Complex microbial inoculant for improving degradation effect of hydroxyl-terminated polybutadiene as well as preparation and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for improving the degradation effect of hydroxyl-terminated polybutadiene as well as preparation and application of the complex microbial inoculant, and belongs to the technical field of biology. According to the present invention, the bacterial flora capable of degrading the hydroxyl-terminated polybutadiene comprises three bacterial strains such as Agromyces sp. HA11, Paracoccus mutanolyticus HC62 and Pseudomonas sp. HM6, and the bacterial flora can be used for degrading the hydroxyl-terminated polybutadiene, such that the bacterial flora can be used for degrading the hydroxyl-terminated polybutadiene, and the bacterial flora can be used for degrading the hydroxyl-terminated polybutadiene, and can be used for degrading the hydroxyl-terminated polybutadiene, and can be used for degrading the hydroxyl-terminated polybutadiene, and can be used for degrading the hydroxyl-terminated polybutadiene; the complex microbial inoculant composed of the three strains can effectively improve the biodegradation effect of HTPB.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a composite bacterial agent for improving the degradation effect of terminal hydroxyl polybutadiene, and a preparation method and application thereof. Background Art

[0002] Hydroxyl-terminated polybutadiene propellant, also known as HTPB propellant, is mainly named after its adhesive system, hydroxyl-terminated polybutadiene. Solid propellants with hydroxyl-terminated polybutadiene as the main adhesive are widely used in aerospace and military fields. The main adhesive of HTPB propellant, hydroxyl-terminated polybutadiene (HTPB), is an artificial polymer material that is prone to aging when coexisting with other components of solid propellants. Therefore, the storage life of HTPB propellant is generally only 5-7 years. Due to the limited storage time of HTPB propellant, a large amount of waste HTPB propellant is produced every year in the world, especially in major military powers, where the amount of waste propellant produced by major military powers is as high as thousands of tons each year. In recent years, with the rapid development of science and technology, countries have stopped using traditional landfill and incineration methods to treat waste propellants, and instead use more environmentally friendly methods to treat waste propellants, such as recovering the active ingredients in the propellant to achieve the recycling of the propellant. After recovering the active ingredients in solid propellants, HTPB adhesives and other auxiliary agents have little content and are difficult to recover, coupled with aging losses during storage, so the recovery of HTPB adhesives is not very meaningful. Hydroxyl-terminated polybutadiene is biologically toxic and flammable, so the traditional physical landfill and open-air incineration methods to treat the propellant bonding system not only have a low safety factor but are also more likely to cause harm to organisms and their environment. To date, scientists from various countries have isolated several microbial strains that can degrade HTPB from different environments. According to existing studies, most of the current research on the microbial degradation of hydroxyl-terminated polybutadiene uses a single strain, but the chemical structure of HTPB is stable, and it is usually difficult for a single bacteria to efficiently degrade HTPB. A large number of studies have shown that composite bacteria can effectively solve the problem of low degradation efficiency of a single bacteria, which has positive significance for the efficient biodegradation of HTPB and environmental protection. Summary of the invention

[0003] The present invention provides a composite bacterial agent for improving the degradation effect of terminal hydroxyl polybutadiene, and a preparation method and application thereof. Combining the degradation characteristics of three strains into a composite bacterial community can significantly improve the degradation effect of hydroxyl polybutadiene.

[0004] According to the purpose of the present invention, the composite bacterial agent for improving the degradation effect of terminal hydroxyl polybutadiene is composed of three strains of Agromyces p. HA11, Paracoccus mutanolyticus HC62, and Pseudomonas sp. HM6, among which Agromyces p. HA11 is in the bacterial community.

[0005] The Agromyces sp. HA11 in the present invention is currently deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on July 10, 2023, with a deposit number of CGMCC No. 27841 and a classification name of Agromyces sp.

[0006] The strain HC62 in the present invention was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on July 10, 2023, with a deposit number of CGMCC No. 27843 and a classification name of Paracoccus mutanolyticus.

