Pseudomonas and methods for degrading alkane organic pollutants

Abstract

This invention relates to the field of organic pollutant degradation, and discloses a strain of Pseudomonas and a method for degrading alkane organic pollutants. The Pseudomonas strain has the accession number CCTCC NO: M 20241106. This Pseudomonas strain can effectively degrade alkane organic pollutants, especially C6-C8 alkanes. The strain can survive stably over a wide pH and temperature range, and efficiently degrades alkane while using n-heptane, 2-methylheptane, and 3-methylpentane as carbon and energy sources for growth and reproduction.

Description

Technical Field

[0001] This invention relates to the field of organic pollutant degradation, specifically to a strain of Pseudomonas and a method for degrading alkane organic pollutants. Background Technology

[0002] Alkane organic compounds are typical volatile organic compounds (VOCs) in the petrochemical industry. They mainly originate from petroleum and natural gas and are important chemical raw materials and energy resources.

[0003] Alkanes are primarily derived from petroleum and natural gas, serving as important chemical raw materials and energy resources. Common alkanes include propane, n-hexane, and n-heptane. Due to the exceptional stability of the CH bonds in alkanes, they are among the most difficult VOCs to degrade catalyzed. Taking n-heptane as an example, it is commonly used as a solvent and a raw material in the production of coatings, plastics, and other polymer materials. It is also released into the environment with industrial waste. Acute inhalation of n-heptane vapors can cause dizziness, nausea, and loss of appetite; in severe cases, it can even lead to loss of consciousness and stupor. It has mild skin irritation; long-term exposure to n-heptane may cause neurasthenia syndrome; it is toxic to aquatic organisms and may have long-term, persistent environmental impacts. Therefore, research on the degradation of n-heptane and other alkane organic pollutants is of significant practical importance.

[0004] Currently, the main methods for treating alkane organic pollutants such as heptane include adsorption, catalytic oxidation, and biodegradation. Examples include photocatalytic ozone oxidation using Cu₂O-CuO / TiO₂, adsorption using zeolite / organometallic frameworks, and biodegradation using activated sludge. Among these, biodegradation is the cleanest method; however, its effectiveness in treating alkane organic pollutants still needs improvement. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem of poor biodegradation efficiency of alkane organic pollutants in existing technologies, and to provide a method for Pseudomonas bacteria to degrade alkane organic pollutants.

[0006] To achieve the above objectives, the present invention provides a strain of Pseudomonas toyotomiensis 414, the preservation number of which is CCTCC NO: M 20241106.

[0007] A second aspect of the present invention provides a microbial agent comprising the aforementioned Pseudomonas bacteria.

[0008] A third aspect of the present invention provides the use of the aforementioned Pseudomonas bacteria or bacterial agents in the degradation of alkanes.

[0009] A fourth aspect of the present invention provides a method for degrading alkane-based organic pollutants, the method comprising: mixing the aforementioned Pseudomonas bacteria and / or bacterial agents with alkane-containing materials, and then carrying out a degradation reaction, wherein the alkane is a C5-C16 alkane.

[0010] The beneficial effects of the present invention through the above technical solution include at least the following:

[0011] The Pseudomonas provided by this invention can survive in a wide range of temperatures (20-40℃) and pH levels (5-9), efficiently degrade alkanes (such as n-heptane, 2-methylheptane and 3-methylpentane), and use them as carbon and energy sources for growth and reproduction.

[0012] Biological Preservation

[0013] The strain provided in this invention is classified as *Pseudomonas toyotomiensis* 414, and was deposited on May 30, 2024, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCCNO: M 20241106, located at Wuhan University, Wuhan, China. In this invention, this strain is referred to as *Pseudomonas toyotomiensis* 414. Attached Figure Description

[0014] Figure 1 This is the phylogenetic tree of Pseudomonas 414 provided by the present invention;

[0015] Figure 2 This is a graph showing the cell growth and n-heptane degradation of Pseudomonas 414 provided by the present invention.

