Microbial mixed inoculant for degrading xylene as well as preparation method and application of microbial mixed inoculant

Abstract

The invention belongs to the technical field of environmental microorganisms, and particularly relates to a mixed microbial agent for degrading xylene as well as a preparation method and application of the mixed microbial agent. In order to improve the degradation efficiency of the xylene, the microbial inoculum is a mixed solution formed by mixing a pokengenia M2 bacterial solution and a protective pseudomonas M9 bacterial solution according to the volume ratio of 1: 1 or 1: 3, and the OD600 of the pokengenia M2 bacterial solution is 0.455 + / -0.005; and the OD600 of the bacterial liquid of the pseudomonas M9 is protected to be 0.455 + / -0.005. The original bacteria liquid of M2 and M9 has the function of degrading xylene, and the compound bacteria agent of M2 and M9 not only can improve the removal rate of paraxylene, but also can shorten the action time. The strain can be used for treating various dimethylbenzene in a water body and enriching a microbial system for degrading aromatic compounds, and is of great significance to development of efficient and stable microbial agents.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and in particular relates to a mixed microbial agent for degrading xylene, and a preparation method and application thereof. Background Art

[0002] Xylene is a common organic solvent widely used in the chemical, paint, rubber and leather industries. However, xylene is highly toxic and volatile, posing a serious threat to the environment and human health. Long-term exposure to xylene may lead to neurological damage, liver and kidney dysfunction, and respiratory diseases. Therefore, it is particularly important to find an efficient and environmentally friendly method for degrading xylene.

[0003] At present, the methods for treating xylene pollution mainly include physical, chemical and biological degradation methods. Although physical methods such as adsorption and membrane separation can remove some xylene, they have problems such as high treatment costs and easy secondary pollution. Although chemical methods such as oxidation and reduction reactions have fast reaction speeds, they also have shortcomings such as high reagent consumption and complex operations.

[0004] In contrast, biodegradation has gradually gained attention due to its advantages of environmental protection, low cost, and no secondary pollution. The biodegradation method uses the metabolic activity of microorganisms to convert xylene into non-toxic or low-toxic substances, thereby achieving the purpose of purifying the environment. Many previous studies have shown that some naturally occurring microorganisms have the ability to degrade xylene, especially Pseudomonas, which is widely used. For example, when K. Sarayu et al. designed a biological contact reactor to treat wastewater containing toluene and xylene, the microorganisms on the biofilm included Pseudomonas sp. LD Collins et al. also used the strain Pseudomonas sp. ATCC 55595 in the distribution bioreactor scheme. This strain can simultaneously degrade benzene, toluene and para-xylene. Patent CN105087429A isolated a Pseudomonas sp.OX5 that can degrade o-xylene from industrially polluted soil. The strain can degrade 80% of o-xylene at a concentration of 1500 mg / L. Patent CN117402774A disclosed a Pseudomonas sp.BO3-4 that can degrade various monocyclic aromatic hydrocarbons including xylene, but the strain's degradation rate of xylene is only 30%. Pseudomonas has been fully utilized in degrading aromatic compound pollution.

[0005] In addition, Rhodococcus sp. ZJUT312 isolated by You et al. has a good degradation rate and a large substrate range for benzene derivatives, and can adapt to an environment of 500 mg / L. The degradation rate of o-xylene after 100 h of culture is as high as 0.117 mmol L -1 h -1 ; Rhodococcus sp.BTO62 of the genus Rhodococcus isolated from the activated sludge of the wastewater treatment plant by Euisoon Jeong et al. can degrade various benzene series, and the degradation efficiency of o-xylene can be maintained at more than 90% after inoculation in a high-load biological trickling filter for 7 days. Patent CN 113151053 A isolated a strain of Klebsiella sp. (KLebsieLLa sp.) from landfill leachate that can degrade various benzene series, and the degradation rate is as high as 100% after culturing in a 5mg / L p-xylene solution for 48h; Patent CN117327613A discloses a benzene series degrading bacterium Rhodococcus aetherivorans, which has a degradation rate of more than 95% for 100mg / L toluene, ethylbenzene and xylene after 48h of cultivation. This shows that in addition to Pseudomonas, there are more bacterial genera that can degrade benzene derivatives, but a single strain is usually unable to cope with complex polluted environments.