[0007] The Pseudomonas sp. HM6 in the present invention, the strain HM6 is currently deposited in the China Microorganism Culture Collection Center on December 27, 2021, with the collection number CGMCC No. 24201, and is classified as Pseudomonas.

[0008] Among them, the address of the General Microbiology Center of China Microbiological Culture Collection Administration is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

[0009] The invention provides a composite bacterial agent, which is composed of three strains: Agromyces sp. HA11, Paracoccus mutanolyticus HC62 and Pseudomonas sp. HM6.

[0010] The present invention also provides the use of the composite bacterial agent for improving the degradation effect of terminal hydroxyl polybutadiene, and the use of the composite bacterial agent in preparing products for degrading terminal hydroxyl polybutadiene.

[0011] The present invention also provides a product for degrading hydroxy-terminated polybutadiene, comprising the above-mentioned bacterial community or the above-mentioned composite bacterial agent.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention provides a composite bacterial agent for improving the degradation effect of terminal hydroxyl polybutadiene and its application. The Agrobacterium HA11, Pseudomonas HM6 and Paracoccus HC62 in the bacterial community can degrade HTPB independently. During the degradation process, the strain HC62 mainly breaks the HTPB long chain into short chains, and uses a small amount of the broken HTPB long chain for growth; the strain HM6 can produce a variety of metabolites during the degradation of HTPB, but the degradation products are still mainly HTPB short chains; the strain HA11 mainly uses the broken HTPB short chain, but its ability to break the HTPB long chain structure is relatively poor. Combining the degradation characteristics of the three strains into a composite bacterial community can significantly improve the degradation effect of hydroxyl polybutadiene. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 is the phylogenetic tree of strain HA1116sDNA sequence.

[0016] Figure 2 It is the phylogenetic tree of strain HM616sDNA sequence.

[0017] Figure 3 It is the phylogenetic tree of strain HC6216sDNA sequence.

[0018] Figure 4 This is the infrared chromatogram of HTPB before degradation.

[0019] Figure 5 This is the infrared chromatogram of HTPB after degradation by the single strain HA11.

[0020] Figure 6 This is the infrared chromatogram of HTPB after degradation by the single strain HM6.

[0021] Figure 7 This is the infrared chromatogram of HTPB after degradation by the single strain HC62.

[0022] Figure 8 This is the infrared chromatogram of HTPB after degradation by bacterial flora (HA11, HM6 and HC62).

[0023] Fig. 9 This is the NMR spectrum of HTPB before degradation.

[0024] Fig.10 This is the nuclear magnetic resonance spectrum of HTPB after degradation by the single strain HA11.

[0025] Fig.11 This is the nuclear magnetic resonance spectrum of HTPB after degradation by the single strain HM6.

[0026] Fig.12 This is the nuclear magnetic resonance spectrum of HTPB after degradation by the single strain HC62.

[0027] Fig.13 This is the nuclear magnetic resonance spectrum of HTPB after degradation by bacterial flora (HA11, HM6 and HC62).

[0028] Specific implementation methods for screening and identifying HTPB-degrading strains

[0029] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the screening process of the strains and the preferred embodiments of the strains in the present invention are described in detail below to facilitate the understanding of technicians.

[0030] The reagents and consumables involved in the present invention can be purchased from commercial channels. If the specific experimental conditions are not specified, they are usually carried out according to conventional experimental conditions or the conditions recommended by the reagent company.

[0031] Example 1

[0032] Isolation and screening of strain HA11

[0033] 1. Preliminary preparations for strain screening:

[0034] Prepare HTPB-inorganic salt solid culture medium, which contains only nitrogen source and no other carbon source required for microbial growth, with HTPB as the only carbon source. The specific preparation process and formula are as follows:

[0035] ①K2HPO 4. 3H2O 0.92g / L; ②KH2HPO40.7 g / L; ③ZnSO4.7H2O 0.002g / L; ④MgSO 4. 7H2O0.7g / L; ⑤MnSO40.001 g / L; ⑥NH4NO31.0 g / L; ⑦NaCl 0.005g / L; ⑧Agar 20g / L; ①② prepared separately, sterilized at 121℃ for 30min, cooled to 50-60℃ and then mixed all reagents together.

