CN115074292A - Pseudomonas for obligately degrading hydroxyl-terminated polybutadiene - Google Patents
Pseudomonas for obligately degrading hydroxyl-terminated polybutadiene Download PDFInfo
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- 238000006731 degradation reaction Methods 0.000 claims abstract description 30
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- A62—LIFE-SAVING; FIRE-FIGHTING
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Abstract
The invention discloses pseudomonas for specifically degrading hydroxyl-terminated polybutadiene, belonging to the technical field of biodegradation. The pseudomonas is pseudomonas HM6, and is preserved in China general microbiological culture Collection center (CGMCC) at 04 th 2022 with the preservation number of CGMCC No. 24201. The pseudomonas HM6 can take hydroxyl-terminated polybutadiene as a unique carbon source, realize the degradation of macromolecular hydroxyl-terminated polybutadiene with the weight-average molecular weight of about 20000g/mol, generate a single-distribution aldehyde micromolecular compound with the molecular weight of about 1500g/mol, and further degrade the micromolecular compound in biochemical treatment and other manners, thereby finally realizing the complete mineralization of the hydroxyl-terminated polybutadiene.
Description
Technical Field
The invention belongs to the technical field of biodegradation, and particularly relates to pseudomonas for specifically degrading hydroxyl-terminated polybutadiene.
Background
Hydroxyl-terminated polybutadiene (HTPB), also called liquid rubber, can form a multi-component three-dimensional net elastic material with various additives through cross-linking and curing reaction in the processing process due to excellent mechanical properties, and is widely applied to adhesives of solid rocket propellants at present. However, with the rapid development of weapons and ammunition and aerospace technologies in our country, the upgrading of solid ammunition along with projectiles generates a large amount of HTPB propellant waste. Conventional disposal methods for waste HTPB propellants include incineration, pyrolysis, and chemical methods, but all of the above methods cause various levels of environmental pollution. The microbial degradation method is low in cost, green and environment-friendly, does not produce secondary pollution, and is an effective method for treating the waste HTPB propellant. However, HTPB belongs to high polymers, and HTPB obligate degrading bacteria are currently lacking, so that HTPB degrades very slowly in the natural environment. The molecular structural formula of HTPB is shown below:
disclosure of Invention
The invention provides pseudomonas HM6, which is preserved in China general microbiological culture Collection center (CGMCC) at 27 th month 12 in 2021 with the preservation number of CGMCC NO. 24201.
The pseudomonas HM6 can take hydroxyl-terminated polybutadiene as a unique carbon source, degrade macromolecular hydroxyl-terminated polybutadiene with a weight-average molecular weight of about 20000g/mol, generate a single-distribution aldehyde micromolecule compound with a molecular weight of about 1500g/mol, and further degrade the micromolecule compound by biochemical treatment (such as an activated sludge method and a biofilm method) and the like, so that complete mineralization of the hydroxyl-terminated polybutadiene can be finally realized.
The invention provides an application of the pseudomonas HM6, which is to use the pseudomonas HM6 for degradation of hydroxyl-terminated polybutadiene.
The invention provides a product for degrading hydroxyl-terminated polybutadiene, which comprises the pseudomonas HM 6. The product may also contain nutrients for maintaining growth and reproduction of Pseudomonas HM6, and other components or strains for degrading hydroxy-terminated polybutadiene.
The invention provides a degradation method of hydroxyl-terminated polybutadiene, which comprises the following steps:
hydroxyl-terminated polybutadiene and an inorganic salt culture medium are mixed and sterilized to prepare a hydroxyl-terminated polybutadiene-inorganic salt liquid culture medium, and then the pseudomonas HM6 is inoculated into the hydroxyl-terminated polybutadiene-inorganic salt liquid culture medium to be cultured under proper culture conditions, so that the degradation of the hydroxyl-terminated polybutadiene can be realized.
In the degradation method, the formula of the inorganic salt culture medium is as follows: KH (Perkin Elmer) 2 PO 4 0.5 part, K 2 HPO 4 .3H 2 O0.655 portion, NaCl 1 portion, NH 4 NO 3 1 part of FeSO 4 .7H 2 O0.02 part, MgSO 4 .7H 2 0.2 portion of O, CaCl 2 0.01 portion, and the pH is adjusted to 7. During the application process, for example, the usage amount of the inorganic salt culture medium is enlarged, the usage amount of each component can be correspondingly adjusted according to the formulaThe proportion is enlarged.
In the degradation method, the content of the hydroxyl-terminated polybutadiene in the hydroxyl-terminated polybutadiene-inorganic salt liquid culture medium is 500 mg/L.