[0006] In order to solve this problem, scientists began to study the application of mixed bacterial agents. Mixed bacterial agents are composed of a variety of microorganisms, and the strains can improve the overall degradation efficiency through metabolic complementarity. In addition, mixed bacterial agents also have the advantages of strong adaptability and good stress resistance, and can play a stable role under changing environmental conditions.

[0007] However, there is a lack of efficient xylene degradation mixed bacterial agents on the market. The mixed bacterial agent prepared by patent CN110591984B with Brachymonas sp. LM-R and Beijerinckia case sp. LM-W can degrade the mixed wastewater of toluene, xylene and acrylic acid, and the degradation rate of each substrate can reach 99.99% under the environment of 1000mg / L. In addition, the strains in the above-mentioned research are mostly from the soil and water bodies of industrial land, and there are few studies on the strains of xylene degradation in other environments. In view of this current situation, the present invention extracts two strains of xylene degradation bacteria from kitchen waste, configures a new mixed bacterial agent for degrading xylene, and introduces its preparation method and application. The mixed bacterial agent significantly improves the degradation efficiency of xylene through scientific strain screening and optimized combination, and provides a new solution for the bioremediation of xylene pollution. Summary of the invention

[0008] In view of the above problems, the present invention provides a composite bacterial agent for degrading xylene, a preparation method and application thereof, which can achieve the purpose of efficiently degrading xylene.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a mixed microbial agent for degrading xylene, comprising a bacterial solution of a Delftia spp. M2 strain and a bacterial solution of a Pseudomonas proteoglycans M9 strain.

[0011] The M2 strain of the present invention has a similarity of 99.86% with Delftia and pond-dwelling Delftia through morphological identification, physiological and biochemical identification and genome sequencing, and can be identified as pond-dwelling Delftia. The M9 strain has a similarity of 100% with Pseudomonas and Pseudomonas protection through morphological identification, physiological and biochemical identification and genome sequencing, and can be identified as Pseudomonas protection.

[0012] Furthermore, the volume ratio of the bacterial solution of the Escherichia coli M2 strain to the bacterial solution of the Pseudomonas proteoglycans M9 strain is 1:1 or 1:3.

[0013] Further, the OD of the bacterial solution of the Escherichia coli M2 strain is 600 The OD of the bacterial solution of the Pseudomonas sp. M9 strain was 0.455±0.005. 600 It is 0.455±0.005.

[0014] Furthermore, the Eubacterium proliferatum M2 strain and the Pseudomonas proteoglycans M9 strain were both isolated from meat and bone waste, and the nucleotide sequences of their 16S rDNA were shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0015] In a second aspect, the present invention provides a method for preparing a mixed microbial agent for degrading xylene, comprising the following steps:

[0016] Step 1, culturing a bacterial solution of the pond-born Delftia M2 strain: inoculating the pond-born Delftia M2 strain into an LB liquid culture medium at an inoculation amount of 10% (volume ratio), culturing at 30° C. and 120 r / min for 3 days to obtain a bacterial solution of the pond-born Delftia M2 strain;

[0017] Step 2, culturing a bacterial solution of the Pseudomonas protection M9 strain: inoculating the Pseudomonas protection M9 strain into an LB liquid culture medium at a 10% inoculation rate, culturing at 30° C. and 120 r / min for 3 days to obtain a bacterial solution of the Pseudomonas protection M9 strain;

[0018] Step 3: Use LB liquid medium to adjust the OD of the bacterial solution of the Delftia spp. M2 strain and the Pseudomonas spp. M9 strain to600 Adjust to 0.455 ± 0.005 to ensure the same concentration of bacteria in the single bacterial solution;

[0019] Step 4, mixing the single bacterial solutions with the same bacterial concentration obtained in step 3 in proportion to obtain a mixed microbial agent.

[0020] In a third aspect, the present invention provides an application of a mixed microbial agent for degrading xylene, which is used to degrade xylene in the environment.

[0021] Furthermore, the environment is a body of water.

[0022] Furthermore, the application method is: inoculating the bacterial agent into a water body containing xylene, the inoculation amount is 10%, and the applicable temperature is 30°C.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention combines two isolated bacteria that can significantly reduce the concentration of xylene in different proportions, performs a significance analysis on xylene in LB culture medium by a substrate method, and then analyzes the degradation of xylene by different proportion combinations, thereby determining the optimal proportion combination for xylene degradation. This is of great significance for enriching the microbial community that degrades aromatic hydrocarbon compounds and developing efficient and stable microbial agents.