[0036] Weigh 10 g of HTPB liquid glue, dissolve it in 50 mL of n-hexane, add 1 mL (about 0.01 g / mL) to each culture dish, and place it in a sterile operating table to air-dry for 3-4 days until the n-hexane is completely evaporated and HTPB forms a thin film on the surface of the culture medium.

[0037] 2. Screening of HTPB-degrading bacteria:

[0038] Sludge samples were collected from the sewage treatment plant, 0.01 g of sludge sample was weighed into a sterile 1.5 mL centrifuge tube, 1 mL of sterile LB culture medium was added, and the microorganisms in the sludge sample were enriched by shaking and incubating at 32°C and 140 rpm / min for 24 h. The sludge sample microorganism enrichment culture medium was diluted to 10 -4 , 100 μL of each dilution was applied to the prepared HTPB-inorganic salt solid culture medium, and cultured in a 32°C constant temperature incubator for 30 days to screen HTPB-degrading bacteria.

[0039] 3. Purification of HTPB-degrading bacteria:

[0040] LB solid medium was prepared to streak and purify the colonies of strain HA11. After purification for 3-4 times, a pure strain was finally obtained and preserved, and named HA11.

[0041] Example 2

[0042] Identification of strain HA11

[0043] 1. Morphological identification:

[0044] like Figure 2 As shown, the screened HA11 strain has a fast growth rate, large colonies, light yellow color, smooth surface and neat edges in LB solid culture dishes.

[0045] 2. Molecular biological identification of strains:

[0046] The DNA of HA11 strain was extracted, PCR amplified and the amplified products were sequenced.

[0047] Primer: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′;

[0048] 1492R(5'-TACGACTTAACCCCAATCGC-3'.

[0049] Reaction system: 2×TaqPCR Master Mix 12.5μL, upstream and downstream primers 1μL each, DNA template 1μL, ddH2O10μL; reaction program: 95℃5min; 94℃1min, 55℃1min, 72℃90s, 35 cycles; 72℃10min.

[0050] The PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the sequencing data were spliced ​​and compared with NCBI to identify the species of the degrading strain.

[0051] The results of sDNA sequencing and assembly of strain HA1116 are as follows:

[0052] The 16sDNA fragment of strain HA11 was amplified by PCR and sequenced by the first generation sequencing technology. The sequencing results were compared with the data in the NCBI database. The comparison results showed that the similarity between strain HA11 and Agromyces p. 3098BRRJ was 99.21%. Figure 2 The phylogenetic tree was constructed using 16sDNA sequencing data, and the results showed that the HTPB-degrading strain HA11 was closely related to strains such as Agromyces sp. strain UASWS3226AD1SH4 and Agromyces agglutinans strain CFH90414, so they were clustered in the same large branch; mnibacterium sp. VN08A0400, Crocebacterium sp. SS6.7, and Diaminobutyricimonas massiliensis strain FF2 were distantly related to strain HA11.

[0053] Deposit information: The strain HA11 is currently deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number CGMCC No.27841 and the classification name of Agrobacterium spp.

[0054] Example 3

[0055] Isolation and screening of strain HM6 (the strain was obtained from Qingdao Science and Technology)

[0056] 1. Preliminary preparations for strain screening:

[0057] Add 100 mg of HTPB liquid gel into a conical flask and add 150 mL of inorganic salt culture medium (KH2PO40.5 g,

[0058] K2HPO4.3H2O 0.655g, NaCl 1g, NH4NO31g, FeSO4.7H2O 0.02g, MgSO4.7H2O 0.2g, CaCl20.01g, adjust pH to 7) to prepare HTPB-inorganic salt liquid culture medium. Take 10mL of aerobic activated sludge (MLSS: 3500-4000mg / L) from a petrochemical plant in a conical flask and culture it at 35°C and 140rpm for 7 days. Use the quantitative transfer method (volume ratio 4%) to transfer the enriched culture fluid to fresh HTPB-inorganic salt culture medium in sequence, and transfer it 5 times in succession to further enrich the degrading bacteria. Add 10% agar to the HTPB-inorganic salt liquid culture medium and sterilize it at 121°C for 20min to prepare HTPB-inorganic salt solid culture medium. After the enriched culture fluid is gradient diluted, take a dilution gradient of 10 -4 , 10 -5 10 μL of the culture solution was evenly spread on the HTPB-inorganic salt solid culture medium. After the colonies grew out, a strain that could grow with HTPB as the only carbon source and energy source was obtained, which was named HM6.

