degrading enzymes to decompose the surface layer of sludge flocs, releasing encapsulated phenolic substances; b1. Changes in the concentration of phenols in the liquid phase drive the conversion of benzene compounds adsorbed by sludge into a free state; c1. Free pollutants are mineralized into CO2 and water by the strain through the β-ketoadipic acid pathway.

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