Method for improving tire rubber desulfurization capability of desulfurization strain
By employing DBT and DS as inducers and overexpressing the b0840 transcription factor, the desulfurization capability of bacteria strains is enhanced, addressing inefficiencies in existing methods and enabling effective tire rubber degradation for industrial use.
Patent Information
- Application Number
- CN202510533101.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-25
- Publication Date
- 2025-07-15
AI Technical Summary
The prior art has problems of low efficiency, high cost and poor stability in improving the desulfurization capacity of desulfurization of tire rubber in tires, and it is difficult to meet industrial needs.
By adding dibenzothiophene (DBT) and/or diallyl sulfide (DS) as inducers to the culture medium after the activation of the desulfurization strain, the sulfur metabolism regulation system of the strain is activated, the synthesis of desulfurization enzymes is promoted, and the effective transcription factor b0840 is screened for overexpression, improving the desulfurization efficiency.
It significantly improves the desulfurization efficiency of desulfurization strains, reduces costs, and achieves an efficient, economical and environmentally friendly desulfurization effect, which is suitable for industrial production.
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Figure CN120310697A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for improving the desulfurization ability of strains, and specifically to a method for improving the desulfurization ability of desulfurizing strains on tire rubber. Background Art
[0002] At present, rubber (Tire Rubber, TR) is widely used and is one of the essential polymer materials in life. The amount of waste rubber generated in China has been increasing year by year, reaching more than 17 million tons, and about 65% of it is vehicle tire rubber. Due to the three-dimensional cross-linked structure of rubber and the presence of related additives contained therein, the degradation period of waste rubber is prolonged, posing a huge potential environmental risk. Therefore, the effective management of waste tire rubber has become an important issue of global concern. At present, only a small part of waste tire rubber has been successfully recycled and properly treated, while most waste tire rubber is disposed of by landfill and incineration, which will undoubtedly harm human health and the environment.
[0003] Due to its stable three-dimensional network structure, waste rubber is difficult to degrade naturally, causing a series of environmental and resource waste problems. Rubber raw materials and raw rubber do not contain sulfur, but due to the vulcanization treatment in the production process of rubber, sulfur elements are added to rubber as vulcanizing agents. Many microorganisms can not only utilize rubber as a source of carbon and energy, but also consume sulfur. During the biodegradation process of rubber, some microorganisms can break the sulfur bonds (S-S and C-S bonds) in rubber. Through their metabolic pathways, these microorganisms can convert sulfur compounds in vulcanized rubber into other forms, such as elemental sulfur or sulfates, thereby achieving sulfur consumption and causing the cross-linking breakage or main-chain polymer breakage of rubber, and converting inorganic sulfur into organic sulfur to enter amino acid metabolism (cysteine synthesis); moreover, the microbial desulfurization method has the advantages of mild reaction conditions, low energy consumption, environmental friendliness, and is conducive to the reuse or recycling of waste rubber as new products. Therefore, the microbial desulfurization of waste rubber has become a hot research direction.
[0004] At present, many studies have isolated and identified effective desulfurizing bacteria, but their desulfurization ability still needs to be improved. The methods for improving the desulfurization ability of desulfurizing bacteria mainly include strain screening and optimization, fermentation condition optimization, and co-culture and synergy: 1) Strain screening and optimization: Screening efficient desulfurizing strains from natural environments or using mutagenesis breeding techniques to improve the desulfurization ability, but the screening process is time-consuming and inefficient, and mutagenesis breeding is random and may introduce adverse mutations; 2) Fermentation condition optimization: Adjusting parameters such as temperature, pH, and dissolved oxygen content to improve the desulfurization efficiency, but the optimization process requires a large number of experiments, is time-consuming and costly, and the optimal conditions of different strains vary greatly and are difficult to be universal; 3) Co-culture and synergy: Utilize the synergistic effect of multiple strains to enhance the desulfurization efficiency. However, the interactions between strains are complex, and there may be competitive relationships, resulting in the growth inhibition of some strains. Moreover, the stability of the co-culture system is poor, making it difficult to apply on a large scale. Although the above methods have certain effects, they still have limitations and need further research and improvement.
[0005] Therefore, with the increasing industrial demand for desulfurization, the limitations of traditional technologies have become more obvious. It is urgent to find a method to improve the desulfurization ability of desulfurizing strains through inducers or overexpression of specific genes to meet the requirements of efficient, economical, and environmentally friendly desulfurization. Summary of the Invention
[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects existing in the prior art and provide a method for improving the desulfurization ability of desulfurizing strains for tire rubber, which has a high desulfurization rate, is efficient, economical, and environmentally friendly, has a simple process, low cost, and is suitable for industrial production.
[0007] The technical solution adopted by the present invention to solve its technical problems is as follows: A method for improving the desulfurization ability of desulfurizing strains for tire rubber. After inoculating the activated culture solution of the desulfurizing strain into the fermentation medium and fermenting to obtain the stationary-phase fermentation broth, add sterilized tire rubber powder and dibenzothiophene and / or diallyl sulfide, and carry out desulfurization fermentation to obtain the product. As an organic analog compound, dibenzothiophene (DBT) is used in the present invention to utilize the sensitivity of the strain to DBT to induce the strain to produce a large amount of desulfurization enzymes, thereby improving the desulfurization efficiency. And diallyl sulfide (DS) may act as a sulfur source signal molecule to activate the sulfur metabolism regulation system of the strain and promote the synthesis of desulfurization enzymes, thereby improving the desulfurization efficiency.
[0008] Preferably, the screening method for using dibenzothiophene and / or diallyl sulfide as inducers to improve the desulfurization ability of desulfurizing strains for tire rubber is as follows: Divide the stationary-phase fermentation broth obtained by inoculating the activated culture solution of the desulfurizing strain into the fermentation medium and fermenting into different conical flasks, add sterilized tire rubber powder to all of them, and add different inducers respectively, carry out desulfurization fermentation, measure the growth of the desulfurizing strain and the weight loss rate of the tire rubber after desulfurization fermentation, and screen out the effective inducers.