[0059] 2. Purification of HTPB-degrading bacteria:

[0060] LB solid medium was prepared to streak and purify the colonies of strain HA16. After purification for 3-4 times, a pure strain was finally obtained and preserved, and named HA16.

[0061] Example 4

[0062] Identification of strain HM6

[0063] 1. Morphological identification:

[0064] Colony photos of HM6 strain are shown in Figure 1 As shown, the colonies on the solid plate are round, milky white, translucent, smooth and shiny, and easy to pick.

[0065] 2. Molecular biological identification of strains:

[0066] The DNA of HA16 strain was extracted, PCR amplified and the amplified products were sequenced.

[0067] Primer: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′;

[0068] 1492R(5'-TACGACTTAACCCCAATCGC-3'.

[0069] Reaction system: 2×TaqPCR Master Mix 12.5μL, upstream and downstream primers 1μL each, DNA template 1μL, ddH2O10μL; reaction program: 95℃5min; 94℃1min, 55℃1min, 72℃90s, 35 cycles; 72℃10min.

[0070] The PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the sequencing data were spliced ​​and compared with NCBI to identify the species of the degrading strain.

[0071] The results of sDNA sequencing and assembly of strain HM616 are as follows:

[0072] The phylogenetic tree of HM6 strain showed that it had the highest homology with Pseudomonas strain HL22-2 (support rate 70%), so they were clustered in the same branch. Pseudomonas stutzeri CCUG11256 and Pseudomonas stutzeri NBRC14165 were distantly related species, so these two were clustered in a small branch alone.

[0073] Deposit information: The strain HM6 was currently deposited at the General Microbiology Center of the China Culture Collection Administration on December 27, 2021, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number CGMCC No. 24201 and the classification name Pseudomonas.

[0074] Example 5

[0075] Isolation and screening of strain HC62

[0076] 1. Preliminary preparations for strain screening:

[0077] Prepare HTPB-inorganic salt solid culture medium, which contains only nitrogen source and no other carbon source required for microbial growth, with HTPB as the only carbon source. The specific preparation process and formula are as follows:

[0078] ①K2HPO 4. 3H2O 0.92g / L; ②KH2HPO40.7 g / L; ③ZnSO4.7H2O 0.002g / L; ④MgSO 4. 7H2O0.7g / L; ⑤MnSO40.001 g / L; ⑥NH4NO31.0 g / L; ⑦NaCl 0.005g / L; ⑧Agar 20g / L; ①② prepared separately, sterilized at 121℃ for 30min, cooled to 50-60℃ and then mixed all reagents together.

[0079] Weigh 10 g of HTPB liquid glue, dissolve it in 50 mL of n-hexane, add 1 mL (about 0.01 g / mL) to each culture dish, and place it in a sterile operating table to air-dry for 3-4 days until the n-hexane is completely evaporated and HTPB forms a thin film on the surface of the culture medium.

[0080] 2. Screening of HTPB-degrading bacteria:

[0081] Sludge samples were collected from the sewage treatment plant, 0.01 g of sludge sample was weighed into a sterile 1.5 mL centrifuge tube, 1 mL of sterile LB culture medium was added, and the microorganisms in the sludge sample were enriched by shaking and incubating at 32°C and 140 rpm / min for 24 h. The sludge sample microorganism enrichment culture medium was diluted to 10 -4 For each dilution, 100 μL was applied to the prepared HTPB-inorganic salt solid culture medium and cultured in a constant temperature incubator at 32°C for 30 days to screen HTPB-degrading bacteria.

[0082] 3. Purification of HTPB-degrading bacteria:

[0083] LB solid medium was prepared to streak-purify the colonies of strain HC62. After purification for 3-4 times, a pure strain was finally obtained and preserved, and named HC62.

