A method for improving polyvinyl alcohol degradation ability by altering the dispersibility of sphingosine bacteria

Abstract

This invention discloses a method for improving the polyvinyl alcohol (PVA) degradation ability of sphingosine bacteria by altering their dispersibility, comprising the following steps: Step 1, preparing LB liquid culture medium; Step 2, culturing sphingosine bacteria seed culture; Step 3, culturing sphingosine bacteria (with different glycosidases or proteases added simultaneously) and measuring and analyzing their PVA degradation ability and cell dispersibility. This invention improves the PVA degradation ability of sphingosine bacteria by adding different enzymes to alter the cell dispersibility during the PVA degradation process.

Description

Technical Field

[0001] This invention belongs to the field of environmental engineering, and specifically relates to a method for improving the polyvinyl alcohol degradation ability by changing the dispersibility of sphingosine bacteria. Background Technology

[0002] Polyvinyl alcohol (PVA) is a highly water-soluble macromolecular polymer with wide applications in industries such as textiles. However, the highly polymerized macromolecular PVA has poor biodegradability, making wastewater treatment difficult. Sphingosine monophosphate bacteria are important environmental microorganisms with a wide range of abilities to degrade organic pollutants and high-molecular-weight PVA.

[0003] Since PVA is a man-made polymer compound that has only been widely used in recent decades, while some microorganisms have evolved the ability to degrade PVA, the degradation efficiency is low. PVA-degrading enzymes are intracellular enzymes, meaning PVA needs to enter the microbial cell to be degraded. On the surface of microorganisms, there are often complex complexes of sugars and proteins that form capsules separating the cell from the external environment or cause cell aggregation to form cell communities. These conditions hinder the exchange of substances between microorganisms and the environment, thus affecting PVA degradation. Summary of the Invention

[0004] Purpose of the invention: To solve the above problems, the present invention provides a method for improving the polyvinyl alcohol (PVA) degradation ability by adding glycosidases or proteases to change the dispersibility of sphingosine monophosphate bacteria. Glycosidases or proteases can decompose the glycoprotein mixture on the surface of microorganisms, reduce surface viscosity, enhance the dispersion of microorganisms, strengthen mass transfer, and thus improve the degradation ability of PVA.

[0005] Technical solution: A method for improving the ability of sphingosine monophosphate bacteria to degrade polyvinyl alcohol, characterized by the following steps:

[0006] Step 1: Prepare LB liquid culture medium with the following formula: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, 121 o Sterilize at C for 15 min, and add 2% agar to the solid culture medium;

[0007] Step 2: Cultivation of Sphingosine Bacteria Seeds. The specific procedures are as follows: Inoculate one loopful of Sphingosine Bacteria into the LB liquid medium prepared in Step 1 and incubate at 37°C. o Incubate at 200 rpm in a constant temperature shaker for 48 hours;

[0008] Step 3: Determination and analysis of PVA degradation ability and cell dispersibility of Sphingosine monophosphate bacteria: The bacterial culture that had been cultured for 48 hours was inoculated into a PVA-containing medium at a 1% inoculation rate (PVA1799 was added to a final concentration of 1 g / L). Glycosidase and / or protease were then added. Samples were taken periodically and centrifuged at 10,000 rpm for 1 min. The PVA content in the sample was measured and the PVA degradation rate was calculated. Cell dispersibility was measured after culturing for 20 hours with the addition of glycosidase and / or protease.

[0009] In one embodiment of the present invention, in step three, the PVA culture medium consists of: PVA1799 1 g / L, NH4Cl 2 g / L, urea 1 g / L, K2HPO4 1.6 g / L, KH2PO4 0.2 g / L, FeSO4·7H2O 0.02 g / L, NaCl 0.02 g / L, CaCl2 0.1 g / L, MgSO4 0.024 g / L, and the pH of the PVA culture medium is 7.5.

[0010] In one embodiment of the present invention, in step three, the glycosidase and / or protease includes any one or a mixture of the following: neutral protease, alkaline protease, alpha-glucosidase, and beta-glucanase.

[0011] In one embodiment of the present invention, the amount of glycosidase and / or protease added is 10-20 U / mL.

