Alcaligenes faecalis YZ-1 and application thereof in degrading succinonitrile
By using fecal alkali-producing bacteria YZ-1 (Alcaligenes faecalis) to efficiently degrade succinate, the problem of low biodegradation efficiency in existing technologies has been solved, achieving low-cost, pollution-free succinate treatment that is suitable for industrial applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINJIANG JURONG ENERGY (GROUP) CO LTD
- Filing Date
- 2026-04-07
- Publication Date
- 2026-06-02
AI Technical Summary
In existing technologies, the biodegradation efficiency of succinate is limited, and there is a lack of highly efficient degradative strains that can use succinate as the sole carbon and nitrogen source. This limits the application of biological methods in the treatment of succinate-containing waste gas. Furthermore, traditional physicochemical methods suffer from problems such as incomplete treatment, easy generation of secondary pollution, high operating costs, and high energy consumption.
Using the fecal alkali-producing bacterium YZ-1 (Alcaligenes faecalis) as the degrading strain, it can efficiently degrade succinic acid in the pH range of 5 to 9. The degradation rate can reach 92% when the initial concentration is as high as 2.5 g/L. It is simple to operate, low in cost, and highly adaptable, making it suitable for the treatment of low-concentration nitrile-containing waste gas.
It achieves efficient, low-cost, and pollution-free degradation of succinic anionyl nitrile, has wide adaptability, is suitable for industrial applications, and has good environmental friendliness.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biodegradation technology, and in particular to a novel strain of alkali-producing bacteria YZ-1 and its use in the degradation of succinic acid. Background Technology
[0002] Succinonitrile (C4H4N2), also known as succinyl nitrile or 1,2-dicyanoethane, is a colorless and odorless waxy solid commonly used as a raw material in organic synthesis, a solvent for extracting aromatic hydrocarbons, and in the preparation of quinacrine pigments, nylon-4, and pharmaceutical intermediates, among other applications. However, succinonitrile exhibits moderate biotoxicity, primarily manifesting as corrosive and irritating effects on human skin and eyes, and causing respiratory discomfort. Furthermore, it partially transforms into highly toxic cyanide within the body, posing serious health risks with prolonged or excessive exposure. It also poses a potential pollution risk to aquatic environments.
[0003] Industries such as petrochemicals, acrylic fiber production, and rubber manufacturing often generate waste gas containing succinic anionylene during production processes and wastewater treatment. Currently, conventional treatment methods for this type of organic waste gas are mainly physicochemical, such as adsorption, alkaline scrubbing, high-temperature incineration, and regenerative thermal oxidizer (RTO). However, these traditional methods either suffer from incomplete treatment and the potential for secondary pollution, or they have shortcomings such as high operating costs, high energy consumption, and high carbon emission intensity, making it difficult to meet the needs of green and low-carbon industrial development. Therefore, developing green, efficient, and low-carbon technologies for treating succinic anionylene-containing waste gas has become a research hotspot and challenge in the field of environmental pollution control.
[0004] Biological purification technology has advantages such as mild reaction conditions, no secondary pollution, and low operating costs. Furthermore, most microorganisms can simultaneously degrade multiple organic pollutants and exhibit strong environmental adaptability, making it particularly suitable for treating low-concentration nitrile-containing waste gases. However, currently reported strains capable of degrading succinic acid (SNA) have limited degradation efficiency, and there is a lack of highly efficient strains that can use SNA as the sole carbon and nitrogen source. These issues collectively restrict the application of biological methods in the efficient and large-scale treatment of SNA-containing waste gases. Summary of the Invention
[0005] To address the aforementioned technical problems in degrading succinate, and more specifically, to efficiently degrade succinate, this invention provides a fecal alkali-producing bacterium YZ-1 and its application in degrading succinate.
[0006] The specific technical solution of this invention is as follows:
[0007] As a first aspect of the present invention, a fecal alkaloid bacterium (Alcaligenes faecalis) YZ-1 is provided, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026471, deposit date March 19, 2026, and deposit address Wuhan University, Wuhan, China, 430072, China.
[0008] In this invention, experimental verification showed that the alkali-producing bacterium YZ-1 exhibits good succinate degradation efficiency in the application of succinate degradation, achieving high-efficiency degradation of succinate with an initial concentration as high as 2.5 g / L. In one experiment, it was verified that after 60 h of cultivation, the alkali-producing bacterium YZ-1 achieved a degradation rate of over 75% for succinate in different pH fermentation environments. After 108 h of fermentation, the degradation rate of alkali-producing bacterium YZ-1 reached over 92%.