[0009] The inventive concept of the screening method of the present invention is: Add TR to the stationary-phase fermentation broth obtained by inoculating the activated culture solution of the desulfurizing strain into the fermentation medium, carry out desulfurization fermentation under the action of different inducers, and by measuring the growth of the desulfurizing strain and the weight loss of the tire rubber after desulfurization fermentation after adding the inducers, use the inducer that promotes the growth of the strain and helps the decomposition and weight loss of the tire rubber as the effective inducer.
[0010] Preferably, the inducer includes one or more of dibenzothiophene (abbreviated as DBT) and / or diallyl sulfide (abbreviated as DS), dimethyl sulfoxide (abbreviated as DMSO), Na2SO4, or Tween-20, etc.
[0011] Preferably, the desulfurizing strain includes Escherichia coli ( Escherichia sp. ), named CSUFT-2024-Rubbr desulfurization, which was deposited on October 21, 2024 at the General Microbiology Center of the China Center for Type Culture Collection, with the deposit number CGMCC No. 32280.
[0012] Preferably, the sulfur content of the sterilized tire rubber powder is 2-4%, and the particle size is below 80 mesh. The tire rubber used in the present invention is from waste tires of Huayi Rubber Factory.
[0013] Preferably, the dosage of the sterilized tire rubber powder is 4-6 g / 100 mL of fermentation medium. Too much or too little dosage of the sterilized tire rubber powder is not conducive to the growth of the strain and desulfurization.
[0014] Preferably, the dosage of dibenzothiophene and / or diallyl sulfide, or other single inducer is 2-4 mmol / L of fermentation medium. If the dosage is too much, it may lead to too low production of desulfurase and the desulfurization efficiency improvement is not obvious. If the dosage is too little, it is not conducive to the growth of the strain.
[0015] Preferably, the temperature of the desulfurization fermentation is 28-32 °C, the rotation speed is 150-250 rpm, and the time is 140-300 h.
[0016] Preferably, the fermentation method of the stable-phase fermentation broth is: inoculating the desulfurizing strain into the strain activation medium for activation, and then inoculating the obtained activated culture solution into the fermentation medium for fermentation to the stable stage to obtain the stable-phase fermentation broth.
[0017] Preferably, the inoculation amount of the desulfurizing strain is 0.08-0.12% (v / v). This inoculation amount can maximize the secretion and activity of desulfurase while ensuring the rapid growth of the strain. If the inoculation amount is too low, it may lead to slow growth of the strain in the initial stage of fermentation and it is difficult to form sufficient biomass, thus affecting the desulfurization efficiency. If the inoculation amount is too high, it may lead to the accumulation of metabolic by-products (such as organic acids), changing the fermentation environment and affecting the activity of desulfurase.
[0018] Preferably, the activation temperature is 28-32 °C, the rotation speed is 150-250 rpm, and the activation time is 18-30 h.
[0019] Preferably, the OD of the activated culture solution600 is 0.6 to 1.0.
[0020] Preferably, the inoculation amount of the activated culture solution is 8-12% (v / v).
[0021] Preferably, the temperature of the fermentation is 28-32 °C, the rotation speed is 150-250 rpm, and the time is 10-14 h.
[0022] Preferably, the OD of the fermentation broth in the stationary phase after fermentation 600 is 0.6 to 1.0.
[0023] Preferably, the formula of the strain activation medium is: in every 1000 mL of deionized water, it contains 8-12 g of polypeptone, 1.5-2.5 g of yeast powder, and 0.5-1.5 g of MgSO4·H2O.
[0024] Preferably, the formula of the fermentation medium is: in every 1000 mL of deionized water, it contains 3-5 g of KH2PO4, 3-5 g of K2HPO4·H2O, 0.6-1.0 g of MgSO4·7H2O, 0.2-0.6 g of NH4Cl, 8-12 g of Na2S2O3·5H2O, 0.005-0.015 g of CaCl2, 1.5-2.5 g of glucose, 0.8-1.2 g of peptone, and 0.08-0.12 g of yeast powder.
[0025] Preferably, the treatment method of the sterilized tire rubber powder is: first soak the ground tire rubber powder with ethanol, then place the soaked tire rubber powder in a ventilated place to volatilize the ethanol, and finally perform high-pressure sterilization.
[0026] Preferably, the soaking time is 36-48 h.
[0027] Preferably, the volatilization time is 3-4 h.
[0028] Preferably, the temperature of the high-pressure sterilization is 115-125 °C, the pressure is 0.10-0.12 MPa, and the time is 0.8-1.2 h.
[0029] The technical solution adopted by the present invention to further solve its technical problems is as follows: A method for improving the desulfurization ability of desulfurizing strains to tire rubber, screening out the transcription factor b0840 that effectively enhances the desulfurization ability of desulfurizing strains to tire rubber from the desulfurizing strains after desulfurization fermentation, inoculating the activated culture solution of the overexpressing desulfurizing strains of transcription factor b0840 into the fermentation medium, fermenting to obtain the stationary-phase fermentation broth, adding sterilized tire rubber powder, and performing desulfurization fermentation, then it is completed. b0840 belongs to the DeoR (Deoxyribonucleoside operon repressor) family, is widely distributed in the bacterial kingdom, and is involved in nucleotide metabolism, sugar metabolism and virulence regulation.
[0030] Preferably, the fermentation method of the stationary-phase fermentation broth is the same as the fermentation method of the stationary-phase fermentation broth described above.
[0031] Preferably, the treatment method of the sterilized tire rubber powder is the same as the treatment method of the sterilized tire rubber powder described above.
[0032] Preferably, the dosage of the sterilized tire rubber powder is 4 - 6 g / 100 mL of fermentation medium.
[0033] Preferably, the temperature of the desulfurization fermentation is 28 - 32 °C, the rotation speed is 150 - 250 rpm, and the time is 140 - 300 h.