[0084] Example 6

[0085] Identification of strain HA11

[0086] 1. Morphological identification:

[0087] like Figure 2 As shown, the screened HC62 strain has a fast growth rate, large colonies, light yellow color, smooth surface and neat edges in LB solid culture dishes.

[0088] 2. Molecular biological identification of strains:

[0089] The DNA of HC62 strain was extracted, PCR amplified and the amplified products were sequenced.

[0090] Primer: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′;

[0091] 1492R(5'-TACGACTTAACCCCAATCGC-3'.

[0092] Reaction system: 2×TaqPCR Master Mix 12.5μL, upstream and downstream primers 1μL each, DNA template 1μL, ddH2O10μL; reaction program: 95℃5min; 94℃1min, 55℃1min, 72℃90s, 35 cycles; 72℃10min.

[0093] The PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the sequencing data were spliced ​​and compared with NCBI to identify the species of the degrading strain.

[0094] like Figure 3 As shown, the results of strain HC6216 sDNA sequencing and assembly are as follows:

[0095] The 16sDNA sequencing results of the strain showed that it was a bacterium of the genus Paracoccus, family Paracoccus, of the phylum Pseudomonas, class α-Proteobacteria, order Rhodobacterales, family Paracoccus, and genus Paracoccus. Figure 1 The phylogenetic tree was constructed using 16sDNA sequencing data, and the results showed that the HTPB-degrading strain HC62 was closely related to strains such as Paracoccus yeei strain GFK11, Paracoccus sp. strain JLT1531, and Paracoccus yeei strain R0-21. Paracoccus alkenifer was also clustered in the same branch; Aquicoccus porphyridii strain L18-17. Albimonas donghaensis strain DS2 and strain HC62 belong to the same family but different genera. Roseovarius aestuarii strain M100 of the Rhodobacterales and Antarcticirhabdusaurantiaca strain R10 of the Chinenseraceae are species of different families from strain HC62. Uncultured alphaproteobacterium NAC1-3 and Hyphomicrobium-like organism US-353 of the Hyphomicrobialales and Hyphomicrobiumaceae are all alphaproteobacteria, as are HTPB-degrading strain HC62.

[0096] Deposit information: The strain HC62 was currently deposited in the China Microbiological Culture Collection Center on July 10, 2023, with the deposit number CGMCC No.27843 and the classification name Paracoccus mutanolyticus. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0097] Example 7

[0098] Test on the effect of composite bacterial flora on HTPB

[0099] Example 1:

[0100] 1. Prepare LB liquid culture medium, the composition ratio of which is: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, pH 7.0-7.4.

[0101] 2. Prepare sterile HTPB-inorganic salt liquid culture medium with the following formula: K2HPO4·3H2O 0.92g / L, KH2HPO4 0.7g / L, FeSO4·7H2O 0.002g / L, MgSO4·7H2O 0.7g / L, MnSO4 0.001 g / L, NH4NO3 1.0g / L, NaCl 0.005g / L, ZnSO4·7H2O 0.002g / L. Prepare 150mL conical flasks, spread 0.1g HTPB liquid gel in each flask, sterilize at 120℃ for 30min, and add 100mL sterile HTPB-inorganic salt liquid culture medium in a sterile environment.

[0102] The purified strain HA11 single colony obtained in Example 1-6 and the strains HM6 and HC62 were picked and inoculated into LB liquid medium for culture, and cultured at 32°C and 140 rpm / min for 24 h until OD 600 When the pH value is ≈1.0, add 1 mL of the bacterial suspension of strains HA11, HM6, and HC62 to 100 mL of inorganic salt liquid culture medium containing 0.1 g HTPB in a ratio of 1:1:1. Seal the bottle mouth with sealing film and tin foil. Culture at 32°C and 140 rpm / min for 28 days. Extract the fermentation liquid with n-hexane ether for 3 times, then extract it with chloroform for 3 times, and place it in a ventilated medium until the n-hexane and chloroform are completely evaporated. All the extraction products are placed in the same sample bottle.