[0009] Furthermore, the alkali-producing bacterium YZ-1 exhibits a wide adaptability to growth pH range, demonstrating good growth and efficient degradation of succinate within a pH range of 5–9. Simultaneously, YZ-1 displays strong tolerance to the succinate substrate. Therefore, YZ-1 shows promising potential for industrial application in the degradation of succinate.
[0010] As a second aspect of the present invention, the application of fecal alkali-producing bacteria YZ-1 in the degradation of succinic acid is provided.
[0011] As a preferred method, fecal alkali-producing bacteria YZ-1 are added to the sample of succinic acid to be degraded to carry out the degradation reaction.
[0012] As a third aspect of the present invention, a method for degrading succinic acid nitrile based on the above-mentioned fecal alkaloid-producing bacteria YZ-1 is provided, comprising the following steps:
[0013] (1) Provide a sample containing succinic anion;
[0014] (2) Add fecal alkali-producing bacteria YZ-1 to the sample and ferment it.
[0015] As a preferred embodiment of the above method, the fermentation is carried out in an environment with a pH of 5 to 9.
[0016] Further preferably, the fermentation is carried out in an environment with a pH of 8 to 9.
[0017] As a preferred embodiment of the above method, the concentration of succinic anionylene in the sample is below 2.5 g / L.
[0018] As a preferred embodiment of the above method, the amount of fecal alkaloid-producing bacteria YZ-1 added is based on the OD of the fermentation system. 600 The amount added is to make OD 600 The value is 0.05~0.25.
[0019] As a preferred embodiment of the above method, fecal alkaloid-producing bacteria YZ-1 is added in the form of a bacterial suspension, the preparation method of which includes the following steps:
[0020] The fecal alkali-producing bacteria YZ-1 was inoculated into the culture medium, and succinic acid was added as a carbon source. The culture was then carried out at 25-35℃ and 100-200 r / min for 12-36 h.
[0021] Further preferably, the culture medium is an inorganic salt culture medium, and the concentration of succinic anion is 1~3 g / L.
[0022] The inorganic salt culture medium can be a conventional inorganic salt culture medium for culturing strains in this field, with the aim of preparing bacterial suspensions. In this invention, the inorganic salt culture medium consists of: Na₂HPO₄ 4.5 g / L, KH₂PO₄ 1.0 g / L, (NH₄)₂SO₄ 2.5 g / L, MgSO₄·7H₂O 0.2 g / L, CaCl₂ 0.023 g / L, and trace element stock solution 1 mL / L, with a pH of 7.0 and deionized water as the solvent. The trace element stock solution consists of: FeSO₄·7H₂O 1.0 g / L, CuSO₄·5H₂O 0.02 g / L, H₃BO₃ 0.014 g / L, MnSO₄·4H₂O 0.10 g / L, ZnSO₄·7H₂O 0.10 g / L, Na₂MoO₄·2H₂O 0.02 g / L, and CoCl₂·6H₂O 0.02 g / L, with deionized water as the solvent.
[0023] Compared with the prior art, the present invention has the following technical effects:
[0024] (1) The alkali-producing bacteria YZ-1 has a good degradation effect on succinate in the application of succinate degradation. In an experiment, it was verified that after culturing for 60 h, the alkali-producing bacteria YZ-1 could achieve a degradation rate of more than 75% for succinate in different pH fermentation environments. After fermentation for 108 h, the degradation rate of alkali-producing bacteria YZ-1 could reach more than 92%. When alkali-producing bacteria YZ-1 degraded succinate with an initial concentration as high as 2.5 g / L, it could also achieve the aforementioned high degradation efficiency.
[0025] (2) The fecal alkali-producing bacteria YZ-1 has a wide range of suitable pH for growth. It can grow well and efficiently degrade succinate in a pH range of 5 to 9. At the same time, the fecal alkali-producing bacteria YZ-1 has a strong tolerance to the substrate succinate.
[0026] (3) The degradation of succinate by fecal alkali-producing bacteria YZ-1 is simple to operate, stable in operation, low in cost, and the reaction conditions are mild, with no secondary pollution to the environment, thus exhibiting good environmental friendliness. Fecal alkali-producing bacteria YZ-1 has good application prospects for industrial degradation of succinate. Attached Figure Description
[0027] Figure 1 Phylogenetic tree of alkali-producing bacteria YZ-1.
[0028] Figure 2 Transmission electron microscopy image of fecal alkali-producing bacteria YZ-1.
[0029] Figure 3 The experimental results show the degradation characteristics of succinate by fecal alkali-producing bacteria YZ-1. Figure A shows the broad-spectrum analysis of fecal alkali-producing bacteria YZ-1, and Figure B shows the cell growth curve.