[0034] Preferably, the screening method of the transcription factor b0840 includes the following steps: (1) Performing transcriptome analysis on the desulfurizing strains after desulfurization fermentation by RNA-seq, measuring the expression levels of different genes in the desulfurizing strains, and screening out multiple transcription factors and 4S pathway genes that specifically respond to dibenzothiophene induction; (2) Respectively constructing overexpressing desulfurizing strains of different transcription factors screened in step (1), inoculating them into the strain activation medium respectively for activation, and then inoculating the obtained activated culture solution into the fermentation medium for fermentation to the stationary stage to obtain the stationary-phase fermentation broth; (3) Respectively adding sterilized tire rubber powder to the stationary-phase fermentation broth obtained in step (2) for desulfurization fermentation, comparing the effects of overexpressing desulfurizing strains of different transcription factors on the expression levels of 4S pathway genes, and at the same time, measuring the growth conditions of the overexpressing desulfurizing strains and the weight loss rate of the tire rubber after desulfurization fermentation, and screening out the transcription factor that effectively enhances the desulfurization ability of the desulfurizing strains to tire rubber.
[0035] The inventive concept for screening in the present invention is as follows: The desulfurization of desulfurizing strains follows the 4S pathway and employs four enzymes (DszA, DszB, DszC, and DszD). It has been found through research that after induction by DBT, the desulfurization ability of desulfurizing strains is significantly improved. Based on the regulatory effect of transcription factors on gene expression, it is speculated that DBT may regulate the expression of genes related to the 4S desulfurization pathway by activating specific transcription factors. To verify this hypothesis, first, RNA-seq is used for transcriptome analysis to identify genes in desulfurizing strains during the fermentation process that specifically respond to DBT induction, and transcription factors and 4S pathway genes that may respond to DBT induction and thus regulate the desulfurization pathway are screened out from them; then, strains overexpressing different transcription factors are constructed in desulfurizing strains, the effects of different overexpressed transcription factors on the desulfurization ability of desulfurizing strains are studied, and the growth conditions of overexpressed desulfurizing strains and the weight loss of TR after desulfurization fermentation in the presence of TR are tested, so as to screen out transcription factors that effectively enhance the desulfurization ability of TR.
[0036] Preferably, in step (3), the method for comparing the effects of transcription factors on the expression levels of 4S pathway genes is as follows: RNA is extracted from overexpressed desulfurizing strains of multiple different transcription factors and reverse-transcribed into cDNA. Using 16S rRNA as an internal reference gene, the qRT-PTR technique is adopted to compare the relative expression levels of different 4S pathway genes in overexpressed desulfurizing strains of different transcription factors.
[0037] Preferably, in step (2), the fermentation method of the stationary-phase fermentation broth is the same as the fermentation method of the stationary-phase fermentation broth described above.
[0038] Preferably, in step (3), the treatment method of the sterilized tire rubber powder is the same as the treatment method of the sterilized tire rubber powder described above.
[0039] Preferably, in step (3), the dosage of the sterilized tire rubber powder is 4 - 6 g / 100 mL of fermentation medium.
[0040] Preferably, in step (3), the temperature of the desulfurization fermentation is 28 - 32 °C, the rotation speed is 150 - 250 rpm, and the time is 140 - 300 h.
[0041] The beneficial effects of the present invention are as follows: (1) In the method of the present invention, after desulfurization fermentation of the WT strain induced by DBT or DS, the weight loss rates of TR can reach 5.16% and 5.03% respectively, which are 2.16 times and 2.10 times the weight loss rates of TR after desulfurization fermentation of the WT strain; after desulfurization fermentation of the WT strain or the WT strain induced by DBT, the relative sulfur contents are 1.71% and 1.34% respectively, and the desulfurization rates are 37.13% and 50.74% respectively; this shows that after the WT strain is induced by DBT or DS, its desulfurization ability is significantly enhanced; (2) In the method of the present invention, after the desulfurization fermentation of the OE-b0840 strain, the weight loss rate of TR can reach 5.63%, which is 3.73 times that of the WT strain (1.51%) after desulfurization fermentation; after the desulfurization fermentation of the WT strain or the OE-b0840 strain, the relative sulfur content is 1.71% and 1.24% respectively, and the desulfurization rates are 37.13% and 54.41% respectively, indicating that overexpression of b0840 in the WT strain can also enhance the desulfurization effect; (3) The method of the present invention significantly improves the desulfurization ability of desulfurizing strains by using specific inducers or overexpressing specific genes, not only improving the desulfurization efficiency, but also providing a new technical approach for the environmental protection treatment of tire rubber. It is efficient, economical, environmentally friendly, with a simple process and low cost, and is suitable for industrial production. Description of the Drawings
[0042] Figure 1 is the growth situation diagram of Escherichia coli ( Escherichia sp. ) after 168 h of desulfurization fermentation without any addition, only adding TR, and adding TR + 5 inducers respectively in Example 1 of the method of the present invention; Figure 2 is the weight loss rate diagram of TR after 168 h of desulfurization fermentation of Escherichia coli ( Escherichia sp. ) without any addition and adding 5 inducers respectively in Example 1 of the method of the present invention in the presence of TR; Figure 3 is the result diagram of transcription factors and 4S pathway genes that respond to induction and regulate the desulfurization pathway in the strain after 168 h of desulfurization fermentation of Escherichia coli ( Escherichia sp. ) without any addition, only adding TR, and adding DBT in Example 1 of the method of the present invention; Figure 4 is the growth situation diagram of Escherichia coli ( Escherichia sp. ) without any addition and adding DBT in Example 1 of the method of the present invention, and Escherichia coli ( Escherichia sp. ) overexpressing 6 transcription factors after 168 h of desulfurization fermentation in the presence of TR; Figure 5 is the weight loss rate diagram of TR after 168 h of desulfurization fermentation of Escherichia coli ( Escherichia sp. ) without any addition and adding DBT in Example 1 of the method of the present invention, and Escherichia coli ( Escherichia sp. ) overexpressing 6 transcription factors after 168 h of desulfurization fermentation in the presence of TR; Figure 6It is the functional analysis result diagram of three 