[0103] The experimental results are as follows:

[0104] like Figure 4-7As shown, the fermentation broth after degradation of single strains (strain HA11, strain HM6, strain HC62) and composite bacterial consortium (HA11, HA16, HA11; HM6 and HC62) was extracted with n-hexane and chloroform, respectively, and the HTPB degradation products were recovered. After the extraction solvents (chloroform and n-hexane) were evaporated, infrared chromatography analysis was performed. Figure 4 This is the infrared spectrum of HTPB without degradation strain treatment. 3600~3500cm -1 It is α-hydrogen bond and polyol, 2917cm -1 It is the asymmetric stretching vibration of -CH2, 1649cm -1 The stretching vibration of C=C double bond is 1280cm -1 and 1062cm -1 are the in-plane bending vibration and stretching vibration of C-OH, respectively. Figure 5 As shown in Figure 2, after degradation by strain HA11, the peak of the degradation product increased significantly at 3681.90 cm -1 3584.16cm -1 Signals were detected at all locations, indicating that strain HA11 produced polyols after degrading HTPB. -1 The signal at the position is alcohol-phenol polymer, and the peak shape is relatively wide. Figure 6 After being degraded by strain HM6, the -1 The signal at 1680-1660cm indicates the presence of alcohol and phenol in the degradation products. Compared with HTPB without biodegradation treatment, it also produces a variety of degradation products. -1 α,β-unsaturated aldehyde, 1639.90cm -1 The vibration of indicates the presence of aldehyde compounds in the degradation products. Figure 7 In the infrared spectrum shown, 2975 cm -1 The peak width of the unsaturated hydrocarbon (=CH2) at 1640cm-1 was significantly narrowed after being treated with strain HC62, indicating that strain HC62 mainly destroyed the unsaturated hydrocarbon structure in HTPB to reduce its concentration, thus narrowing the peak width. The stretching of the C=O bond of β-diketone is between 1640 and 1540cm-1. -1 The peak width at 1000-1300 cm -1 The area is alcohol expansion, and after being treated with strain HC62, it is 1079cm -1 The peak width at 805 cm / s increased, indicating that the degradation products contained alcohols. -1 The peak at this position disappeared after the strain degraded it, indicating that strain HC62 also has a certain degradation efficiency for heterocyclic aromatic substances.

[0105] Compared with the degradation of HTPB by single strain, the number of infrared chromatographic peaks was significantly reduced after degradation by composite bacteria HA11, HM6, and HC62 ( Figure 6 ), indicating that the types of degradation products decreased and the degradation effect was improved. After degradation by the bacterial community, the signals at 911.44cm-1, 966.42cm-1 and 1640.05cm-1 were relatively high, indicating that the main products of HTPB after degradation by the composite bacterial community were unsaturated hydrocarbons (about 1675-1600cm-1 for C=C; 990cm-1 and 910cm-1 for -CH=CH2; 965cm-1 for trans-dihydrogen); another major product was saturated carboxylic acid, and the infrared chromatogram of the degradation product showed a strong signal at 1437.20cm-1 (1440-13 95cm-1 is a saturated carboxylic acid); 1237.98cm-1 is an α,β-unsaturated carboxylic acid ester (1300~1250cm-1); 1310.88cm-1 and 1350.42cm-1 are carboxylates (between 1420 and 1300cm-1); weak signals were detected at 1049.09cm-1 and 1077.61cm-1 in the infrared chromatogram of the degradation product, indicating that a small amount of saturated and chain anhydrides (about 1170~1045cm-1) are also present in the degradation product. Fig. 9 This is the NMR spectrum of HTPB without degradation strain treatment. Fig.10 The nuclear magnetic resonance spectrum showed that strain HA11 would slightly interrupt the long-chain structure of HTPB during the degradation of HTPB, so some -CH3 structures could be detected at about 1.5ppm; the signal intensity was about 5.01E+07;