[0016] Figure 3(A) shows the n-heptane degradation rate of Pseudomonas 414 in culture media with different pH values;

[0017] Figure 3(B) shows the effect of different pH values ​​on the mineralization of Pseudomonas 414;

[0018] Figure 4(A) shows the n-heptane degradation rate of Pseudomonas 414 at different temperatures;

[0019] Figure 4(B) shows the effect of different temperatures on the growth of Pseudomonas 414;

[0020] Figure 4(C) shows the effect of different temperatures on the mineralization of Pseudomonas 414;

[0021] Figure 5 The degradation curves of Pseudomonas 414 against different initial concentrations of n-heptane provided by this invention are shown.

[0022] Figure 6 This is the growth curve of Pseudomonas 414 provided by the present invention under different initial n-heptane concentrations. Detailed Implementation

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] In this invention, unless otherwise specified, "degradation capacity" refers to the rate of degradation per 10 units of time. 6 The maximum weight of alkanes that a live CFU strain can degrade; "alkane degradation rate" refers to 10 per unit time. 6 The weight of alkanes degraded by CFU live bacteria strains is understandable; the degradation rate of alkanes is usually less than or equal to the degradation capacity of the strain.

[0025] During the decomposition of alkanes, the strains also use alkanes as a carbon source and energy source to reproduce, meaning that the number of viable bacteria will increase as the decomposition process proceeds. Therefore, unless otherwise stated, the amount of strains used, the number of viable bacteria, the amount of viable bacteria inoculated, and other expressions related to the amount of strains mentioned in this invention are all assumed to be the "initial amount" of strains or inoculants used when culturing or decomposing alkanes.

[0026] The first aspect of the present invention provides a strain of Pseudomonas toyotomiensis 414, the preservation number of which is CCTCC NO: M 20241106.

[0027] The inventors of this invention accidentally isolated a bacterial strain from activated sludge in a petrochemical plant in Ningbo, Zhejiang Province. Research revealed that this strain possesses a good ability to degrade alkanes. This strain is an aerobic, Gram-negative bacterium. Its colonies are small, white, opaque, plump, smooth, and moist, easily picked up, and grow along streaks. 16S rDNA sequencing identified it as a Pseudomonas bacterium. The phylogenetic tree of this strain is shown below. Figure 1 As shown, its 16S rDNA is as shown in SEQ ID NO.1.

[0028] SEQ ID NO.1:

[0029]

[0030] The *Pseudomonas* strain provided by this invention can produce a large number of live *Pseudomonas* cells after cultivation. The cultivation method is not particularly demanding, as long as it enables the *Pseudomonas* to proliferate. For example, live *Pseudomonas* cells can be inoculated into a liquid culture medium at an inoculum of 1-5 vol%, and cultured at 30-40°C for 9-24 hours to obtain a culture solution. The culture medium can be any culture medium known in the art suitable for culturing *Pseudomonas*, such as R2A medium.

[0031] This invention can further isolate live Pseudomonas cells from the above-mentioned culture medium. The method of separation is not particularly limited, as long as it can enrich the cells from the culture medium. For example, it can be achieved by centrifugation and / or filtration. The conditions for centrifugation and filtration can be known conditions, which will not be elaborated here.

[0032] A second aspect of the present invention provides a microbial agent comprising the aforementioned Pseudomonas bacteria.

[0033] In this invention, the form of the microbial agent can be a conventional form of microbial agent in the art, such as a solid, liquid or semi-solid form.

[0034] In some preferred embodiments of the present invention, the bacterial agent contains live cells of the Pseudomonas bacteria.

[0035] The number of live bacteria in the bacterial agent can be selected within a wide range, as long as it meets the requirements of relevant standards; for example, it can be 10. 9 CFU / g of bacterial agent or higher.

[0036] The preparation method of the bacterial agent can refer to the conventional preparation methods in this field, and will not be described in detail here.

[0037] A third aspect of the present invention provides the use of the aforementioned Pseudomonas bacteria or bacterial agents in the degradation of alkanes.

[0038] A fourth aspect of the present invention provides a method for degrading alkane-based organic pollutants, the method comprising: mixing the aforementioned Pseudomonas bacteria or bacterial agent with alkane-containing materials, and then carrying out a degradation reaction.

