Complex microbial inoculant for degrading 2, 3-dichloropropanol as well as preparation method and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for degrading 2, 3-dichloropropanol (DCP) as well as a preparation method and application of the complex microbial inoculant. The invention relates to the technical field of biology, and discloses a complex microbial inoculant. The complex microbial inoculant is prepared from a rhodococcus strain DC-20, a rhodococcus bayden strain DC-21, a rhodococcus strain DT-18 and bacillus cereus DBC-6. The complex microbial inoculant can degrade DCP and has good degradation efficiency, and after continuous degradation is conducted for 14 days, the degradation efficiency of DCP of 60 mg / L, the degradation efficiency of DCP of 90 mg / L and the degradation efficiency of DCP of 150 mg / L are 100%, 94.78% and 91.903% respectively. The complex microbial inoculant disclosed by the invention can be used for enhancing the biodegradation of 1, 2, 3-trichloropropane (TCP) so as to realize efficient and thorough removal of the TCP.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a composite bacterial agent for degrading 2,3-dichloropropanol, and a preparation method and application thereof. Background Art

[0002] Dichloropropanol is a widely used chemical raw material and the main intermediate product produced in the production process of epichlorohydrin. 2,3-dichloropropanol (DCP) and 1,3-dichloro-2-propanol can be produced in the classic or alternative epichlorohydrin production process. In addition, dichloropropanol is also produced as a degradation product in the biological metabolism of 1,2,3-trichloropropane, a priority pollutant. Both DCP and 1,3-dichloropropanol have highly toxic effects such as teratogenicity, carcinogenicity, and mutagenicity, but DCP is significantly more toxic to the human body than 1,3-dichloropropanol, and is more stable and more difficult to degrade than 1,3-dichloropropanol. Even low concentrations of DCP exposure can pose a huge threat to the ecological environment and human health. Therefore, the prevention, control and governance of DCP pollution have important scientific and social significance, and relevant research work is urgently needed.

[0003] Commonly used remediation technologies mainly include physical remediation technology, chemical remediation technology and biological remediation technology. Among them, biological remediation technology is an ideal means to control DCP pollution because of its advantages such as being green, economical, free of secondary pollution, and suitable for large-scale pollution remediation. In bioremediation technology, bioaugmentation technology based on the addition of high-efficiency bacterial agents is considered to be the fundamental way to completely remove pollutants from the environment. However, due to the high chemical stability and strong resistance to biodegradation of DCP, only a small number of strains that can grow and metabolize with DCP as the only carbon source and energy source have been reported. Therefore, if you want to achieve efficient degradation of DCP into H2O, CO2 or other non-toxic and harmless substances, it is crucial to isolate and purify efficient degradation strains from the environment and construct efficient degradation bacterial communities. Summary of the invention

[0004] In view of the above situation, in order to make up for the above existing defects, the present invention provides a method for preparing a composite bacterial agent for degrading 2,3-dichloropropanol, which is used to solve the problem of how to efficiently biodegrade 2,3-dichloropropanol.

[0005] Another object of the present invention is to provide a composite bacterial agent for degrading 2,3-dichloropropanol.

[0006] The above-mentioned purpose of the present invention is achieved by the following technologies:

[0007] In order to solve the above technical problems, the present invention first provides a composite bacterial agent (referred to as composite bacterial agent 1), wherein the composite bacterial agent 1 contains (or its active ingredients are) Rhodococcus sp. strain DC-20, Rhodococcus baikonurensis strain DC-21, Rhodococcus sp. strain DT-18 and Bacillus cereus strain DBC-6;

[0008] The Rhodococcus sp. strain DC-20 is deposited at the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No. 30357.

[0009] The Rhodococcus baikonurensis strain DC-21 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.30416;

[0010] The Rhodococcus sp. strain DT-18 is deposited at the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 30417.

[0011] The Bacillus cereus strain DBC-6 has a deposit number of CGMCC No. 30356 in the General Microbiological Center of China National Microbiological Culture Collection Administration.