In the above degradation method, the inoculum size of the Pseudomonas HM6 is 4% of the volume of the hydroxyl-terminated polybutadiene-inorganic salt liquid culture medium.
In the above degradation method, the culture conditions are: shaking and culturing at 35 deg.C and 150 rpm.
The invention has the beneficial effects that:
the pseudomonas HM6 is obtained by screening, the strain can take hydroxyl-terminated polybutadiene as a unique carbon source, the degradation of macromolecular hydroxyl-terminated polybutadiene with the weight-average molecular weight of about 20000g/mol is realized, a single-distribution aldehyde micromolecule compound with the molecular weight of about 1500g/mol is generated, and the micromolecule compound can be further degraded by conventional biochemical treatment and other modes, so that the complete mineralization of the hydroxyl-terminated polybutadiene can be finally realized. The whole degradation process is low in cost, green and environment-friendly, and secondary pollution cannot be generated.
Drawings
FIG. 1 is a photograph of a colony of HM6 strain;
FIG. 2 is a photomicrograph of the HM6 strain;
FIG. 3 is a phylogenetic tree of strain HM6 based on the 16S rDNA sequence;
FIG. 4 is a graph of the change in the degradation rate of HTPB during degradation;
FIG. 5 is an infrared spectrum of original HTPB and its degradation products, wherein the upper diagram is an infrared spectrum of the original HTPB and the lower diagram is an infrared spectrum of the degradation products;
FIG. 6 is a NMR spectrum of HTPB and its degradation products, wherein the upper diagram is the NMR spectrum of HTPB and the lower diagram is the NMR spectrum of the degradation products;
FIG. 7 is a graph showing the results of the Schiff test.
Detailed Description
The terms used in the present invention have generally the meanings that are commonly understood by those of ordinary skill in the art, unless otherwise specified. The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
1. Strain screening and identification
(1) Screening
100mg of HTPB liquid gel was added to the flask, and 150mL of mineral salt medium (KH) 2 PO 4 0.5g， K 2 HPO 4 .3H 2 O 0.655g，NaCl 1g，NH 4 NO 3 1g，FeSO 4 .7H 2 O 0.02g，MgSO 4 .7H 2 O 0.2g，CaCl 2 0.01g, pH was adjusted to 7), to prepare an HTPB-inorganic salt liquid medium. 10mL of aerobic activated sludge (MLSS: 3500-4000mg/L) from a petrochemical plant was cultured in a conical flask at 35 ℃ and 140rpm for 7 days. The enrichment culture solution is sequentially transferred to a fresh HTPB-inorganic salt culture medium by adopting a quantitative transfer (volume ratio is 4%), and the enrichment degradation bacteria are further enriched by continuously transferring for 5 times. Adding 10% agar into HTPB-inorganic salt liquid culture medium, and sterilizing at 121 deg.C for 20min to obtain HTPB-inorganic salt solid culture medium. After the culture solution obtained by enrichment is diluted in a gradient way, the dilution gradient is taken to be 10 -4 、10 -5 The 10 mu L of culture solution is evenly coated in an HTPB-inorganic salt solid culture medium, after the bacterial strain grows out, a bacterial strain which can grow by taking the HTPB as a unique carbon source and energy source is obtained and is named as HM 6.
(2) Identification
The colony photograph of HM6 strain is shown in FIG. 1, and the colony is round on solid plate, and is milky white, translucent, smooth and glossy, and easy to pick. The HM6 strain was observed in more detail by transmission electron microscopy, and as a result, the strain had a short rod shape, flagella, and a cell size of about 1.2. mu.m, as shown in FIG. 2. Phylogenetic tree of HM6 Strain As shown in FIG. 3, the strain has the highest homology with Pseudomonas strain HL22-2, and it is inferred that HM6 strain is Pseudomonas strain and is therefore named Pseudomonas sp. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 27 days 12 months 2021 with the preservation number of CGMCC NO. 24201.
2. HTPB degradation test
Preparing an HTPB-inorganic salt liquid culture medium, wherein the HTPB content is 500mg/L, and sterilizing for 20min by high-pressure steam at 121 ℃. The strain HM6 is activated by LB culture medium, washed by equal volume of sterile water, inoculated into HTPB-inorganic salt liquid culture medium according to the inoculation amount of 4% (v/v), and cultured in an air bath constant temperature oscillator of 150rpm at 35 ℃ by taking HTPB as a unique carbon source. Extracting the culture solution with n-hexane of equal volume when culturing at 3 rd, 7 th, 14 th, 21 th and 28 th days, filtering the extractive solution with 0.22 μm filter membrane, and volatilizing the solvent to obtain HTPB degradation product.