[0039] In this invention, the alkane can be a branched alkane or a straight-chain alkane, preferably a C5-C16 alkane, more preferably a C6-C8 alkane. In the most preferred embodiment of this invention, the C7 alkane is a straight-chain n-heptane. In another preferred embodiment of this invention, the C7 alkane is octane (2-methyl-heptane).

[0040] In this invention, the method for degrading alkanes further includes: activating and expanding the strain in a culture medium before inoculating the strain into a system containing alkane.

[0041] In this invention, the culture medium is any one of R2A medium, LB medium and PDB medium, preferably R2A medium.

[0042] In this invention, the activation and amplification conditions include: a temperature of 20-35℃, preferably 28-32℃; and a time of 1-60h, preferably 45-50h.

[0043] According to the present invention, the activated culture medium is R2A solid slant culture medium.

[0044] According to the present invention, the culture medium for expansion is R2A liquid culture medium.

[0045] According to the present invention, during expansion culture, the inoculum size of *Pseudomonas* in the culture medium can be selected within a wide range, as long as it meets the requirements of relevant standards. Preferably, the inoculum size of *Pseudomonas* in the culture medium results in an OD value of [missing value] for the inoculated solution. 600 It is 0.01-0.02.

[0046] According to some preferred embodiments of the present invention, the activation and expansion methods are as follows: Activation: Pseudomonas 414 is inoculated onto R2A solid slant culture medium and cultured at 28-32℃ for 30-40 hours to obtain slant cells. Expansion: The slant cells are picked up with an inoculation loop and inoculated into R2A liquid culture medium, the amount of cells used being such that the viable count in the culture medium is 10-1. 5 -10 6 CFU / mL medium, incubated at 28-32℃ for 45-50 h to obtain OD 600 =0.05-0.2% bacterial solution.

[0047] According to the present invention, the R2A solid culture medium comprises: yeast extract (0.4-0.5 g / L), soluble starch (0.4-0.5 g / L), MgSO4 (0.1-0.5 g / L), tryptone (0.4-0.5 g / L), glucose (0.4-0.5 g / L), sodium pyruvate (0.2-0.4 g / L), K2HPO4 (0.1-0.5 g / L), and agar (17-19 g / L). The solvent can be deionized water. According to the angle of the culture medium surface relative to the horizontal ground, it can be divided into planar culture medium and slant culture medium. The liquid culture medium, compared with the solid culture medium, does not contain agar.

[0048] Inorganic salt culture medium: pH = 6-8, Na2HPO4 (1-5 g / L), KH2PO4 (0.5-1.5 g / L), (NH4)2SO4 (2-3 g / L), MgSO4 (0.01-0.3 g / L), CaCl2 (0.02-0.03 g / L), FeSO4 (0.1-1.5 mg / L), CuSO4 (0.005-0.03 mg / L), H3BO3 (0.01-0.02 mg / L), MnSO4 (0.01-0.15 mg / L), ZnSO4 (0.01-0.15 mg / L), Na2MoO4 (0.001-0.03 mg / L), CoCl2 (0.05-0.03 mg / L), solvent can be deionized water.

[0049] In this invention, the amount of *Pseudomonas* or the bacterial agent used relative to 1 mg of alkane results in a total viable count of 10-1 *Pseudomonas*. 2 -10 6 CFU, preferably 5×10 3 -5×10 4 CFU.

[0050] In this invention, the degradation temperature is 15-45℃, preferably 20-40℃, and more preferably 26-39℃.

[0051] In this invention, the degradation time is 1-60 hours, preferably 8-30 hours.

[0052] According to the present invention, the degradation time is not the time required to completely degrade the alkane, but the time required to degrade half of the initial amount of alkane, i.e., the half-life of alkane degradation.

[0053] According to the present invention, the degradation time and the amount of strain can be flexibly adjusted according to the amount of alkanes to be degraded and actual needs. According to some preferred embodiments of the present invention, when the amount of n-heptane is 65-350 mg / L, within the half-life of n-heptane decomposition (approximately 9-15 h), the degradation rate of alkanes continuously increases with the increase of the amount of alkanes. That is, the strain of the present invention has a strong ability to degrade alkanes, and the degradation rate of alkanes is always less than or equal to the degradation capacity of the strain. Therefore, it can be reasonably inferred that within the total degradation time (48 h) of the present invention, every 10... 6 The strain provided by this invention can degrade alkanes in amounts exceeding 342 mg, for example, 350 mg, 400 mg, 450 mg, 500 mg, 1000 mg, 2000 mg, or any value within a range formed by any two of these values. Therefore, any use of the strain provided by this invention to degrade alkanes, regardless of the degradation rate, falls within the protection scope of this invention.