[0012] In one embodiment of this invention, the specific method for determining the PVA content in step three is as follows:

[0013] a. The PVA-containing sample in the shake flask was centrifuged at 10,000 rpm for 1 min to remove bacterial cells, and the supernatant was used to determine the PVA content;

[0014] b. Add 1 mL of the sample to be tested, 7.5 mL of 25 g / L boric acid, and 0.75 mL of I2-KI to a 25 mL colorimetric tube, and bring the volume to 25 mL. Let the tube stand for 10 min away from light, and measure the absorbance at 690 nm. Calculate the PVA content based on the standard curve. The PVA degradation rate β (%) is calculated using the following formula:

[0015] β(%)=[(α0-α1)] / α0×100%;

[0016] Where α0 represents the PVA content before the reaction, and α1 represents the PVA content after the reaction.

[0017] In one embodiment of the present invention, the I2-KI solution is prepared as follows: 10 g / L I2, 80 g / L KI.

[0018] In one embodiment of this invention, the specific method for determining the bacterial cell dispersion in step three is as follows: take a loop of bacterial culture medium, gently apply it to the surface of a glass slide, allow it to dry naturally, fix it, stain it with crystal violet, decolorize it, and then observe it under a 100x oil immersion microscope.

[0019] In one embodiment of the present invention, the protease and / or glycosidase can hydrolyze the polysaccharide and protein complex on the surface of sphingosine bacteria, improve cell dispersibility, and thereby enhance the degradation ability of sphingosine bacteria for PVA.

[0020] Beneficial effects: This invention effectively alters the dispersibility of sphingosine bacteria by adding glycosidase or protease during the degradation of PVA by sphingosine bacteria, thereby improving the ability of sphingosine bacteria to degrade polyvinyl alcohol. Attached Figure Description

[0021] Figure 1 This is a schematic diagram showing the relationship between the content of degraded PVA and time obtained by adding different types of enzymes in this invention;

[0022] Figure 2 This invention relates to the microscopic morphological observation of sphingosine bacteria obtained after culturing with different types of enzymes for 20 hours. Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Example

[0024] A method for improving the polyvinyl alcohol degradation ability by altering the dispersibility of sphingosine monophosphate bacteria includes the following steps:

[0025] Step 1: The LB liquid medium is prepared as follows: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, 121 o Sterilize at C for 15 min, and add 2% agar to the solid culture medium.

[0026] Step 2: Cultivation of Sphingosine Bacteria Seeds. The specific procedures are as follows: Inoculate one loopful of Sphingosine Bacteria (from the China Center for Type Culture Collection, strain preservation number CCTCC NO: M 2016302) into LB liquid medium and incubate at 37°C. o Incubate at 200 rpm in a constant temperature shaker at C for 48 hours.

[0027] Step 3: Determination and analysis of PVA degradation ability and cell dispersibility of Sphingosine monophosphate bacteria: The bacterial culture that had been cultured for 48 hours was inoculated into a PVA-containing medium (PVA medium composition includes PVA1799 1 g / L, NH4Cl 2 g / L, urea 1 g / L, K2HPO4 1.6 g / L, KH2PO4 0.2 g / L, FeSO4·7H2O 0.02 g / L, NaCl 0.02 g / L, CaCl2 0.1 g / L, MgSO4 0.024 g / L, PVA medium pH is 7.5) at a 1% inoculation rate. Different enzymes were added at the same time. Samples were taken at regular intervals and centrifuged at 10,000 rpm for 1 min. The supernatant was taken to determine the PVA content in the sample and calculate the PVA degradation rate. After culturing for 20 hours with the added enzymes, the cell dispersibility of the bacterial culture was determined.

[0028] The method for determining PVA content is as follows:

[0029] a. The PVA-containing sample in the shake flask was centrifuged at 10,000 rpm for 1 min to remove bacterial cells, and the supernatant was used to determine the PVA content;

[0030] b. Add 1 mL of the sample to be tested, 7.5 mL of 25 g / L boric acid, and 0.75 mL of (10 g / L I2, 80 g / L KI)I2-KI to a 25 mL colorimetric tube, and bring the volume to 25 mL. Let the tube stand for 10 min away from light, and measure the absorbance at 690 nm (the control group should be treated the same way). Calculate the PVA content based on the standard curve. The PVA degradation rate beta (%) is calculated using the following formula:

[0031] beta(%)=[(alpha0-alpha1)] / alpha0×100%;

[0032] Wherein, alpha0 represents the PVA content before the reaction, and alpha1 represents the PVA content after the reaction.