4S pathway genes screened in Example 2 of the method of the present invention (wherein, Figure (a) shows the relationship between the dszA gene of the reported desulfurizing bacterium and the 4S pathway gene b1012; Figure (b) shows the relationship between the dszC gene of the reported desulfurizing bacterium and the 4S pathway genes b0039 and b1695); Figure 7 It is the relative expression level diagram of the 4S pathway genes dszA, dszC1, and dszC2 in the wild-type strain, Escherichia coli in Example 1 of the method of the present invention ( Escherichia sp. ), after adding DBT, and Escherichia coli in Example 2 of the method of the present invention ( Escherichia sp. ), in the strain overexpressing six transcription factors; Figure 8 It is the comparative diagram of the relative sulfur content of TR after 168 h of desulfurization fermentation in the case of containing TR, without adding strains, only adding Escherichia coli ( Escherichia sp. ), adding DBT in Example 1 of the method of the present invention, and Escherichia coli in Example 2 of the method of the present invention ( Escherichia sp. ), in the strain overexpressing b0840; Figure 9 It is the scanning electron energy spectrometer diagram of TR after 168 h of desulfurization fermentation in the case of containing TR, without adding strains, only adding Escherichia coli ( Escherichia sp. ), adding DBT in Example 1 of the method of the present invention, and Escherichia coli in Example 2 of the method of the present invention ( Escherichia sp. ), in the strain overexpressing b0840; Figure 10 It is the scanning electron microscope diagram of TR (magnification is 300 times and 1000 times) after 168 h of desulfurization fermentation in the case of containing TR, without adding strains (Figure (a), (b)), only adding Escherichia coli ( Escherichia sp. )(Figure (c), (d)), adding DBT in Example 1 of the method of the present invention (Figure (e), (f)), and Escherichia coli in Example 2 of the method of the present invention ( Escherichia sp. ), in the strain overexpressing b0840 (Figure (g), (h)); Figure 11 It is the Fourier transform infrared spectrum diagram of the surface of TR after 168 h of desulfurization fermentation in the case of containing TR, without adding strains, only adding Escherichia coli ( Escherichia sp. ), adding DBT in Example 1 of the method of the present invention, and Escherichia coli in Example 2 of the method of the present invention ( Escherichia sp. ), in the strain overexpressing b0840; In the present invention, WT represents the wild-type strain of Escherichia coli ( Escherichia sp. ), TR represents tire rubber, DBT represents dibenzothiophene, and OE represents overexpression. Detailed implementation mode
[0043] The present invention will be further described below in conjunction with embodiments and the accompanying drawings.
[0044] The waste tire rubber powder used in the embodiments of the present invention (surface analysis sulfur content is 2.67%, relative sulfur content is 2.72%) was purchased from Huayi Rubber Factory; the Escherichia coli used ( Escherichia sp. ), named CSUFT-2024-Rubbr desulfurization, was deposited on October 21, 2024 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 32280; the formula of the strain activation medium used in the embodiments of the present invention is: in every 1000 mL of deionized water, it contains 10 g of polypeptone, 2 g of yeast powder and 1 g of MgSO4·H2O; the formula of the fermentation medium used in the embodiments of the present invention is: in every 1000 mL of deionized water, it contains 4 g of KH2PO4, 4 g of K2HPO4·H2O, 0.8 g of MgSO4·7H2O, 0.4 g of NH4Cl, 10 g of Na2S2O3·5H2O, 0.01 g of CaCl2, 2 g of glucose, 1 g of peptone and 0.1 g of yeast powder; the LB (Luria-Bertani) agar medium used in the embodiments of the present invention is a general nutrient medium and is prepared according to the standard formula; all media in the embodiments of the present invention are first autoclaved at 121°C and 0.1 MPa for 20 min before use; the raw materials or chemical reagents used in the embodiments of the present invention, unless otherwise specified, are all obtained through conventional commercial channels.
[0045] Example 1 The activated culture solution of Escherichia coli ( Escherichia sp. ), the sterilized tire rubber powder is added at a dosage of 5 g / 100 mL of the fermentation medium, and DBT or DS is added at a dosage of 3 mmol / L of the fermentation medium respectively, and desulfurization fermentation is carried out at 30°C and a rotation speed of 200 rpm for 168 h to obtain the product; The fermentation method of the stationary-phase fermentation broth is as follows: the Escherichia coli ( Escherichia sp. ) strain is inoculated into the strain activation medium at an inoculation amount of 0.10% (v / v), and activation is carried out at 30°C and a rotation speed of 200 rpm for 24 h until OD 600 reaches 0.80, and then the obtained activated culture solution is inoculated into the fermentation medium at an inoculation amount of 10% (v / v), and fermentation is carried out at 30°C and a rotation speed of 200 rpm for 12 h until the stationary phase OD 600 reaches 0.75 to obtain the stationary-phase fermentation broth; The treatment method of the sterilized tire rubber powder is as follows: First, soak the ground tire rubber powder in ethanol for 42 h, then place the soaked tire rubber powder in a ventilated place to volatilize ethanol for 3.5 h, and finally carry out high-pressure sterilization at 121 °C and 0.1 MPa for 1.0 h.
[0046] The screening method for using DBT or DS as an inducer to improve the desulfurization ability of desulfurizing strains on tire rubber is as follows: The culture solution after activation of Escherichia coli ( Escherichia sp. is inoculated into the fermentation medium, and the stationary-phase fermentation broth obtained by fermentation is placed in different conical flasks. The sterilized tire rubber powder is added at a dosage of 5 g / 100 mL of the fermentation medium, and DBT, DS, DMSO, Na2SO4 or Tween-20 are added at a dosage of 3 mmol / L of the fermentation medium respectively. Desulfurization fermentation is carried out at 30 °C and a rotation speed of 200 rpm for 168 h. The growth of the desulfurizing strains and the weight loss rate of the tire rubber after desulfurization fermentation are measured, and the effective inducers are screened out as DBT or DS; the fermentation method of the stationary-phase fermentation broth and the treatment method of the sterilized tire rubber powder are the same as before.
[0047] As Figure 1 shown, except for DMSO and Na2SO4 (indicating that not all sulfur-containing reagents can be used as inducers to increase biomass), under the action of Tween-20, DS or DBT, the growth of the desulfurizing strains is increased compared with the group without adding inducers. Among them, DBT or DS are the inducers that can most promote the growth of the desulfurizing strains and obtain the maximum biomass. The biomass OD 600 of the desulfurizing strains can reach 1.41 and 1.26 respectively, which are 1.26 times and 1.13 times of the biomass (OD 600 1.12) of the desulfurizing strains when only TR is added without adding inducers, indicating that compared with other inducers, adding DBT or DS can better promote the growth of the desulfurizing strains.