[0054] In this invention, the pH of the degradation is 4-10. In order to allow Pseudomonas 414 to multiply more rapidly and further improve the degradation rate of alkane, the pH is preferably 5-9, and more preferably 6-8.

[0055] According to a preferred embodiment of the present invention, at pH = 6.5-7.5, the Pseudomonas 414 is ultimately able to degrade 90-99% of n-heptane. According to other preferred embodiments of the present invention, under alkaline conditions (pH = 7.5-9.5), the ability of Pseudomonas 414 to degrade alkanes is stronger than that under acidic conditions. Therefore, the Pseudomonas 414 provided by the present invention can degrade alkanes not only in a neutral environment, but also under alkaline and acidic conditions, and the degradation ability is ranked as follows: neutral > alkaline > acidic.

[0056] According to the present invention, in order to promote the degradation of n-heptane, the degradation reaction can be carried out in a stirring apparatus, such as a shaker, preferably at a shaker speed of 140-180 r / min.

[0057] According to a preferred embodiment of the present invention, the method for degrading n-heptane is as follows: after activating and expanding Pseudomonas 414, it is inoculated into a culture medium containing n-heptane, wherein the inoculation amount of the strain is 10 mg / mg n-heptane. 3 -10 4 CFU is then subjected to degradation reaction in an environment with a temperature of 28-32℃ and a pH of 6.5-7.5.

[0058] The present invention will be described in detail below through embodiments.

[0059] The contents of n-heptane, 2-methylheptane, 3-methylpentane, and carbon dioxide were determined using an Agilent gas chromatograph.

[0060] R2A solid slant culture medium composition: yeast extract 0.5 g / L, soluble starch 0.5 g / L, MgSO4·7H2O 0.50 g / L, tryptone 0.5 g / L, glucose 0.5 g / L, sodium pyruvate 0.3 g / L, K2HPO4·3H2O 0.45 g / L, agar 18 g / L, solvent is deionized water; liquid culture medium, compared to solid culture medium, does not contain agar.

[0061] Inorganic salt culture medium: pH=7, Na2HPO4·12H2O (4.5g / L), KH2PO4 (1g / L), (NH4)2SO4 (2.5g / L), MgSO4·7H2O (0.2g / L), CaCl2 (0.023g / L), FeSO4·7H2O (1mg / L), CuSO4·5H2O (0.02mg / L), anhydrous H3BO3 (0.014mg / L), MnSO4·4H2O (0.1mg / L), ZnSO4·7H2O (0.1mg / L), Na2MoO4·2H2O (0.02mg / L), CoCl2·6H2O (0.02mg / L), solvent is deionized water.

[0062] The 16S rDNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd.

[0063] Example 1

[0064] First, the strains are inoculated sequentially into the culture medium for activation and expansion.

[0065] Slant activation culture: Pseudomonas 414 was inoculated into R2A solid slant medium and cultured at 30℃ for 36 h to obtain slant cells.

[0066] Expanded culture: Pick up the slant cells with an inoculation loop and inoculate them into R2A liquid medium. Incubate at 30°C for 48 hours to obtain a bacterial suspension with a viable cell concentration of OD600 = 0.1.

[0067] The bacterial culture was inoculated into 50 mL of fresh inorganic salt medium (pH 7) containing 137 mg / L n-heptane. The inoculation volume of the bacterial culture was such that the OD... 600 =0.01. Three parallel samples and one blank control group without bacterial inoculation were designed. The samples were cultured in a shaker at 30℃ and 160 rpm. Every 5-15 hours, 5 mL of bacterial solution was drawn using a syringe to measure the bacterial OD value and n-heptane concentration. The results are as follows: Figure 2 As shown (the “heptane” in the figure is “n-heptane”).