[0033] The specific steps for observing the dispersion of sphingosine bacteria using an optical microscope are as follows:

[0034] Take one loop of bacterial culture solution after 20 hours of incubation, gently spread it on the surface of a glass slide, allow it to dry naturally, fix it, stain it with crystal violet, decolorize it, and observe it under a 100x oil immersion microscope.

[0035] Specific Example 1: Degradation of PVA by Sphingosine Bacteria without the addition of any enzymes (Control Example)

[0036] A cyclosphingosine seed was inoculated from LB solid medium into LB liquid medium and placed at 37°C. oThe culture was carried out in a constant temperature shaker at 200 rpm for 48 hours. Then, the fermentation broth was taken and inoculated into a PVA-containing medium at a 1% inoculum. The PVA content was measured at regular intervals, and the cell dispersibility was observed after 20 hours of culture.

[0037] Specific Example 2: Degradation of PVA by Sphingosine Bacteria after Adding Neutral Protease

[0038] A cyclosphingosine seed was inoculated from LB solid medium into LB liquid medium and placed at 37°C. o The culture was carried out in a constant temperature shaker at 200 rpm for 48 hours. Then, the fermentation broth was taken and inoculated into a PVA-containing medium at a 1% inoculum size, and 15 U / mL of neutral protease (produced by Xiasheng Enzyme Biotechnology Co., Ltd.) was added. The PVA content was measured at regular intervals, and the cell dispersibility was observed after 20 hours of culture.

[0039] Specific Example 3: Degradation of PVA by Sphingosine Bacteria after Adding Alkaline Protease

[0040] A cyclosphingosine seed was inoculated from LB solid medium into LB liquid medium and placed at 37°C. o The culture was carried out in a constant temperature shaker at 200 rpm for 48 hours. Subsequently, the fermentation broth was taken and inoculated into a PVA-containing medium at a 1% inoculum size, and 15 U / mL of alkaline protease (produced by Xiasheng Enzyme Biotechnology Co., Ltd.) was added. The PVA content was measured at regular intervals, and the cell dispersibility was observed after 20 hours of culture.

[0041] Specific Example 4: Degradation of PVA by Sphingosine Bacteria after Addition of Alpha-Glucosidase

[0042] A cyclosphingosine seed was inoculated from LB solid medium into LB liquid medium and placed at 37°C. o The culture was carried out in a constant temperature shaker at 200 rpm for 48 hours. Then, the fermentation broth was taken and inoculated into a PVA-containing medium at a 1% inoculum size, and 15 U / mL of alpha-glucosidase (produced by Xiasheng Enzyme Biotechnology Co., Ltd.) was added. The PVA content was measured at regular intervals, and the cell dispersibility was observed after 20 hours of culture.

[0043] Specific Example 5: Degradation of PVA by Sphingosine Bacteria after Adding Beta-glucanase

[0044] A cyclosphingosine seed was inoculated from LB solid medium into LB liquid medium and placed at 37°C. o The culture was carried out in a constant temperature shaker at 200 rpm for 48 hours. Subsequently, the fermentation broth was taken and inoculated into a PVA-containing medium at a 1% inoculum size, and 15 U / mL of beta-glucanase (produced by Xiasheng Enzyme Biotechnology Co., Ltd.) was added. The PVA content was measured at regular intervals, and the cell dispersibility was observed after 20 hours of culture.

[0045] Specific Example 6: Degradation of PVA by Sphingosine Bacteria after Adding Neutral Protease + Alpha-Glucosidase

[0046] A cyclosphingosine seed was inoculated from LB solid medium into LB liquid medium and placed at 37°C. o The culture was carried out in a constant temperature shaker at 200 rpm for 48 hours. Subsequently, the fermentation broth was taken and inoculated into a PVA-containing medium at a 1% inoculum size. 15 U / mL of neutral protease and 15 U / mL of alpha-glucosidase (produced by Xiasheng Enzyme Biotechnology Co., Ltd.) were added. The PVA content was measured at regular intervals, and the cell dispersibility was observed after 20 hours of culture.

[0047] from Figure 1 As can be seen from Examples 1-6, different types of proteases or glycosidases were added. The relationship between the amount of PVA degraded and time leads to the following conclusions: alkaline proteases are not conducive to the degradation of PVA by sphingosine bacteria; neutral proteases, alpha-glucosidase, and beta-glucanase are all conducive to the degradation of PVA by sphingosine bacteria, among which the combined use of neutral proteases and alpha-glucosidase has the best effect.