[0012] In a further embodiment, the ratio of the number of live bacteria of the Rhodococcus sp. strain DC-20, the Rhodococcus baikonurensis strain DC-21, the Rhodococcus sp. strain DT-18 and the Bacillus cereus strain DBC-6 in the above-mentioned composite bacterial agent 1 is 1:2:2:2.

[0013] Secondly, the present invention also provides a composite bacterial agent (referred to as composite bacterial agent 2), wherein the composite bacterial agent 2 contains (or its active ingredients are) the Rhodococcus sp. strain DC-20 and the Rhodococcus baikonurensis strain DC-21, or contains the Rhodococcus sp. strain DC-20 and the Rhodococcus sp. strain DT-18 described in claim 1, or contains the Rhodococcus sp. strain DC-20 described in claim 1 and Bacillus cereus strain DBC-6.

[0014] The ratio of the number of live bacteria of the Rhodococcus sp. strain DC-20 to the Rhodococcus baikonurensis strain DC-21 in the above-mentioned composite bacterial agent 2 may be 1:2; the ratio of the number of live bacteria of the Rhodococcus sp. strain DC-20 to the Rhodococcus sp. strain DT-18 may be 1:2; the ratio of the number of live bacteria of the Rhodococcus sp. strain DC-20 to the Bacillus cereus strain DBC-6 may be 1:2.

[0015] In a further embodiment, the composite bacterial agent 1 and the composite bacterial agent 2 may be bacterial agents having the following uses 1) or 2) or 3) or 4):

[0016] 1) Degradation of 2,3-dichloropropanol;

[0017] 2) preparing a product for degrading 2,3-dichloropropanol;

[0018] 3) Repair the environment polluted by 2,3-dichloropropanol;

[0019] 4) Prepare products for repairing the environment polluted by 2,3-dichloropropanol.

[0020] In a further embodiment, the composite bacterial agent 1 and the composite bacterial agent 2 may further include a carrier.

[0021] Preferably, the carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol. The liquid carrier may be a phosphate buffer, a culture medium, a vegetable oil, a mineral oil or water. In the bacterial agent, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture or a mixture of cells and a filtrate. The dosage form of the composition may be a variety of dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0022] The phosphate buffer contains 21.759 g K2HPO4·H2O, 33.48 g Na2HPO4·12H2O, 8.79 g KH2PO4, 5.09 g NH4Cl per liter, and the pH is 7.4.

[0023] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0024] The present invention also provides any of the following applications of the composite bacterial agent 1 and the composite bacterial agent 2:

[0025] 1) Degradation of DCP;

[0026] 2) preparing products for degradation of DCP;

[0027] 3) Repair the DCP polluted environment;

[0028] 4) Prepare products for repairing DCP-polluted environments.

[0029] Specifically, preferably, the preparation method of the composite bacterial agent 1 comprises the following steps:

[0030] The Rhodococcus sp. strain DC-20 or a fermentation product thereof, the Rhodococcus baikonurensis strain DC-21 or a fermentation product thereof, the Rhodococcus sp. strain DT-18 or a fermentation product thereof, and the Bacillus cereus strain DBC-6 or a fermentation product thereof are mixed to obtain a composite bacterial agent 1.

[0031] Specifically, preferably, the preparation method of the composite bacterial agent 2 comprises the following steps:

[0032] The Rhodococcus sp. strain DC-20 or its fermentation product is mixed with the Rhodococcus baikonurensis strain DC-21 or its fermentation product, or the Rhodococcus sp. strain DC-20 or its fermentation product is mixed with the Rhodococcus sp. strain DT-18 or its fermentation product, or the Rhodococcus sp. strain DC-20 or its fermentation product is mixed with the Bacillus cereus strain DBC-6 or its fermentation product to obtain composite bacterial agent 2.

[0033] The method can obtain the mixture by directly mixing the Rhodococcus sp. strain DC-20, the Rhodococcus baikonurensis strain DC-21, the Rhodococcus sp. strain DT-18 and the Bacillus cereus strain DBC-6, or by mixing the three bacteria in the carrier.

[0034] The present invention also provides a method for degrading 2,3-dichloropropanol, which comprises: adding the composite bacterial agent 1 or the composite bacterial agent 2 to a sample containing 2,3-dichloropropanol to carry out a reaction, thereby achieving degradation of 2,3-dichloropropanol.