[0068] from Figure 2 It can be seen that within 0-30 hours, the concentration of n-heptane gradually decreased, with a half-life of approximately 9 hours, while the cell concentration gradually increased. At 20 hours, the OD... 600 The absorbance reached 0.15. This result indicates that Pseudomonas 414 can utilize n-heptane as a carbon and energy source for growth and reproduction, and has a stable and efficient ability to degrade n-heptane.

[0069] Example 2

[0070] The inorganic salt culture medium was adjusted to different pH values ​​(5, 6, 7, 8, 9) using 1 mol / L NaOH aqueous solution or 1 mol / L H2SO4 aqueous solution; the bacterial suspension prepared in Example 1 was inoculated into 50 mL of fresh inorganic salt culture medium containing 68 mg / L n-heptane, and the inoculation amount of the bacterial suspension was such that the OD 600 =0.01. Three parallel samples and one blank control group without bacterial strain were designed for each pH group. The samples were cultured in a constant temperature shaker at 30℃ and 160r / min for 20h. After culturing, samples were taken and the degradation rate of n-heptane and CO2 concentration in the reaction solution were measured. The results are shown in Figure 3(A) and Figure 3(B) ("heptane" in the figure is "n-heptane").

[0071] As shown in Figure 3(A), strain 414 was able to degrade n-heptane within a pH range of 5-9, indicating that this strain has good acid and alkali tolerance. With increasing pH, the degradation rate of n-heptane initially increased and then decreased, with the optimal pH for degradation being 7. Furthermore, it was found that strain 414's ability to degrade n-heptane under alkaline conditions was stronger than its ability under acidic conditions. As shown in Figure 3(B), the CO2 value also peaked at pH 7, similar to the trend of degradation efficiency.

[0072] Example 3

[0073] The bacterial culture prepared in Example 1 was inoculated into 50 mL of fresh inorganic salt medium containing 68 mg / L n-heptane (pH = 7), with the inoculation amount ensuring an OD600 of 0.01. Each culture medium was incubated at 20°C, 25°C, 30°C, 35°C, and 40°C using a shaker at 160 rpm. Three parallel samples and one blank control group without inoculation were designed for each temperature. After 20 h of incubation, samples were taken, and the n-heptane degradation rate, OD600 value, and CO2 content in the reaction solution were measured. The results are shown in Figures 4(A), 4(B), and 4(C) (in the figures, "heptane" refers to "n-heptane").

[0074] As shown in Figure 4(A), within the temperature range of 20-40℃, *Pseudomonas* 414 can grow and reproduce using n-heptane as both a carbon and energy source, and degrade n-heptane; within the temperature range of 25-35℃, the degradation rate of n-heptane by *Pseudomonas* 414 reaches over 99%. Figure 4(B) shows that the growth rate of *Pseudomonas* 414 is consistent with the trend of n-heptane degradation rate. Figure 4(C) shows that the growth rate of *Pseudomonas* 414 and the CO2 produced also show a consistent trend with the n-heptane degradation rate.

[0075] Example 4

[0076] Heptane was added to several fresh inorganic salt culture media at pH 7, resulting in initial concentrations of heptane of 68, 137, 205, 274, and 342 mg / L, respectively. The bacterial suspension prepared in Example 1 was then inoculated into the culture media, with the inoculation amount resulting in an OD... 600 =0.01. Three parallel samples and one blank control group without inoculation were designed. The samples were cultured in a shaker at 30℃ and 160 rpm. Samples were taken every 5-15 minutes to determine the concentration of n-heptane and the OD of Pseudomonas 414. 600 The results are shown below. Figure 5 and Figure 6 (The “heptane” in the diagram is “n-heptane”).

[0077] from Figure 5 and Figure 6 It can be seen that when the concentration of n-heptane is 68-342 mg / L, Pseudomonas 414 can multiply rapidly within 20 hours, and the degradation rate of n-heptane reaches more than 80% within 30 hours. Among them, n-heptane with a concentration of 68-274 mg / L is almost completely degraded.