[0025] The pool-grown Delftia M2 strain and the Pseudomonas M9 strain with xylene degradation ability provided by the present invention are obtained by separating meat and bone waste in kitchen waste as the bacterial source, and will not cause harm to the surrounding environment and ecological balance. Through cultivation in LB medium with xylene added, it is found that the M2 strain, the M9 strain and the bacterial agents compounded in different proportions of M2 and M9 all have good ability to degrade xylene, and the compounded bacterial agent has better removal effect and shorter action time. The composite bacterial agent can be applied to ecological environment restoration, can degrade xylene in water bodies, enriches the microbial system for degrading aromatic compounds, and is of great significance to the development of efficient and stable microbial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A xylene concentration standard curve was set for bacterial tolerance;

[0027] Figure 2 This is a graph showing the degradation effect of the pond-grown Delftia M2 strain on xylene;

[0028] Figure 3 This is a diagram showing the degradation effect of xylene by Pseudomonas proteoglycans M9 strain;

[0029] Figure 4The morphological identification results of the pool-born Delftia M2 strain, where (a) is the colony morphology of the M2 strain on LB solid medium, (b) is the Gram staining oil microscopic observation of the M2 strain, and (c) is the flagella staining oil microscopic observation of the M2 strain;

[0030] Figure 5 To protect the morphological identification results of Pseudomonas sp. M9 strain, (a) is the colony morphology of M9 strain on LB solid medium, (b) is the Gram staining oil microscopic observation of M9 strain, and (c) is the flagella staining oil microscopic observation of M9 strain;

[0031] Figure 6 This is the phylogenetic tree of the pool-borne Delftia M2 strain;

[0032] Figure 7 To protect the phylogenetic tree of Pseudomonas sp. M9 strain;

[0033] Figure 8 The results of the antagonism test between the pool-borne Delftia M2 strain and the Pseudomonas proteoglycans M9 strain;

[0034] Fig. 9 The growth curves of the pool-grown Delftia M2 strain, the Pseudomonas proteoglycans M9 strain, and the co-culture of the M2 strain and the M9 strain;

[0035] Fig.10 This is the degradation effect diagram of xylene when the ratio of composite bacterial agents M2 and M9 for degrading xylene is 1:1;

[0036] Fig.11 This is the degradation effect diagram of xylene when the ratio of composite bacterial agents M2 and M9 for degrading xylene is 1:2;

[0037] Fig.12 This is the degradation effect diagram of xylene when the ratio of composite bacterial agents M2 and M9 for degrading xylene is 2:1;

[0038] Fig.13 This is the degradation effect diagram of xylene when the ratio of composite bacterial agents M2 and M9 for degrading xylene is 1:3;

[0039] Fig.14 This is the degradation effect diagram of xylene when the ratio of composite bacterial agents M2 and M9 for degrading xylene is 3:1;

[0040] Fig.15 This is the degradation effect diagram of xylene when the ratio of composite bacterial agents M2 and M9 for degrading xylene is 1:5;

[0041] Fig.16 This is the degradation effect diagram of xylene when the ratio of the composite bacterial agents M2 and M9 for degrading xylene is 5:1. DETAILED DESCRIPTION

[0042] In order to further illustrate the technical solution of the present invention, the present invention is further described below through embodiments.

[0043] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0044] The culture medium formula used in the following examples is:

[0045] Xylene liquid selection medium: peptone 2.0g, beef extract 0.4g, NaCL 1.0g, C8H 10 100μL, ultrapure water 1000mL. 10 The other components were dissolved in ultrapure water, and the pH was adjusted to 6.9-7.1 with 5 mol / L HCL solution and 5 mol / L NaOH solution. The culture medium was sterilized at 121℃ for 20 min. After the culture medium was cooled to room temperature, C8H 10 , to prevent excessive temperature from causing C8H 10 of volatilization.

[0046] Xylene solid screening medium: Add 20g agar to the xylene liquid selection medium, heat to dissolve the agar, sterilize at 121℃ for 20min, and add C8H2O after the medium cools to 50-60℃. 10 , shake well and pour onto the plate.