[0035] In the above method, the reaction can be carried out at 30° C. and pH 7.0. The reaction can be carried out under anaerobic conditions.

[0036] The present invention also provides a method for achieving complete degradation of 1,2,3-trichloropropane (TCP), the method comprising: adding the composite bacterial agent 1 or the composite bacterial agent 2 to TCP hydrolyzed by a halogenated alkane dehalogenase to achieve efficient and complete degradation of TCP.

[0037] The present invention proposes a composite bacterial agent for degrading 2,3-dichloropropanol, and a preparation method and application thereof. The beneficial effects achieved by the present invention using the above scheme are as follows:

[0038] 1. Using efficient bacterial flora to degrade DCP can not only remove DCP from the environment efficiently, but also solve the practical problem that the priority pollutant 1,2,3-trichloropropane is difficult to completely degrade, forming an efficient, green, economical, and secondary pollution-free microbial remediation preparation, providing functional bacterial agents and technical reserves for the sustainable development of green pollution control technology.

[0039] 2. The composite bacterial agent of the present invention can be used to degrade DCP, and can also be used in combination with halogenated alkane dehalogenase to degrade TCP, a priority pollutant. It has the significant advantages of being green, economical, and free of secondary pollution, and has important research significance and application value.

[0040] 3. The composite bacterial agent of the present invention can also be combined with halogenated alkane dehalogenase to degrade the preferential pollutant 1,2,3-trichloropropane (TCP) and has a good degradation efficiency. Compared with the 53.92% TCP degradation efficiency of halogenated alkane dehalogenation, the TCP degradation efficiency is increased by 18.28% after adding the composite bacterial agent of the present invention. The composite bacterial agent of the present invention can promote the efficient and thorough degradation of TCP. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The growth of the strain in the inorganic salt medium with DCP as the carbon source;

[0042] Figure 2 The effect of the amount of bacterial species added during the bacterial community construction on the DCP degradation efficiency;

[0043] Figure 3 is the effect of temperature on the DCP degradation efficiency of the bacterial community;

[0044] Figure 4 The effect of pH on the DCP degradation efficiency of the bacterial community;

[0045] Figure 5 is the effect of initial DCP concentration on the degradation efficiency of bacterial community;

[0046] Figure 6 is the dynamic changes of DCP concentration and degradation efficiency over time;

[0047] Figure 7 This is the kinetic characteristic curve of bacterial degradation of DCP. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technology in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0049] Description of biological material deposit

[0050] Abbreviation of depository unit: CGMCC

[0051] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0052] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0053] Deposit date: April 16, 2024

[0054] CGMCC Registration Number: CGMCC No.30357

[0055] Classification and nomenclature: Rhodococcus sp. strain

[0056] Strain ID: DC-20

[0057] Description of biological material deposit

[0058] Abbreviation of depository unit: CGMCC

[0059] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0060] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0061] Deposit date: April 24, 2024

[0062] CGMCC Registration Number: CGMCC No.30416

[0063] Classification and nomenclature: Rhodococcus baikonurensis strain

[0064] Strain ID: DC-21

[0065] Description of biological material deposit

[0066] Abbreviation of depository unit: CGMCC

[0067] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0068] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0069] Deposit date: April 24, 2024

[0070] CGMCC Registration Number: CGMCC No.30417

[0071] Classification and nomenclature: Rhodococcus sp. strain

[0072] Strain ID: DT-18

[0073] Description of biological material deposit

[0074] Abbreviation of depository unit: CGMCC

[0075] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0076] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0077] Deposit date: April 16, 2024

[0078] CGMCC Registration Number: CGMCC No.30356

[0079] Classification name: Bacillus cereus strain

[0080] Strain ID: DBC-6

[0081] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0082] Inorganic salt culture medium: Na2HPO4·7H2O 7g, KH2PO4 3g, (NH4)2SO4 5g, NaCl 3g, MgSO4·7H2O 0.7g, distilled water to 1L, pH 7.0; add 20g agar powder to the solid culture medium, sterilize with high pressure steam at 121℃ for 30min and set aside.