[0048] As Figure 2 shown, after the co-desulfurization fermentation of TR with different inducers and desulfurizing strains, the weight loss rate of TR has increased. Among them, after the desulfurization fermentation of the WT strain induced by DBT or DS, the weight loss rates of TR are 5.16% and 5.03% respectively, which are significantly higher than those of other inducers for desulfurization fermentation, and are 2.16 times and 2.10 times of the weight loss rate (2.39%) of TR after only desulfurization fermentation by desulfurizing strains respectively. This shows that adding DBT or DS can further promote the desulfurization effect, probably because the desulfurizing strains are highly sensitive to DBT or DS, and DBT or DS can induce the desulfurizing strains to produce a large amount of desulfurase, and these enzymes act on the three-dimensional cross-linked network structure of rubber to promote its desulfurization.
[0049] As Figure 4As shown, after the desulfurization fermentation of the DBT-induced WT strain, the biomass in the TR increased compared to the WT strain, and the final biomass OD 600 was 1.41, which was 1.33 times that of the WT strain (OD 600 1.06), indicating that the addition of DBT could better promote the growth of the strain.
[0050] As Figure 5 shown, after the desulfurization fermentation of the DBT-induced WT strain, the weight loss rate of the TR was 6.03%, which was 3.99 times that of the WT strain after desulfurization fermentation (1.51%), indicating that the addition of DBT could improve the desulfurization effect.
[0051] As Figure 7 shown, compared with the WT strain, the expression levels of the three 4S pathway genes in the DBT-induced strain were significantly up-regulated by 12 - 18 times, indicating that the addition of DBT could positively regulate the expression of the 4S pathway genes.
[0052] As Figure 8 shown, after the desulfurized tire rubber powder after desulfurization fermentation was fully burned in a high-temperature pure oxygen environment, the relative sulfur content changes of the TR without adding the strain, only adding Escherichia coli ( Escherichia sp. ), and adding DBT for desulfurization fermentation for 168 h were measured by organic elemental analysis (Vario MACRO cube, Elementar). Among them, after the desulfurization fermentation of the WT strain and the DBT-induced WT strain, the relative sulfur content in the TR continued to decrease, and the relative sulfur contents were 1.71% and 1.34% respectively, and the desulfurization rates were 37.13% and 50.74% respectively, indicating that after the co-fermentation of the WT strain and the TR, the relative sulfur content in the TR could be significantly reduced; while after the co-fermentation of the strain induced by DBT and the TR, the relative sulfur content in the TR was further reduced compared with the WT strain, indicating that DBT induction could effectively improve the desulfurization effect of the strain.
[0053] As Figure 9 shown, using an EDS spectrometer to scan the surface of the TR without adding the strain, only adding Escherichia coli ( Escherichia sp. ), and adding DBT for desulfurization fermentation for 168 h in the presence of the TR. The results showed that after the desulfurization fermentation in the presence of the desulfurizing strain, the surface element contents of the TR all changed. Among them, after the desulfurization fermentation of the WT strain and the DBT-induced strain, the sulfur contents on the TR surface decreased from 2.67% to 2.21% and 1.95% respectively, and the DBT-induced WT strain was 1.57 times the desulfurization rate of the WT strain during desulfurization fermentation. This trend was also consistent with Figure 8 the change trend of the relative sulfur content tested by the organic elemental analysis method in
[0054] As Figure 10 shown, using a scanning electron microscope, the surface morphology changes of TR were observed at two magnifications of 300 times and 1000 times in the case of containing TR, without adding strains, only adding Escherichia coli ( Escherichia sp. ), and after adding DBT and performing desulfurization fermentation for 168 h, in order to study the effect of DBT induction on the surface structure of TR. The results showed that the surface of TR without adding strains was relatively smooth and flat, with a dense structure, while the dense structure on the surface of TR after desulfurization fermentation with the WT strain was damaged, showing small holes. After desulfurization fermentation of the WT strain induced by DBT, the surface of TR was rougher and had more holes, probably because DBT induction enhanced the desulfurization ability of the strain, thus more effectively degrading the sulfide in TR.
[0055] As Figure 11 shown, FTIR spectroscopy was used to further determine the cleavage of the main chain of the TR polymer and the sulfur bridge during the desulfurization process. The results showed that in the case of containing TR, without adding strains, only adding Escherichia coli ( Escherichia sp. ), and after desulfurization fermentation of the WT strain induced by DBT for 168 h, there were significant differences in the FTIR spectra of TR: after desulfurization fermentation with the WT strain and the WT strain induced by DBT, the peak intensity of the characteristic C=C bond of the rubber main chain at 650 cm -1 gradually decreased, indicating that its surface structure was broken; after desulfurization fermentation with the WT strain and the WT strain induced by DBT, compared with TR without adding strains, the peak intensity of the C=O bond at 1401 cm -1 gradually increased, indicating that the strain had an oxidation reaction; the order of the peak intensity of the C-S characteristic peak at 1641 cm -1 was WT+TR+DBT < WT+TR, indicating that the WT strain induced by DBT more effectively degraded the C-S bond in TR during the fermentation process, resulting in a decrease in the intensity of the C-S characteristic peak, further indicating that DBT induction enhanced the desulfurization ability of the strain; compared with TR without adding strains, under the desulfurization fermentation of the WT strain and the WT strain induced by DBT, the intensities of the characteristic peaks of the S=O bond and the O=S=O bond at 1074 cm -1 gradually increased, indicating that the intermediate product of sulfur-containing oxides formed during the desulfurization process increased, indicating that the strain had strong desulfurization ability, and DBT induction further promoted this oxidation process.