[0047] LB liquid medium: peptone 10.0 g, yeast powder 5.0 g, NaCL 5.0 g, pH 6.9-7.1, ultrapure water 1000 mL, autoclave at 121 °C for 20 min.

[0048] LB solid medium: Add 20 g agar to LB liquid medium, heat to dissolve the agar, and sterilize at 121°C for 20 min.

[0049] Example 1: Enrichment, separation and purification of functional bacteria

[0050] 1. Enrichment of functional bacteria:

[0051] (1) Use an electronic balance to weigh 10 g of meat and bone waste from Xinyuan Restaurant, Dongshan Campus of Shanxi University, into a 250 mL sterile conical flask, add 90 mL of sterile water, and culture in a shaking incubator at 30 °C and 120 rpm for 30 min to prepare a bacterial suspension.

[0052] (2) Use a sterile pipette to draw 10 mL of bacterial suspension and inoculate it into 100 mL of xylene liquid for selective culture. Cultivate in a shaking incubator at 30°C and 120 rpm for 30 days. Add 10 μL of C8H 10 Reagent to eliminate microorganisms that cannot utilize xylene.

[0053] 2. Isolation and purification of functional bacteria:

[0054] (1) Bacterial dilution: Take 100 μL of enriched bacterial solution and add 900 μL of LB liquid medium to make 10 -1 Dilute bacterial solution; take 100 μL 10 -1 Add 900 μL LB liquid medium to dilute the bacterial solution, that is, 10 -2 Dilute the bacterial solution and then obtain 10 -3 , 10 -4 Dilute the bacterial solution.

[0055] (2) Isolation and purification of bacterial strains: Take 1 μL of the diluted bacterial solution and streak it into the xylene solid screening medium, and incubate it upside down in a 30°C constant temperature incubator for 24 hours. After the colonies grow, pick the strains with good growth and repeatedly streak and purify them until a pure strain is obtained. Pick the pure strain and inoculate it into a sterilized EP tube containing 1 mL of LB liquid medium, and culture it at 30°C and 120 r / min for 2 days to make a single bacterial solution, which is stored in a 4°C refrigerator for later use.

[0056] Example 2: Screening of functional bacteria for degrading xylene

[0057] The single bacterial liquid stored at 4°C was cultured at 30°C and 120r / min for 2 days to activate the strain, and the single bacterial liquid was inoculated into LB liquid culture medium with an inoculum of 10% (volume ratio) to expand the culture, and the bacterial liquid was cultured at 30°C and 120r / min for 2 days. Take 0.3mL of bacterial liquid, i.e., 10% (volume ratio) of the inoculum, and inoculate the bacterial liquid into 3mL of xylene liquid selection culture medium. Prepare 12 test tubes for each strain on the inoculation day to prevent sampling errors during culture. After mixing evenly, the test tubes were covered with silicone plugs and cultured continuously in a constant temperature incubator at 30°C for 6 days. Samples were taken on the 0th, 2nd, 4th, and 6th days, respectively. After cooling to room temperature, they were diluted 5 times with methanol and filtered through a 0.22μm filter head. The filtrate was set aside.

[0058] The concentration of xylene in the test tube was detected using a high performance liquid chromatograph, and the chromatographic conditions were:

[0059] Temperature: Room temperature.

[0060] Mobile phase: 100% methanol (chromatographic grade).

[0061] Injection volume: 10 μL.

[0062] Detector wavelength: 254nm.

[0063] Flow rate: 1.0mL / min.

[0064] Sample residence time: 10 min.

[0065] According to the total amount of xylene added during bacterial culture, the strain tolerance is 1 mg / mL, and methanol is diluted 5 times to 0.2 mg / mL, so a xylene gradient standard curve of 0.01-0.32 mg / mL is made. Take 5.78 μL, 11.65 μL, 25.7 μL, 46.2 μL and 92.4 μL of xylene in a 25 mL volumetric flask, dilute to the scale with methanol, and obtain a standard series of solutions with a concentration gradient of 0.01 mg / mL, 0.02 mg / mL, 0.08 mg / mL, 0.16 mg / mL and 0.32 mg / mL. Filter with a 0.22 μm filter head and the filtrate is set aside. Under the above chromatographic conditions, inject the sample after the baseline is stable, record the chromatographic peak area, and use the mass concentration of xylene (mg / mL) as the horizontal axis and the corresponding chromatographic peak area as the vertical axis to draw a standard curve, such as Figure 1 shown.