[0083] Example 1, screening of DCP-degrading strains:

[0084] 1) Take 5 mL of groundwater sample and add it to an inorganic salt liquid culture medium containing 150 mg / L 2,3-dichloropropanol (DCP), and culture it in a constant temperature shaker at 30°C and 160 r / min for 5 days.

[0085] 2) Take 5 mL of the turbid culture medium and transfer it into fresh 150 mg / L DCP inorganic salt liquid culture medium. Repeat this process 3 times.

[0086] 3) The plate dilution method was used for screening and separation. The culture solution was diluted step by step with phosphate buffer solution, and 100 μL of the dilution solution was spread on a solid culture medium containing DCP inorganic salts, and cultured in a constant temperature incubator at 30°C for 48 h. After colonies grew on the plate, colonies of different morphologies were picked up and repeatedly streaked for separation, and 9 DCP-degrading strains were initially obtained.

[0087] 4) The above 9 DCP-degrading strains were inoculated into an inorganic salt liquid medium containing 150 mg / L DCP, and cultured at 30°C, 120 r / min, and away from light for 7 days. The growth of the strains was measured every other day (OD 600 Calculation), DCP-degrading bacteria were screened again, and the experimental results are as follows Figure 1 shown.

[0088] Depend on Figure 1 It can be seen that the growth of strain DBC-6 was the best, followed by DC-20, DC-4, DC-21, DC-7 and DT-18, and the growth of DC-1, DC-2 and DC-6 was poor. Therefore, the DCP-degrading strains DBC-6, DC-20, DC-4, DC-21, DC-7 and DT-18 were selected in subsequent experiments to construct efficient bacterial communities.

[0089] The composition of LB medium is: NaCl 10g, tryptone 10g, yeast extract 5g, distilled water to 1L, pH 7.2-7.4; 20g agar powder is added to the solid medium; high-pressure steam sterilization is carried out at 121°C for 30min before use.

[0090] The composition of the inorganic salt culture medium is: Na2HPO4·7H2O 7g, KH2PO43.0 g, (NH4)2SO45 g, NaCl 3g, MgSO4·7H2O 0.7g, distilled water to 1L, pH 7.0; 20g agar powder is added to the solid culture medium; it is sterilized by high-pressure steam at 121°C for 30min and set aside.

[0091] Example 2: Construction of a highly efficient DCP-degrading bacterial community:

[0092] 1) The 6 strains (DBC-6, DC-20, DC-4, DC-21, DC-7 and DT-18) described in Example 1 were activated and inoculated into LB liquid medium. The culture medium was shaken at 30°C and 160 rpm for 24 h. The OD of the culture medium was adjusted using phosphate buffer. 600 All were diluted to 0.8.

[0093] 2) On this basis, taking the degradation efficiency of DCP as the response value, an orthogonal experimental design with six factors (6 strains) and three levels (the amount of bacteria added was 0 mL, 1 mL and 2 mL) was used to construct the bacterial community. There were a total of 18 designs. The experimental designs and results are shown in Table 1.

[0094] 3) According to the orthogonal experimental design, mixed bacterial communities were constructed and inoculated into 30 mL of inorganic salt culture medium containing 150 mg / DCP. The culture was shaken at 30°C and 160 r / min in the dark for 21 days. Four replicates were set for each treatment. The concentration of DCP in each culture medium was determined. The experimental results are shown in Table 1.

[0095] Table 1 Strain combination experimental design and results

[0096]

[0097] SPSS was used to perform variance analysis on the above experimental results. The analysis results are shown in Table 2.

[0098] Table 2 Test of between-subject effects

[0099]

[0100] R 2 =0.823 (adjusted R 2 =0.998)

[0101] The variance analysis showed that DBC-6 and DC-21 had the most significant effect on DCP degradation efficiency, followed by DC-20 and DT-18, and DC-7 and DC-4 had the least effect. The primary and secondary relationships of the six factors were: DBC-6>DC-21>DT-18>DC-20>DC-7>DC-4.