[0056] Example 2 From the Escherichia coli after desulfurization fermentation described in Example 1 ( Escherichia sp.), the transcription factor b0840 that can effectively enhance the desulfurization ability of the desulfurizing strain to tire rubber is screened out. The culture solution after activation of the overexpressing desulfurizing strain of the transcription factor b0840 is inoculated into the fermentation medium for fermentation to obtain the fermentation broth in the stationary phase. Sterilized tire rubber powder is added at a dosage of 5 g / 100 mL of the fermentation medium, and desulfurization fermentation is carried out at 30 °C and a rotation speed of 200 rpm for 168 h to obtain the product; the fermentation method of the fermentation broth in the stationary phase and the treatment method of the sterilized tire rubber powder are the same as those in Example 1; The screening method of the transcription factor b0840 includes the following steps: (1) Escherichia coli after the desulfurization fermentation described in Example 1 ( Escherichia sp. ) is subjected to transcriptome analysis by RNA-seq to measure the expression levels of different genes in the desulfurizing strain, and 6 transcription factors that specifically respond to dibenzothiophene induction and 3 4S pathway genes are screened out; (2) Overexpressing desulfurizing strains of the 6 different transcription factors screened in step (1) are respectively constructed, inoculated into the strain activation medium for activation, and then the obtained activated culture solution is inoculated into the fermentation medium for fermentation to the stationary stage to obtain the fermentation broth in the stationary phase; the fermentation method of the fermentation broth in the stationary phase is the same as that in Example 1; (3) In each fermentation broth in the stationary phase obtained in step (2), sterilized tire rubber powder is added at a dosage of 5 g / 100 mL of the fermentation medium, and desulfurization fermentation is carried out at 30 °C and a rotation speed of 200 rpm for 168 h. The effects of the overexpressing desulfurizing strains of 6 different transcription factors on the expression levels of 3 4S pathway genes are compared. At the same time, the growth conditions of the overexpressing desulfurizing strains and the weight loss rate of the tire rubber after desulfurization fermentation are measured. The transcription factor that can effectively enhance the desulfurization ability of the desulfurizing strain to tire rubber is screened out as b0840; the method for comparing the effects of transcription factors on the expression levels of 4S pathway genes is as follows: RNA of the overexpressing desulfurizing strains of 6 different transcription factors is extracted and reverse transcribed into cDNA. Using 16S rRNA as an internal reference gene, the qRT-PTR technology is used to compare the relative expression levels of 3 different 4S pathway genes in the overexpressing desulfurizing strains of 6 different transcription factors; the treatment method of the sterilized tire rubber powder is the same as that in Example 1.
[0057] As Figure 3 shown, using RNA-seq for transcriptome analysis in Example 1, after the combined desulfurization fermentation of DBT and WT strains in TR, the genes in Escherichia coli ( Escherichia sp. ) that specifically respond to DBT induction are screened, and 6 transcription factors that may respond to DBT induction to regulate the desulfurization pathway and 3 4S pathway genes are screened out.
[0058] As Figure 4As shown, except for the OE-b1187 strain, the biomass of all other overexpressed desulfurization strains in TR increased compared to the WT strain. Among them, the final biomass (OD 600 1.36) of the OE-b0840 strain was 1.28 times that of the WT strain (OD 600 1.06).
[0059] As Figure 5 shown, after the desulfurization fermentation of the OE-b1187 strain, the weight loss rate of TR was similar to that of the WT strain during desulfurization fermentation. After the desulfurization fermentation of other overexpressed strains, the weight loss rate of TR increased. Among them, after the desulfurization fermentation of the OE-b0840 strain, the weight loss rate of TR (5.63%) increased significantly, which was 3.73 times that of the WT strain during desulfurization fermentation (1.51%). In addition, after the desulfurization fermentation of the OE-b0840 strain, the weight loss rate of TR was 5.63%, which was similar to the weight loss rate of TR (6.03%) after the desulfurization fermentation of the DBT-induced WT strain.
[0060] In summary, by comparing the growth of overexpressed desulfurization strains and the weight loss of TR, it was comprehensively concluded that overexpression of b0840 helped the strain utilize TR for its own growth, thereby increasing the desulfurization rate of TR.
[0061] The microbial desulfurization pathway of the present invention is the 4S pathway, which is catalyzed by three enzymes, DszA, DszB, and DszC encoded by the dsz operon. In order to identify Figure 3 whether the three 4S pathway genes screened out can play a desulfurization role, phylogenetic analysis was carried out.
[0062] As Figure 6 (a) shows, b1012 (dszA) is closely related to the dszA of the reported desulfurization strains Bacillus subtilis and LuteimiTRobium xylanilyticum , and has the same superfamily Flavin of monooxygenase, indicating that b1012 (dszA) can play a desulfurization role; as Figure 6 (b) shows, b0039 (dszC1) and b1695 (dszC2) are closely related to the dszC of the reported desulfurization strains Klebsiella pneumoniae , and have the same superfamily ACAD, indicating that both b0039 (dszC1) and b1695 (dszC2) can play a desulfurization role; the 4S pathway genes b1012, b0039, and b1695 have the same domain as dszA and dszC, can encode the dszA and dszC genes, and can perform the same desulfurization function.
[0063] In order to explore the role of overexpressed transcription factors in regulating the transcriptional expression of 4S pathway genes, Figure 3Functional analysis was performed on the three selected 4S pathway genes: RNA was extracted from the overexpressing desulfurizing strains of six different transcription factors and reverse transcribed into cDNA. Using 16S rRNA as the internal reference gene and qRT-PTR technology, the relative expression levels of the three different 4S pathway genes in the overexpressing desulfurizing strains of six different transcription factors were compared. Combining with Figure 4 the growth of the overexpressing desulfurizing strains in Figure 5 and the weight loss rate of tire rubber after desulfurization fermentation in
[0064] As Figure 7 shown, compared with the WT strain, the expression levels of the 4S pathway genes in each overexpressing strain increased to varying degrees. Among them, in the OE-b0840 strain, the expression level of the 4S pathway gene was significantly higher than that of other overexpressing strains. Compared with the WT strain, the expression level was upregulated by 13-18 times, similar to the WT strain induced by DBT. The upregulation of the expression level in the OE-b0840 strain was also consistent with the increase in the biomass of TR in Figure 4 and the increase in the weight loss rate of TR in Figure 5 after desulfurization fermentation of the OE-b0840 strain. Therefore, it was further proved that overexpression of b0840 in Escherichia coli ( Escherichia sp. ) could positively regulate the expression of 4S pathway genes.