[0066] To prevent the possible volatilization of xylene in the culture medium, a portable xylene detector produced by Shenzhen Xinyangwei Technology Development Co., Ltd. was used to detect whether there was xylene gas in the test tube. No volatilization of xylene gas was found. The strains that can significantly remove xylene were screened out.

[0067] The standard curve is as follows:

[0068] S=2428.1c+3.0688

[0069] The calculation formula of removal rate is as follows:

[0070]

[0071] Figure 2 and Figure 3 The degradation effect of xylene by M2 and M9 strains alone. For M2 strain, the degradation rate was 41.20% on the second day, and reached 94.36% and 82.05% on the fourth and sixth days respectively; for M9 strain, the degradation rate was 62.87% on the second day, and reached more than 76.87% on the fourth and sixth days.

[0072] The above results show that both strain M2 and strain M9 have the ability to degrade xylene significantly. Strain M2 and strain M9 were stored in a 4℃ refrigerator for further study.

[0073] Example 3: Identification of strains

[0074] 1. Morphological identification of strains: Figure 4 As shown in (a), the M2 strain was isolated and screened by streaking culture on LB solid culture medium. The colony morphology was observed and it was found that the colonies of the M2 strain on LB solid culture medium were white, round in shape, with neat edges and were opaque.

[0075] Gram staining, flagella staining and motility observation were performed on the M2 strain, and the test results were: Gram staining negative, no flagella, and no motility. Figure 4 (b) is the Gram staining oil microscope observation of the M2 strain. Figure 4 (c) is the flagella staining oil microscope observation image of the M2 strain.

[0076] like Figure 5 As shown in (a), the present invention obtains the M9 strain by streaking culture, separation and screening on LB solid culture medium. By observing its colony morphology, it is found that the colonies of the M9 strain on the LB solid culture medium are white, circular in shape, with neat edges and are translucent.

[0077] Gram staining, flagella staining and motility observation were performed on the M9 strain, and the test results were: Gram staining negative, flagella, and motility. Figure 5 (b) is the Gram staining oil microscope observation of M9 strain. Figure 5 (c) is the flagella staining oil microscope observation image of M9 strain.

[0078] 2. Physiological and biochemical identification of strains: Referring to the Manual of Identification of Common Bacterial Systems, physiological and biochemical identification of strains M2 and M9 was carried out. The identification indicators and results are shown in Table 1:

[0079] Table 1: Physiological and biochemical characteristics of strains

[0080]

[0081] Note: The reaction status is divided into positive and negative, the positive trait is coded as "+" and the negative trait is coded as "-".

[0082] 3. Molecular biological identification of strains: 16S rDNA identification of M2 and M9 strains: M2 and M9 strains were expanded and cultured in LB liquid medium with an inoculation of 10%, 30°C, 120r / min for 2 days, and then sent to Shenzhen Micro-Tech Alliance Technology Group for sequencing. The sequencing results of M2 strain are shown in SEQ ID NO: 1, and the sequencing results of M9 strain are shown in SEQ ID NO: 2. The results of the 16S rDNA strain sequence comparison of the strains are shown in Table 2.

[0083] Table 216S rDNA strain sequence comparison results

[0084]

[0085] Figure 6 This is the phylogenetic tree of the M2 strain. It was found that the M2 strain and Delftia lacustris strain ABBid12-01 (KP119498.1) were clustered into one branch, and the result was reliable.

[0086] Figure 7 This is the phylogenetic tree of the M9 strain. It was found that the M9 strain and the Pseudomonas protegens strain H407 (MH669312.1) were clustered into one branch, and the result was reliable.

[0087] Combined with the above-mentioned morphological identification, physiological and biochemical identification and 16S rDNA gene sequence results, it is shown that the M2 strain isolated and screened by the present invention is Delftia lacustris, named as Delftia lacustris M2 strain; the M9 strain isolated and screened by the present invention is Pseudomonas protegens, named as Pseudomonas protegens M9 strain.

[0088] Example 4: Preparation of a composite bacterial agent for degrading xylene

[0089] 1. Test for strain antagonism: Figure 8 As shown, M2 and M9 were streaked in a tic-tac-toe pattern on LB solid culture medium to observe whether the bacteria at the intersection grew well. The results showed that there was no antagonism between the two bacteria, and the bacteria at the intersection grew well, which could be used for the preparation of composite bacterial agents.