[0102] On this basis, the Duncan test was used to analyze the effect of the level factor (amount of bacterial strain added) on the degradation efficiency of DCP. The experimental results are as follows: Figure 2 As shown. From the analysis results, it can be seen that the best composition of the bacterial community is 2mL DBC-6, 1mL DC-20, 2mL DC-21, 2mL DT-18, 0mL DC-4 and 0mL DC-21. Therefore, the best composition of the bacterial community is 2mL DBC-6, 1mL DC-20, 2mL DC-21, 2mL DT-18. The degradation efficiency of DCP by the optimized bacterial community CDCP-9 is 90.6%.

[0103] Example 3, molecular biological identification of bacteria:

[0104] The total DNA of DC-20, DC-21, DT-18 and DBC-6 was extracted using the Omega 200T bacterial DNA extraction kit. The specific extraction steps were carried out according to the instructions of the kit. The extracted genome was subjected to a 16s rDNA PCR amplification experiment in a PCR instrument to determine the species of the bacteria. The primers used in the amplification experiment were P1-27 F (5'-AGAGTTTGATCCTGGCTCAG-3') and P2-1492 R (5'-GGTTACCTTGTTACGACTT-3'), respectively.

[0105] The obtained PCR products were sequenced, and after the sequencing results were returned, the sequences were submitted to NCBI for BLAST (Basic Local Alignment Search Tool) comparison, and the bacterial species tested were determined by searching the database for identical or similar strain sequences.

[0106] According to the sequencing results (SEQ ID NO: 1), strain DC-20 was identified as a Rhodococcus sp. strain and recorded as Rhodococcus sp. strain DC-20. The strain was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​April 25, 2024, with the deposit number CGMCC No. 30357.

[0107] According to the sequencing results (SEQ ID NO: 2), strain DC-21 was identified as Rhodococcus baikonurensis strain, recorded as Rhodococcus baikonurensis strain DC-21. The strain was deposited in the General Microbiology Center of China National Microbiological Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​May 8, 2024, with the deposit number CGMCC No. 30416.

[0108] According to the sequencing results (SEQ ID NO: 3), strain DT-18 was identified as a Rhodococcus sp. strain and recorded as Rhodococcus sp. strain DT-18. The strain was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​May 8, 2024, with the deposit number CGMCC No. 30417.

[0109] According to the sequencing results (SEQ ID NO: 4), strain DBC-6 was identified as Bacillus cereus strain and recorded as Bacillus cereus DBC-6. The strain was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​April 25, 2024, with the deposit number CGMCC No. 30356.

[0110] Example 4: Evaluation of the environmental adaptability of efficient bacterial flora:

[0111] (1) Effect of temperature on DCP degradation efficiency

[0112] 150 mg / L DCP was added to 30 mL of inorganic salt medium, and the bacterial community constructed in Example 2 was inoculated into the above DCP medium. The culture was shaken at 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C for 21 days at 160 r / min. The degradation effect of bacterial community CDCP-9 on DCP at different temperatures (20°C to 40°C) was investigated. The experimental results are as follows: Figure 3 shown.

[0113] The bacterial community CDCP-9 had a DCP degradation efficiency of more than 70% in the range of 25℃ to 35℃, and the highest DCP degradation efficiency was 91.07% at 30℃. The DCP degradation efficiency of the bacterial community decreased at 20℃ and 40℃, which were 56.93% and 46.73%, respectively.

[0114] The above research results show that the bacterial community CDCP-9 has a high degradation effect in the range of 20℃~40℃, but when the temperature continues to rise or fall, it is not conducive to the degradation of DCP by the bacterial community, and the effect of high temperature on the bacterial community is more significant.

[0115] (2) Effect of pH on DCP degradation efficiency

[0116] 150 mg / L DCP was added to 30 mL of inorganic salt medium, and the pH of the medium was adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 using an acid-base buffer. The bacterial community constructed in Example 2 was inoculated into the above DCP medium and cultured in a constant temperature shaker at 30°C and 160 r / min for 21 days to investigate the changes in the DCP degradation efficiency of the bacterial community CDCP-9 under different pH (5.0-9.0) conditions. The experimental results are as follows Figure 4 shown.