[0078] In addition, analysis Figure 5 The data show that when the concentration of n-heptane is 68, 137, 205, 274 and 342 mg / L, its half-life is approximately 9.5, 9, 13, 15 and 15 h, respectively. It can be seen that the degradation rate of alkanes by the strain continuously increases within the half-life.

[0079] Example 5

[0080] 2-Methylheptane was added to a fresh inorganic salt culture medium at pH 7 to achieve an initial concentration of 69.8 mg / L. The bacterial suspension prepared in Example 1 was then inoculated, with the inoculation amount resulting in an OD of [missing value]. 600 =0.01. The culture was carried out under isothermal shaking at 30℃ and 160 r / min, and the OD was measured after 18 h of culture. 600 The value was 0.058, and the degradation rate of 2-methylheptane was 74%, indicating that the strain of the present invention can also use 2-methylheptane as a carbon source and energy source for growth and reproduction, and degrade 2-methylheptane.

[0081] Example 6

[0082] 3-Methylpentane was added to a fresh inorganic salt culture medium at pH 7 to achieve an initial concentration of 68 mg / L. The bacterial suspension prepared in Example 1 was then inoculated, with the inoculation amount resulting in an OD of [missing value]. 600 =0.01. The culture was carried out under isothermal shaking at 30℃ and 160 r / min, and the OD was measured after 18 h of culture. 600The concentration was 0.042 and the degradation rate of 3-methylpentane was 68%, indicating that the strain of the present invention can also use 3-methylpentane as a carbon source and energy source for growth and reproduction, and degrade 3-methylpentane.

[0083] Comparative Example 1

[0084] The method of Example 5 was used to degrade n-heptane, except that the strains used were Mycobacterium SLQ-1 (CCTCC NO: M 2024107, disclosed in CN202410563266.0), Oligotrophozoites HY-2 (CCTCC NO: M 2018714, disclosed in ZL201811570344.0), and Mycobacterium lysate (CCTCC NO: M2023883, disclosed in CN117384787A). After 9 hours of degradation, the degradation rate and OD of n-heptane were measured. 600 The values ​​are shown in Table 1.

[0085] Table 1

[0086] strain number Degradation rate of n-heptane, % <![CDATA[OD 600 Value CCTCC NO: M 2024107 34.5 0.058 CCTCC NO: M 2018714 29.1 0.044 CCTCC NO: M 2023883 14.6 0.023

[0087] As shown in Table 1, compared with other strains, the strains provided by this invention have a stronger ability to degrade alkanes (such as n-heptane).

[0088] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas toyotomiensis 414, characterized in that, The preservation number of the Pseudomonas is CCTCC NO: M 20241106.

2. A microbial agent, characterized in that, The bacterial agent comprises the Pseudomonas as described in claim 1.

3. The application of the Pseudomonas bacillus of claim 1 or the bacterial agent of claim 2 in the degradation of alkanes.

4. A method for degrading alkane-based organic pollutants, characterized in that, The method includes: mixing the Pseudomonas aeruginosa of claim 1 or the inoculum of claim 2 with an alkane-containing material, and then carrying out a degradation reaction.

5. The method according to claim 4, wherein, The alkane is a C5-C16 alkane, preferably a C6-C8 alkane.

6. The method according to claim 4, wherein, The method further includes: activating and expanding the strain in a culture medium before inoculating the strain into a system containing alkane.

7. The method according to claim 6, wherein, The culture medium is any one of R2A medium, LB medium and PDB medium, preferably R2A medium.

8. The method according to claim 6, wherein, The activation and expansion conditions include: a temperature of 20-35℃, preferably 28-32℃; and a time of 1-60h, preferably 45-50h.

9. The method according to claim 4, wherein, The amount of *Pseudomonas* or the bacterial agent used relative to 1 mg of alkane results in a total viable count of 10-1 *Pseudomonas*. 2 -10 6 CFU, preferably 5×10 3 -5×10 4 CFU.

10. The method according to any one of claims 4-9, wherein, The degradation temperature is 15-45℃, preferably 20-40℃, and more preferably 26-39℃; And / or, the degradation time is 1-60 hours, preferably 8-30 hours; And / or, the pH of the degradation is 5-9, preferably 6-8.

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