Measuring the molecular weight and distribution of the degradation product by gel permeation chromatography; representing HTPB degradation products by a Fourier infrared spectroscopy method and a nuclear magnetic resonance method; and verifying whether the degradation product contains aldehyde group by adopting a Schiff test. The above test results are shown below:
(1) molecular weight distribution characteristics of HTPB
As can be seen from Table 1, the number average molecular weight (Mn) of HTPB as such was 8726g/mol, the weight average molecular weight (Mw) was 18768 g/mol, and the polydispersity index was 2.15, indicating that HTPB as such was a broad-distribution polymer compound. The original HTPB degradation by pseudomonas HM6 produced a new component with a significantly lower molecular weight than the initial HTPB, around 1580g/mol, and a polydispersity index close to 1, indicating that the degradation product is a monodispersed high molecular compound.
TABLE 1 molecular weight distribution of HTPB in the degradation process
Only two molecular weight distribution intervals exist after the HTPB is degraded, so that the peak areas corresponding to the two molecular weights represent the content change of the HTPB before and after the degradation, and the degradation rate of the HTPB is calculated to obtain a graph 4. As can be seen from FIG. 4, the degradation rate of 7d is up to 24%, and the degradation rate of 28d is 35%.
(2) Infrared spectroscopic analysis
The results of infrared spectroscopic analysis of the HTPB intact and the degradation products are shown in fig. 5, wherein the upper panel of fig. 5 is the infrared spectrum of the HTPB intact, and the lower panel of fig. 5 is the infrared spectrum of the degradation products.
As can be seen from FIG. 5, 3356.23cm in the original sample -1 is-OH stretching vibration peak at 2917cm -1 And 1469cm -1 Is of the formula-CH 2 Of (4) asymmetric stretching vibration and bending vibration of 1649cm -1 Stretching vibration of C ═ C double bond, 1280cm -1 And 1062 cm -1 The vibration is C-OH in-plane bending vibration and stretching vibration, and the band intensity of the stretching vibration is higher, 902cm -1 Out-of-plane rocking vibration of the vinyl (1, 2-structure) is treated.
Compared with the original HTPB sample, the peak yield of the degradation product is obviously increased. 3357cm -1 is-OH stretching vibration peak at 1040cm -1 And 1079cm -1 Respectively, C-OH (primary alcohol), and thus, alcohol compounds are present in the degradation products. 2920cm -1 And 1469cm -1 Is of the formula-CH 2 1260cm of asymmetric stretching vibration and bending vibration -1 The stretching vibration attributed to the carboxylic acid C-OH. 866cm -1 Where is the out-of-plane bending vibration of C-H on the double bond. The C ═ C stretching vibration frequency is often lower than the C ═ O stretching vibration frequency, and therefore, 1658cm -1 Telescopic vibration of 1632cm -1 The C ═ C stretching vibration indicates the presence of aldehydes in the degradation products.
(3) Nuclear magnetic resonance spectrogram analysis
The results of nmr hydrogen spectra analysis of the HTPB sample and the degradation product are shown in fig. 6, where the upper graph of fig. 6 is the nmr hydrogen spectrum of the HTPB sample, and the lower graph of fig. 6 is the nmr hydrogen spectrum of the degradation product.
FIG. 6 illustrates a typical HTPB 1 H NMR spectrum with deuterated CDCl as solvent 3 (upper diagram). Peak at 7.26ppm is deuterated reagent CDCl 3 Solvent peak of (2). The hydrogen on the unsaturated carbon on the HTPB is concentrated between 4.7 and 5.7ppm, while the hydrogen on the saturated carbon resonates between 1.0 and 2.2 ppm. The molecular weight of the HTPB is sharply reduced after being degraded, but the basic structural unit of the HTPB is still retained, and the nuclear magnetic analysis of the HTPB after being degraded is as follows based on the lower graph of fig. 6: peak at 7.26ppm deuterated reagent CDCl 3 The solvent peak of (1), the peak between 5.3 and 5.6ppm belongs to the hydrogen on the carbon atom at a in the chemical structure, the peak between 4.9 and 5.0ppm belongs to the hydrogen on the carbon atom at c, the peak at 4.09ppm belongs to the hydrogen on the carbon atom at b, the peak between 1.8 and 2.3ppm belongs to the hydrogen on the carbon atom at e, the peak between 0.8 and 0.9ppm belongs to the hydrogen on the carbon atom at d, and the peaks at 8.10 and 6.95ppm of newly added low fields are attributed to the hydrogen oxidized to the aldehyde group (-CHO) after degradation.