[0117] The DCP degradation efficiency of bacterial community CDCP-9 was higher than 50% under the condition of pH 5.0-9.0, and the highest DCP degradation efficiency was 91.80% at pH 7.0. The lowest DCP degradation efficiency was 56.93% at pH 5.0. When the pH continued to increase or decrease, the degradation efficiency of bacterial community on DCP decreased, and the effect of acidic conditions on the degradation of DCP by bacterial community was more significant.

[0118] (3) Effect of initial DCP concentration on degradation efficiency

[0119] 60 mg / L, 90 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L DCP were added to 30 mL of inorganic salt medium, and the bacterial community constructed in Example 2 was inoculated into the above DCP medium. The culture was shaken at 30 ° C and 160 r / min for 21 days in a constant temperature shaker to observe the changes in the bacterial community CDCP-9 degradation efficiency of the bacterial community at different initial DCP concentrations (60 mg / L, 90 mg / L, 150 mg / L, 200 mg / L and 250 mg / L). The experimental results are as follows Figure 5 shown.

[0120] The degradation efficiency of DCP by the bacterial community decreases with the increase of DCP concentration. When the initial concentration of DCP is 60 mg / L, 90 mg / L, 150 mg / L, 200 mg / L and 250 mg / L, the degradation efficiency of DCP by the bacterial community is 100%, 94.78%, 91.903%, 63.45% and 47.2%, respectively. High concentration of DCP has a significant inhibitory effect on the biodegradation of the bacterial community.

[0121] Example 5: Analysis of the degradation performance of bacterial flora on DCP

[0122] The bacterial colony constructed in Example 2 was inoculated into 30 mL of an inorganic salt medium containing 150 mg / L DCP, and cultured at 30°C and 160 r / min in the dark for 21 days. The DCP concentration of the culture medium after degradation for 0, 3, 6, 9, 12, 15, 18, and 21 days was determined. The changes in the DCP concentration and degradation efficiency in the culture medium over time are shown in Table 2. Figure 6 shown.

[0123] The concentration of DCP dropped rapidly after inoculation, and reached the lowest concentration of 14.10 mg / L after 15 days of degradation, at which time the degradation efficiency of DCP reached the maximum, which was 91.27%. Within 15 to 21 days, the concentration of DCP remained basically unchanged, which may be due to the accumulation of metabolites inhibiting the degradation activity of the bacterial community on DCP.

[0124] The microbial degradation process of DCP generally conforms to first-order reaction kinetics and can be fitted according to the first-order reaction equation (1) to obtain the reaction rate k (d- 1 ).

[0125] LnC=-kt+b(1)

[0126] Where C is the DCP concentration after t days of degradation (mg / L), and t is the DCP degradation time (d).

[0127] The kinetic curve of DCP degradation by bacterial flora is as follows Figure 7 The kinetic equation for DCP degradation by the bacterial community is LnC = -0.1605t + 5.0746, which conforms to the first-order reaction kinetics (R 2 =0.9982), the degradation rate constant is 0.1605d- 1 .

[0128] The composite bacterial agent of the present invention can also be combined with halogenated alkane dehalogenase to degrade the preferentially treated pollutant 1,2,3-trichloropropane (TCP), and has a good degradation efficiency. Under the combined action of halogenated alkane dehalogenase and the composite bacterial agent, after continuous degradation for 15 days, the degradation efficiency of 200 mg / L TCP is 72.20%. Compared with the TCP degradation efficiency of 53.92% of halogenated alkane dehalogenation, after adding the composite bacterial agent of the present invention, the degradation efficiency of TCP is increased by 18.28%. The composite bacterial agent of the present invention can promote the efficient and thorough degradation of TCP.

[0129] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, material or apparatus including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, material or apparatus.

[0130] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composite bacterial agent for degrading 2,3-dichloropropanol, characterized in that: Contains Rhodococcus sp. strain DC-20, Rhodococcus baikonurensis strain DC-21, Rhodococcus sp. strain DT-18 and Bacillus cereus strain DBC-6; The Rhodococcus sp. strain DC-20 is deposited at the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No. 30357. The Rhodococcus baikonurensis strain DC-21 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.30416; The Rhodococcus sp. strain DT-18 is deposited at the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 30417. The Bacillus cereus strain DBC-6 has a deposit number of CGMCC No. 30356 in the General Microbiological Center of China National Microbiological Culture Collection Administration.