[0090] 2. Plotting of growth curve: In 20 mL LB liquid medium, 10% (volume ratio) of M2 strain, 10% (volume ratio) of M9 strain and 5% (volume ratio) of M2 strain plus 5% (volume ratio) of M9 strain were inoculated respectively, and cultured continuously for 7 days at 30°C and 120 r / min. The OD600 value was measured with a microplate reader every 2 hours at the beginning of the culture, and then measured every 12 hours. With time as the horizontal axis, OD 600 The value is the vertical axis, and the strain growth curve is drawn.

[0091] Fig. 9The growth curves of M2 strain, M9 strain and co-culture of M2 and M9 strains are shown in Figure 2. The results show that the M2 strain is in a rapid growth period between 12h and 48h, and in a plateau period between 48h and 144h. The M9 strain is in a rapid growth period between 12h and 48h, and in a plateau period between 48h and 144h. The co-culture of M2 strain and M9 strain is in a rapid growth period between 12h and 48h, and in a plateau period between 48h and 144h. It can be seen from the figure that mixed culture does not reduce the growth rate of bacteria. After 96h, the growth of the mixed group is faster than that of the single culture.

[0092] 3. Preparation of mixed bacterial agents and determination of removal efficiency:

[0093] (1) Inoculate the pool-grown Delftia M2 strain and Pseudomonas serovar Pseudomonas M9 strain into LB liquid culture medium at a 10% inoculation rate and culture at 30°C and 120 r / min for 3 days. Take the cultured bacterial liquid and measure its OD 600 value, and adjust the OD with LB liquid medium 600 The M2 single bacterial agent and the M9 single bacterial agent were then mixed evenly at a volume ratio of 1:1 and 1:3 to obtain a mixed bacterial agent.

[0094] (2) Take 10 mL of each mixed bacterial agent, i.e., 10% (volume ratio) of the inoculum volume, and inoculate the bacterial solution in a test tube with a xylene concentration of 1 mg / mL and mix well. Set up three replicates for each treatment. After mixing well, cover the test tube with a silicone stopper and culture it continuously in a constant temperature incubator at 30°C for 6 days. Take samples on the 0th, 2nd, 4th, and 6th days, dilute them 5 times with methanol, and filter them through a 0.22 μm filter. Use a high performance liquid chromatograph to detect the concentration of xylene in the test tube, and screen out bacterial agents with high xylene removal efficiency.

[0095] Fig.10 The degradation effect of xylene when the ratio of composite bacterial agent M2 and M9 is 1:1. The results show that compared with the removal efficiency of M2 and M9 strains alone, the ratio reached 65.59% on the second day, and the removal rates on the fourth and sixth days reached 77.93% and 94.74%, respectively. This ratio significantly shortened the action time and improved the removal efficiency.

[0096] Fig.11 The degradation effect of xylene when the ratio of composite bacterial agent M2 and M9 is 1:2. The results show that compared with the removal efficiency of M2 and M9 strains alone, the efficiency improvement of 1:2 is poor.

[0097] Fig.12The degradation effect of xylene when the ratio of composite bacterial agent M2 and M9 is 2:1. The results show that compared with the removal efficiency of M2 and M9 strains alone, the removal rate of 2:1 did not differ much on the second day, but reached 81.93% on the sixth day, which is higher than the removal efficiency of M9 strain alone.

[0098] Fig.13 The degradation effect of xylene when the ratio of the composite bacterial agent M2 and M9 is 1:3. The results show that the degradation rate of the bacterial agent with a mixing ratio of 1:3 reached 73.24% on the second day, which significantly shortened the action time compared with the action of M2 and M9 alone.

[0099] Fig.14 The degradation effect of xylene when the ratio of the composite bacterial agent M2 and M9 is 3:1. The results show that the degradation rate of the bacterial agent with a mixing ratio of 3:1 is higher than that of M2 but lower than that of M9 on the second day, and the degradation rate reaches more than 80% after the fourth day.