(4) Schiff's test
Preparing a Schiff reagent: 0.05g of basic fuchsin is finely ground and dissolved in 50mL of water containing 0.5mL of concentrated hydrochloric acid, and 0.5g of Na is added 2 SO 3 And (4) stirring the solid, standing the solid until the red color fades, and preparing the Schiff reagent for later use. 1mL of Schiff reagent is added into the HTPB degradation product, and the color development condition is observed after the mixture is kept stand for 2 hours. The results of the test are shown in fig. 7, where purple spots were visible on the surface of HTPB degradation products, indicating the presence of aldehyde groups in the degradation products.
Combining infrared spectroscopy, nuclear magnetic resonance hydrogen spectroscopy and Schiff test results, the following analyses were obtained: pseudomonas HM6 effects the degradation of HTPB by cleaving the double bond in the HTPB molecule. After the HTPB is degraded by pseudomonas HM6, a small-molecule polymer with the molecular weight of about 1500g/mol can be generated, and the polymer contains aldehyde groups.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (8)
1. The pseudomonas HM6 is characterized in that the pseudomonas HM6 is preserved in the China general microbiological culture Collection center of China general microbiological culture Collection center (CGMCC) at 04.01.2022, with the preservation number of CGMCC NO. 24201.
2. Use of pseudomonas HM6 according to claim 1 for the degradation of hydroxyl terminated polybutadiene.
3. A product for degrading hydroxyl-terminated polybutadiene, characterized in that it comprises pseudomonas HM6 according to claim 1.
4. A degradation method of hydroxyl-terminated polybutadiene is characterized by comprising the following steps:
mixing hydroxyl-terminated polybutadiene with an inorganic salt culture medium, sterilizing to prepare a hydroxyl-terminated polybutadiene-inorganic salt liquid culture medium, then inoculating the pseudomonas HM6 described in claim 1 into the hydroxyl-terminated polybutadiene-inorganic salt liquid culture medium, and culturing under appropriate culture conditions to realize degradation of the hydroxyl-terminated polybutadiene.
5. The degradation method according to claim 4, wherein the formula of the inorganic salt medium is as follows: KH (Perkin Elmer) 2 PO 4 0.5 part, K 2 HPO 4 .3H 2 O0.655 portion, NaCl 1 portion, NH 4 NO 3 1 part of FeSO 4 .7H 2 O0.02 part, MgSO 4 .7H 2 0.2 portion of O, CaCl 2 0.01 portion, and the pH is adjusted to 7.
6. The degradation method according to claim 4, wherein the content of the hydroxyl-terminated polybutadiene in the hydroxyl-terminated polybutadiene-inorganic salt liquid medium is 500 mg/L.
7. The degradation method according to claim 4, wherein the Pseudomonas HM6 inoculation amount is 4% of the volume of the hydroxyl-terminated polybutadiene-inorganic salt liquid culture medium.
8. A degradation method according to claim 4, characterized in that the culture conditions are: shaking and culturing at 35 deg.C and 150 rpm.
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| RU2067115C1 (en) * | 1993-06-02 | 1996-09-27 | Инновационное производственно-коммерческое предприятие "Инновационные биотехнологии" | Strain of bacterium pseudomonas fluorescens decomposing styrene and ethylbenzene |
| CN110283354A (en) * | 2019-05-29 | 2019-09-27 | 武汉理工大学 | It is a kind of for the degradation solvent for scrapping end hydroxy butadiene composite solidpropellant and application |
| CN114214233A (en) * | 2021-12-22 | 2022-03-22 | 黄河三角洲京博化工研究院有限公司 | Styrene degrading bacterium and application thereof |
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2022
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| RU2067115C1 (en) * | 1993-06-02 | 1996-09-27 | Инновационное производственно-коммерческое предприятие "Инновационные биотехнологии" | Strain of bacterium pseudomonas fluorescens decomposing styrene and ethylbenzene |
| CN110283354A (en) * | 2019-05-29 | 2019-09-27 | 武汉理工大学 | It is a kind of for the degradation solvent for scrapping end hydroxy butadiene composite solidpropellant and application |
| CN114214233A (en) * | 2021-12-22 | 2022-03-22 | 黄河三角洲京博化工研究院有限公司 | Styrene degrading bacterium and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118620806A (en) * | 2024-08-14 | 2024-09-10 | 青岛科技大学 | Enterobacter pseudo-He and application thereof |
| CN118620806B (en) * | 2024-08-14 | 2024-10-11 | 青岛科技大学 | Enterobacter pseudo-He and application thereof |
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| CN115074292B (en) | 2023-05-19 |
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