2. The composite bacterial agent for degrading 2,3-dichloropropanol according to claim 1, characterized in that: The ratio of the number of live bacteria of Rhodococcus sp. strain DC-20, Rhodococcus baikonurensis strain DC-21, Rhodococcus sp. strain DT-18 and Bacillus cereus strain DBC-6 in the composite bacterial agent is 1:2:2:

2.

3. The composite bacterial agent for degrading 2,3-dichloropropanol according to claim 1, characterized in that: The composite bacterial agent contains the Rhodococcus sp. strain DC-20 and the Rhodococcus baikonurensis strain DC-21 described in claim 1, or contains the Rhodococcus sp. strain DC-20 and the Rhodococcus sp. strain DT-18 described in claim 1, or contains the Rhodococcus sp. strain DC-20 and Bacillus cereus strain DBC-6 described in claim 1.

4. The composite bacterial agent for degrading 2,3-dichloropropanol according to any one of claims 1 to 3, characterized in that: The composite bacterial agent is a bacterial agent having the following uses 1) or 2) or 3) or 4): 1) Degradation of 2,3-dichloropropanol; 2) preparing a product for degrading 2,3-dichloropropanol; 3) Repair the environment polluted by 2,3-dichloropropanol; 4) Prepare products for repairing the environment polluted by 2,3-dichloropropanol.

5. The use of the composite bacterial agent for degrading 2,3-dichloropropanol according to any one of claims 1 to 3, characterized in that: The composite bacterial agent has the following uses 1) or 2) or 3) or 4): 1) Degradation of 2,3-dichloropropanol; 2) preparing a product for degrading 2,3-dichloropropanol; 3) Repair the environment polluted by 2,3-dichloropropanol; 4) Prepare products for repairing the environment polluted by 2,3-dichloropropanol.

6. The use of the composite bacterial agent for degrading 2,3-dichloropropanol according to claim 5, characterized in that: The method for degrading 2,3-dichloropropanol comprises: adding the composite bacterial agent described in any one of claims 1 to 3 to a sample containing 2,3-dichloropropanol to carry out a reaction, thereby achieving the degradation of 2,3-dichloropropanol.

7. The use of the composite bacterial agent for degrading 2,3-dichloropropanol according to claim 6, characterized in that: The reaction was carried out at 30°C, pH 7.0 and anoxic conditions; Under the reaction conditions, the degradation efficiency of the composite bacterial agent on 60 mg / L, 90 mg / L and 150 mg / L 2,3-dichloropropanol is 100%, 94.78% and 91.903% respectively.

8. The use of the composite bacterial agent for degrading 2,3-dichloropropanol according to claim 5, characterized in that: The invention also includes a method for degrading 1,2,3-trichloropropane (TCP), which specifically comprises: adding the composite bacterial agent described in any one of claims 1 to 3 to TCP hydrolyzed by a haloalkane dehalogenase to carry out a reaction, thereby achieving efficient and thorough degradation of TCP.

9. The method for preparing the composite bacterial agent for degrading 2,3-dichloropropanol according to claim 1 or 2, characterized in that: The specific steps include: The composite bacterial agent is obtained by mixing the Rhodococcus sp. strain DC-20 or its fermentation product, the Rhodococcus baikonurensis strain DC-21 or its fermentation product, the Rhodococcus sp. strain DT-18 or its fermentation product and the Bacillus cereus strain DBC-6 or its fermentation product as described in claim 1.

10. The method for preparing the composite bacterial agent for degrading 2,3-dichloropropanol according to claim 3, characterized in that: The specific steps include: The composite bacterial agent is obtained by mixing the Rhodococcus sp. strain DC-20 or its fermentation product described in claim 1 with the Rhodococcus baikonurensis strain DC-21 or its fermentation product, or mixing the Rhodococcus sp. strain DC-20 or its fermentation product described in claim 1 with the Rhodococcus sp. strain DT-18 or its fermentation product, or mixing the Rhodococcus sp. strain DC-20 or its fermentation product described in claim 1 with the Bacillus cereus strain DBC-6 or its fermentation product.

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