[0106] Fig.11 It shows that strain HM6 will interrupt the long chain structure of HTPB in large quantities during the HTPB degradation process. The 1.5ppm signal is enhanced, and the -CH3 structure is detected with a signal intensity of 2.98E+08. However, its efficiency in interrupting the long chain of HTPB is slightly lower than that of strain HC62. At the same time, strain HM6 will also use other substances in HTPB during the HTPB degradation process, so its NMR detection spectrum peaks disappear at 1.4ppm and 1.25ppm; the peaks of HTPB treated with strain HC62 also change at 5.5pp-5.0ppm and 1.5ppm-1.0ppm ( Fig.12 ), the peak of HTPB at 5.5ppm was significantly weakened after being treated with strain HC62, and the -CH3 signal detected at 1.5ppm was the strongest among the three strains, about 4.75E+08. This was because strain HC62 destroyed the original long-chain structure of HTPB and degraded HTPB into a large number of short-chain structures, thus producing a large amount of -CH3.

[0107] Fig.13It is HTPB degraded by a composite bacterial community (HA11, HM6, HC62). The signal intensity of -CH3 after degradation by the composite bacterial community is about 7.5E+07.

[0108] The comprehensive test results (NMR and IR) showed that strains HA11, HM6 and HC62 could all grow by using HTPB with a destroyed long-chain structure. However, although strain HA11 could produce a variety of degradation products in the process of HTPB degradation, the efficiency of breaking the long chain of HTPB was low. HM6 tended to break the long chain of HTPB into medium-long chains, so the -CH3 signal intensity was weaker than that of strain HC62. Combined with the -CH3 signal intensity test results, it showed that the strain also mainly used short-chain HTPB for growth metabolism; strain HC62 could break the long chain of HTPB into a large number of short-chain structures, but the number of short-chain HTPB it used was limited. Therefore, combining the above three types of HTPB into a composite bacterial agent to degrade HTPB can effectively improve the HTPB degradation effect.

[0109] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A composite bacterial agent for improving the degradation effect of hydroxy-terminated polybutadiene, characterized in that: It consists of three strains: Agrobacterium HA11, Paracoccus HC62, and Pseudomonas HM6; all strains are deposited in the China General Microbiological Culture Collection Center; the deposit number of Agrobacterium HA11 is CGMCC No.27841, and the classification name is Agrobacterium; the deposit number of Paracoccus HC62 is CGMCC No.27843, and the classification name is Paracoccus; the deposit number of Pseudomonas HM6 is CGMCC No.24201, and the classification name is Pseudomonas.

2. The composite bacterial agent for improving the degradation effect of hydroxy-terminated polybutadiene according to claim 1, characterized in that: The effective bacterial count ratio of the three strains of Agrobacterium HA11, Paracoccus HC62 and Pseudomonas HM6 is 1:1:

1.

3. The method for preparing the composite bacterial agent for improving the degradation effect of hydroxy-terminated polybutadiene according to claim 1 or 2, characterized in that: The composite bacterial agent is obtained by mixing corresponding strains in a ratio of 1:1:

1.

4. The method for preparing the composite bacterial agent for improving the degradation effect of hydroxy-terminated polybutadiene according to claim 1 or 2, characterized in that: The preparation method of the composite bacterial agent comprises the following steps: S1. Strain selection: Three strains were selected: Agrobacterium HA11, Paracoccus HC62, and Pseudomonas HM6; S2. Medium preparation: Use HTPB-inorganic salt solid medium as the selection medium, wherein HTPB is the only carbon source, and the medium mainly contains a nitrogen source and a small amount of mineral salts; S3. Strain screening: Enrich microorganisms from sludge samples from sewage treatment plants, and then screen strains that can degrade HTPB in HTPB-inorganic salt solid culture medium; S4. Purification and identification: Pure strains were obtained by streaking purification technology, and the strain species were identified by molecular biological methods; S5. Preparation of composite bacterial agent: The three screened strains HA11, HC62 and HM6 were mixed in a ratio of 1:1:1 to form a composite bacterial agent.

5. An application of the composite bacterial agent for improving the degradation effect of hydroxy-terminated polybutadiene according to claim 1, characterized in that: The composite bacterial agent is used in preparing a product for degrading hydroxy-terminated polybutadiene.

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