[0048] from Figure 2 As can be seen from Examples 1-6, when different types of proteases or glycosidases were added, the bacterial cells in the control group showed obvious aggregation. After adding alkaline protease, the aggregation degree increased. The aggregation in the beta-glucan group was slightly weakened. After adding neutral protease and alpha-glucosidase, the aggregation was significantly improved and the dispersion of microorganisms was significantly enhanced.

[0049] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the equivalent meaning and scope of the claims be included within the protection scope of the present invention.

Claims

1. A method for improving the degradation ability of polyvinyl alcohol (PVA) by altering the dispersibility of sphingosine monophosphate bacteria, characterized in that: Includes the following steps: Step 1: Prepare LB liquid culture medium with the following formula: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, 121 o Sterilize at C for 15 min, and add 2% agar to the solid culture medium; Step 2: Cultivation of Sphingosine Bacteria Seeds. The specific procedures are as follows: Inoculate one loopful of Sphingosine Bacteria into the LB liquid medium prepared in Step 1 and incubate at 37°C. o Incubate at 200 rpm in a constant temperature shaker for 48 hours; Step 3: Determination and analysis of PVA degradation ability and cell dispersibility of Sphingosine monophosphate: The bacterial culture that had been cultured for 48 hours was inoculated into a PVA-containing medium at a 1% inoculation rate. Then, 15 U / mL of neutral protease and 15 U / mL of alpha-glucosidase were added. Samples were taken at regular intervals and centrifuged at 10,000 rpm for 1 min. The supernatant was collected to determine the PVA content in the sample and analyze the PVA degradation rate. The cell dispersibility was determined after culturing for 20 hours with the addition of neutral protease and alpha-glucosidase. The bacterial culture was collected for analysis.

2. The method for improving polyvinyl alcohol degradation ability by altering the dispersibility of sphingosine monophosphate bacteria according to claim 1, characterized in that: In step three, the PVA culture medium consists of: PVA1799 1 g / L, NH4Cl 2 g / L, urea 1 g / L, K2HPO4 1.6 g / L, KH2PO4 0.2 g / L, FeSO4·7H2O 0.02 g / L, NaCl 0.02 g / L, CaCl2 0.1 g / L, MgSO4 0.024 g / L, and the pH of the PVA culture medium is 7.

5.

3. The method for improving polyvinyl alcohol degradation ability by altering the dispersibility of sphingosine bacteria according to claim 1, characterized in that: In step three, the specific method for determining the PVA content is as follows: a. The PVA-containing sample in the shake flask was centrifuged at 10,000 rpm for 1 min to remove bacterial cells, and the supernatant was used to determine the PVA content; b. Add 1 mL of the sample to be tested, 7.5 mL of 25 g / L boric acid, and 0.75 mL of I2-KI to a 25 mL colorimetric tube, and bring the volume to 25 mL. Let the tube stand for 10 min away from light, and measure the absorbance at 690 nm. Calculate the PVA content based on the standard curve. The PVA degradation rate β (%) is calculated using the following formula: β（%）=[（α0-α1）] / α0×100%； Where α0 represents the PVA content before the reaction, and α1 represents the PVA content after the reaction.

4. The method for improving polyvinyl alcohol degradation ability by altering the dispersibility of sphingosine bacteria according to claim 3, characterized in that: The I2-KI solution was prepared as follows: 10 g / L I2, 80 g / L KI.

5. The method for improving polyvinyl alcohol degradation ability by altering the dispersibility of sphingosine bacteria according to claim 1, characterized in that: The specific method for determining the bacterial cell dispersion in step three is as follows: Take one loop of bacterial culture medium, gently apply it to the surface of a glass slide, allow it to dry naturally, fix it, stain it with crystal violet, decolorize it, and observe it under a 100x oil immersion microscope.

6. The method for improving polyvinyl alcohol degradation ability by altering the dispersibility of sphingosine monophosphate bacteria according to any one of claims 1-5, characterized in that: The neutral protease and alpha-glucosidase can reduce the aggregation of the strain, thereby increasing the efficiency of PVA crossing the cell membrane into the cell, accelerating decomposition, and improving the degradation ability of PVA by sphingosine bacteria.

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