[0065] To further explore whether the OE-b0840 strain could improve the desulfurization ability of Escherichia coli ( Escherichia sp. ) on TR: As Figure 8 shown, the desulfurized tire rubber powder after desulfurization fermentation treatment was fully burned in a high-temperature pure oxygen environment. The relative sulfur content changes of TR were measured by organic elemental analysis (Vario MACRO cube, Elementar) after 168 h of desulfurization fermentation of the strain without addition, only addition of Escherichia coli ( Escherichia sp. ) and the OE-b0840 strain. Among them, after desulfurization fermentation of the WT strain or the OE-b0840 strain, the relative sulfur content of TR continued to decrease, reaching 1.71% and 1.24% respectively, and the desulfurization rates were 37.13% and 54.41% respectively. The desulfurization rate of the OE-b0840 strain was 1.47 times that of the WT strain. This conclusion was also consistent with the increase in the biomass of the strain in Figure 4 , the increase in the weight loss rate of TR in Figure 5 and the positive regulation of the expression of 4S pathway genes in Figure 7 after desulfurization fermentation of the OE-b0840 strain, and was similar to the desulfurization fermentation effect of the WT strain induced by DBT.
[0066] As Figure 9As shown, using an EDS spectrometer, in the presence of TR, without adding strains, only adding Escherichia coli ( Escherichia sp. ), and the OE-b0840 strain, the surface of TR was scanned after 168 h of desulfurization fermentation to study the effect of b0840 overexpression on the surface properties of TR, and the distribution of C, O, and S on the surface of TR, the corresponding surface spectra, and their weight percentages were determined. The results showed that after desulfurization fermentation by the desulfurizing strains, the surface element contents of TR all changed significantly. Among them, after desulfurization fermentation by the WT strain or the OE-b0840 strain, the sulfur content on the surface of TR decreased from 2.67% to 2.21% and 1.81% respectively, and the desulfurization rate of the OE-b0840 strain was 1.87 times that of the WT strain. This trend was also consistent with Figure 8 the change trend of the relative sulfur content tested by the organic elemental analysis method in
[0067] As Figure 10 shown, using a scanning electron microscope, the surface morphology changes of TR were observed at two magnifications of 300 times and 1000 times in the presence of TR, without adding strains, only adding Escherichia coli ( Escherichia sp. ), and the OE-b0840 strain after 168 h of desulfurization fermentation to study the effect of b0840 overexpression on the surface structure of TR. The results showed that the surface of TR without adding strains was relatively smooth and flat, and the structure was dense. After desulfurization fermentation by the WT strain, the dense structure on the surface of TR was damaged, and small holes appeared. In particular, compared with other desulfurization fermentations, after desulfurization fermentation by the OE-b0840 strain, the surface of TR became looser and more uneven, and the pores that appeared became larger. This may be due to the redox reaction generated by the enzymes secreted by the desulfurizing strains. Especially under the overexpression of b0840, the activity of the enzymes was further enhanced, thus more effectively degrading the sulfides in TR, resulting in changes in the surface structure, thereby exposing different proportions of the elements initially arranged on the surface.
[0068] Microbial activities on the rubber surface can lead to the formation, disappearance, or modification of certain functional groups, indicating changes in chemical groups in the product, such as unsaturated groups, branched chains, comonomers, and additives (antioxidants). As Figure 11 shown, FTIR spectroscopy was used to further determine the cleavage of the main chain and sulfur bridges of the TR polymer during the desulfurization process. The results showed that in the presence of TR, without adding strains, only adding Escherichia coli ( Escherichia sp. ), and the OE-b0840 strain after 168 h of desulfurization fermentation, there were significant differences in the FTIR spectra of TR: in the TR without adding strains, at 650 cm -1A peak was observed at [specific position], corresponding to the C=C bond, which is characteristic of the rubber backbone. After desulfurization fermentation with the WT strain, the DBT-induced WT strain, and the OE-b0840 strain, the peak intensity of the C=C bond gradually decreased, indicating that the surface structure was broken; after desulfurization fermentation with the WT strain, the DBT-induced WT strain, and the OE-b0840 strain, compared with the TR without added strain, the peak intensity of the C=O bond at 1401 cm -1 gradually increased, indicating that an oxidation reaction occurred in the strain; the C-S characteristic peak at 1641 cm -1 is a marker of the rubber cross-linking and vulcanization bridge, and its intensity also varied under different desulfurization fermentation conditions. The order of peak intensity was OE-b0840+TR < WT+TR+DBT < WT+TR, indicating that the OE-b0840 strain and the DBT-induced WT strain more effectively degraded the C-S bond in TR during fermentation, resulting in a decrease in the intensity of the C-S characteristic peak. This shows that the OE-b0840 strain and the DBT-induced WT strain have stronger desulfurization ability, especially the desulfurization effect of the OE-b0840 strain is the most significant; compared with the TR without added strain, under the desulfurization fermentation of the WT strain, the DBT-induced WT strain, and the OE-b0840 strain, the intensity of the characteristic peaks of the S=O bond and the O=S=O bond at 1074 cm -1 gradually increased, indicating that Escherichia coli ( Escherichia sp. ) can not only oxidatively cleave the C=C bond on the TR backbone, but also oxidatively cleave the C-S bond on the cross-linked thioether bridge to generate new carbonyl bonds C=O, sulfone bonds O=S=O, and sulfoxide bonds S=O. In summary, under OE-b0840 desulfurization fermentation, the strain is more intense in both chemical bond cleavage and new functional group formation, which also indicates that overexpression of b0840 can cause Escherichia coli ( Escherichia sp. ) to release more sulfur-containing groups from the surface of TR, significantly enhancing the desulfurization ability of TR.
Claims
1. A method for improving the desulfurization ability of desulfurizing strains on tire rubber, characterized in that: The culture solution after activation of the desulfurizing strain is inoculated into the fermentation medium, and the resulting stationary-phase fermentation broth is added with sterilized tire rubber powder and dibenzothiophene and / or diallyl sulfide, and then desulfurization fermentation is carried out to obtain the product.