[0100] Fig.15 This is the degradation effect diagram of xylene when the ratio of composite bacterial agents M2 and M9 for degrading xylene is 1:5; Fig.16 The degradation effect of xylene when the ratio of M2 and M9 for the composite bacterial agent for degrading xylene is 5:1. The results show that the degradation rates at these two ratios are at most 62.69% and 42.23% after 6 days of cultivation, which are lower than the effects of M2 and M9 strains acting alone. These two ratios are not suitable for the preparation of composite bacterial agents.

[0101] The experimental results of compounding the bacterial agents in multiple ratios showed that the composite bacterial agent has the ability to significantly degrade xylene, with a removal rate of up to 94.74%. The optimal ratios of the composite bacterial agents 1:1 and 1:3 improved the degradation efficiency, and the degradation efficiency on the second day was higher than that of the single bacteria for xylene; the degradation rates of the two ratios of the agents were lower than those of M2 and higher than those of M9 on the fourth day; and the degradation rate of 1:1 was significantly higher than that of M2 and M9 on the sixth day, from a maximum of 76.87% when the M9 strain acted alone to a maximum of 94.74%. At the same time, the action time was shortened, and the shortening effect of the composite bacterial agent of 1:3 was more obvious. The M2 strain and the M9 strain acted alone to reach the highest degradation rate on the sixth day, while the composite bacterial agent of 1:3 shortened the action time by 2 days and reached the highest degradation rate on the fourth day. Our experimental results also show that the degradation rates of the ratios of 1:2 and 2:1 are not much different from those of the strain acting alone, the removal efficiency of the ratio of 3:1 on the second day is lower than that of the strain acting alone, and the ratios of 1:5 and 5:1 are not suitable for the preparation of composite bacterial agents.

[0102] The above shows and describes the main features and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0103] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0104] SEQ NO:1:

[0105]

[0106] SEQ NO:2:

[0107]

Claims

1. A microbial mixed agent for degrading xylene, characterized in that: The bacterial agent includes a bacterial solution of the Delftia spp. M2 strain and a bacterial solution of the Pseudomonas proteoglycans M9 strain.

2. The microbial mixed agent for degrading xylene according to claim 1, characterized in that: The volume ratio of the bacterial solution of the Escherichia coli M2 strain to the bacterial solution of the Pseudomonas proteoglycans M9 strain is 1:1 or 1:

3.

3. The microbial mixed agent for degrading xylene according to claim 2, characterized in that: The OD of the bacterial solution of the Escherichia coli M2 strain 600 The OD of the bacterial solution of the Pseudomonas sp. M9 strain was 0.455±0.

005. 600 It is 0.455±0.

005.

4. A mixed microbial agent for degrading xylene according to any one of claims 1 to 3, characterized in that: The E. coli M2 strain and the Pseudomonas proteoglycans M9 strain are both isolated from meat and bone waste, and the nucleotide sequences of their 16S rDNA are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

5. A method for preparing the mixed microbial agent for degrading xylene according to claim 3, characterized in that: The following steps are involved: Step 1, culturing a bacterial solution of the pond-born Delftia M2 strain: inoculating the pond-born Delftia M2 strain into an LB liquid culture medium at an inoculation volume ratio of 10%, and culturing at 30° C. and 120 r / min for 3 days to obtain a bacterial solution of the pond-born Delftia M2 strain; Step 2, culturing a bacterial solution of the Pseudomonas protection M9 strain: inoculating the Pseudomonas protection M9 strain into an LB liquid culture medium at an inoculation rate of 10% by volume, and culturing at 30° C. and 120 r / min for 3 days to obtain a bacterial solution of the Pseudomonas protection M9 strain; Step 3: Use LB liquid medium to adjust the OD of the bacterial solution of the Delftia spp. M2 strain and the Pseudomonas spp. M9 strain to 600 Adjust to 0.455±0.005 to ensure the same concentration of bacteria in the single bacterial solution; Step 4, mixing the single bacterial solutions with the same bacterial concentration obtained in step 3 in proportion to obtain a mixed microbial agent.

6. An application of the mixed microbial agent for degrading xylene according to claim 3, characterized in that: Used to degrade xylene in the environment.

7. The use of a mixed microbial agent for degrading xylene according to claim 6, characterized in that: The environment is a body of water.

8. The use of a mixed microbial agent for degrading xylene according to claim 7, characterized in that: The application method is: inoculating the bacterial agent into a water body containing xylene, with a volume ratio of 10% of the inoculation amount and an applicable temperature of 30°C.

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