2. The method for improving the desulfurization ability of strain tire rubber according to claim 1, wherein: The screening method for using dibenzothiophene and / or diallyl sulfide as an inducer to improve the desulfurization ability of the desulfurizing strain on tire rubber is as follows: The stationary-phase fermentation broth obtained by inoculating the culture solution after activation of the desulfurizing strain into the fermentation medium is placed in different conical flasks, all added with sterilized tire rubber powder, and different inducers are respectively added, and then desulfurization fermentation is carried out. The growth of the desulfurizing strain and the weight loss rate of the tire rubber after desulfurization fermentation are measured to screen out the effective inducer; the inducer includes dibenzothiophene and / or diallyl sulfide and one or more of dimethyl sulfoxide, Na2SO4 or Tween-20.
3. The method for improving the desulfurization ability of desulfurizing strains for tire rubber according to claim 1 or 2, characterized in that: The desulfurizing strain includes Escherichia coli ( Escherichia sp. ), named CSUFT-2024-Rubbrdesulfurization, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on October 21, 2024, with the deposit number CGMCC No. 32280; the sulfur content of the sterilized tire rubber powder is 2-4%, and the particle size is below 80 mesh; the dosage of the sterilized tire rubber powder is 4-6 g / 100 mL of the fermentation medium; the dosage of dibenzothiophene and / or diallyl sulfide, or other single inducer is 2-4 mmol / L of the fermentation medium; the temperature of the desulfurization fermentation is 28-32 °C, the rotation speed is 150-250 rpm, and the time is 140-300 h.
4. The method for improving the desulfurization ability of desulfurizing strains on tire rubber according to any one of claims 1 to 3, characterized in that: The fermentation method of the fermentation broth in the stationary phase is as follows: inoculate the desulfurizing strain into the strain activation medium for activation, and then inoculate the obtained activated culture solution into the fermentation medium for fermentation to the stationary phase to obtain the fermentation broth in the stationary phase; the inoculation amount of the desulfurizing strain is 0.08-0.12% (v / v); the activation temperature is 28-32 °C, the rotation speed is 150-250 rpm, and the activation time is 18-30 h; the OD 600 of the activated culture solution is 0.6-1.0; the inoculation amount of the activated culture solution is 8-12% (v / v); the fermentation temperature is 28-32 °C, the rotation speed is 150-250 rpm, and the time is 10-14 h; the OD 600 of the fermentation broth in the stationary phase after fermentation is 0.6-1.0; the formula of the strain activation medium is: in every 1000 mL of deionized water, it contains 8-12 g of polypeptone, 1.5-2.5 g of yeast powder, and 0.5-1.5 g of MgSO4·H2O; the formula of the fermentation medium is: in every 1000 mL of deionized water, it contains 3-5 g of KH2PO4, 3-5 g of K2HPO4·H2O, 0.6-1.0 g of MgSO4·7H2O, 0.2-0.6 g of NH4Cl, 8-12 g of Na2S2O3·5H2O, 0.005-0.015 g of CaCl2, 1.5-2.5 g of glucose, 0.8-1.2 g of peptone, and 0.08-0.12 g of yeast powder.
5. The method for improving the desulfurization ability of desulfurizing strains to tire rubber according to any one of claims 1 to 4, characterized in that: The treatment method for the sterilized tire rubber powder is as follows: First, the ground tire rubber powder is soaked in ethanol, then the soaked tire rubber powder is placed in a ventilated place to volatilize the ethanol, and finally high-pressure sterilization is carried out; the soaking time is 36-48 h; the volatilization time is 3-4 h; the temperature of the high-pressure sterilization is 115-125 °C, the pressure is 0.10-0.12 MPa, and the time is 0.8-1.2 h.
6. A method for improving the desulfurization ability of desulfurizing strains on tire rubber, characterized in that: Screen out the transcription factor b0840 that can effectively enhance the desulfurization ability of the desulfurizing strain on tire rubber from the desulfurizing strain after desulfurization fermentation as described in any one of claims 1-5. The culture solution after activation of the overexpressing desulfurizing strain of the transcription factor b0840 is inoculated into the fermentation medium, and the resulting stationary-phase fermentation broth is added with sterilized tire rubber powder, and then desulfurization fermentation is carried out to obtain the product.
7. The method for improving the desulfurization ability of desulfurizing strains for tire rubber according to claim 6, wherein: The screening method for the transcription factor b0840 includes the following steps: (1) Use RNA-seq to perform transcriptome analysis on the desulfurizing strain after desulfurization fermentation as described in claim 1, measure the expression levels of different genes in the desulfurizing strain, and screen out multiple transcription factors and 4S pathway genes that specifically respond to dibenzothiophene induction; (2) Respectively construct overexpressing desulfurizing strains of different transcription factors screened in step (1), inoculate them into the strain activation medium for activation, and then inoculate the resulting activated culture solution into the fermentation medium for fermentation to the stationary stage to obtain the stationary-phase fermentation broth; (3) Respectively add sterilized tire rubber powder to the stationary-phase fermentation broth obtained in step (2) for desulfurization fermentation, compare the effects of overexpressing desulfurizing strains of different transcription factors on the expression levels of 4S pathway genes, and at the same time, measure the growth of the overexpressing desulfurizing strains and the weight loss rate of the tire rubber after desulfurization fermentation to screen out the transcription factor that can effectively enhance the desulfurization ability of the desulfurizing strain on tire rubber.
8. The method for improving the desulfurization ability of desulfurizing strains for tire rubber according to claim 7, characterized in that: In step (3), the method for comparing the effects of transcription factors on the expression levels of 4S pathway genes is as follows: Extract the RNA of overexpressing desulfurizing strains of multiple different transcription factors and reverse transcribe it into cDNA. Using 16S rRNA as an internal reference gene, adopt qRT-PTR technology to compare the relative expression levels of different 4S pathway genes in overexpressing desulfurizing strains of different transcription factors.
9. The method for improving the desulfurization ability of desulfurizing strains on tire rubber according to any one of claims 6 to 8, characterized in that: The fermentation method of the fermentation broth in the stationary phase is the same as the fermentation method of the fermentation broth in the stationary phase described in claim 4; the treatment method of the sterilized tire rubber powder is the same as the treatment method of the sterilized tire rubber powder described in claim 5; the dosage of the sterilized tire rubber powder is 4-6 g / 100 mL of the fermentation medium; the temperature of the desulfurization fermentation is 28-32 °C, the rotation speed is 150-250 rpm, and the time is 140-300 h.