[0030] Figure 4 The results show the effects of different pH environments on the degradation of succinate by fecal alkali-producing bacteria YZ-1. Figure A shows the removal rate of succinate by different pH environments, Figure B is a line graph showing the effect of different pH values on CO2 production, and Figure C is a growth graph of the strain.
[0031] Figure 5 The results show the effect of different substrate concentrations on the degradation of succinate by *Alkali-producing bacteria* YZ-1. Figure A shows the removal rate of succinate by *Alkali-producing bacteria* YZ-1 at different initial concentrations, Figure B is a line graph showing the effect on CO2 production, and Figure C is a growth graph of the strain.
[0032] Figure 6 The effect of different initial bacterial counts on the degradation of succinate by fecal alkali-producing bacteria YZ-1 is shown in Figure A, which shows the removal rate of succinate by different initial bacterial counts; Figure B is a line graph showing the effect of different initial bacterial counts on CO2 generation; and Figure C is a growth graph of the strain. Detailed Implementation
[0033] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.
[0034] In the following examples, the composition of the culture medium used is as follows:
[0035] The inorganic salt culture medium consists of: Na₂HPO₄ 4.5 g / L, KH₂PO₄ 1.0 g / L, (NH₄)₂SO₄ 2.5 g / L, MgSO₄·7H₂O 0.2 g / L, CaCl₂ 0.023 g / L, and trace element stock solution 1 mL / L, with a pH of 7.0 and deionized water as the solvent. The trace element stock solution consists of: FeSO₄·7H₂O 1.0 g / L, CuSO₄·5H₂O 0.02 g / L, H₃BO₃ 0.014 g / L, MnSO₄·4H₂O 0.10 g / L, ZnSO₄·7H₂O 0.10 g / L, Na₂MoO₄·2H₂O 0.02 g / L, and CoCl₂·6H₂O 0.02 g / L, with deionized water as the solvent.
[0036] The R2A solid culture medium consists of: 0.50 g / L yeast extract, 0.50 g / L soluble starch, 0.05 g / L MgSO4·7H2O, 0.50 g / L peptone, 0.50 g / L glucose, 0.30 g / L sodium pyruvate, 0.45 g / L K2HPO4·3H2O, 15–20 g / L agar, pH 7.2, and deionized water as the solvent.
[0037] Example 1: Isolation, purification and identification of Alcaligenes faecalis YZ-1
[0038] (1) Isolation and purification of fecal alkaloid-producing bacteria YZ-1
[0039] Activated sludge was collected from a wastewater treatment plant of a petrochemical company in Zhejiang Province. After the collected sludge was allowed to settle for 2 hours, the supernatant and suspended impurities were removed, and the fine-particle sludge was retained. The lower layer of sludge after 2 hours of settling was taken and mixed with inorganic salt culture medium at a volume ratio of 1:2. Then, 1.5 L of the mixture was introduced into a 2 L sludge acclimation tank, using succinate as the sole carbon and energy source of the system, and acclimation culture was carried out at room temperature (25 ℃).
[0040] During the sludge acclimation process, the acclimation tank was continuously aerated. After aeration was stopped daily, the mixture was allowed to stand for 30 minutes, and the pH of the supernatant was measured and maintained at 7.0 ± 0.5. Half of the supernatant culture medium was discarded daily and replaced with an equal volume of fresh inorganic salt medium. After complete degradation of succinic acid in the system, substrate was added to a concentration of 1.7 g / L. After one month of acclimation, 10 mL of the sludge-water mixture from the acclimation tank was taken and inoculated into a shake flask containing 100 mL of sterile inorganic salt medium. The shake flask experiment was conducted at 30℃ and 160 r / min. The results showed that the acclimated sludge could stably degrade succinic acid. Three acclimated samples were obtained, designated YZ-1, YZ-2, and YZ-3.
[0041] Five mL samples from each shake flask were transferred to new shake flasks for further performance verification. An initial concentration of 1.7 g / L succinate was added, and the samples were incubated at 30℃ and 160 r / min. After 24 h, the substrate concentration was measured. The results showed that the transferred acclimatized sample YZ-1 exhibited the best performance, stably degrading 92% of succinate within 108 h. The degradation rates of succinate for YZ-2 and YZ-3 were 32% and 46%, respectively.
[0042] Sludge sample YZ-1 was continuously subcultured and enriched in shake flasks containing 100 mL of inorganic salt medium. Each subculture added an initial concentration of 1.7 g / L succinate, for a total of 6 subcultures (5 mL for the first 3 subcultures, 2 mL for the 4th, and 1 mL for the last two). The enriched bacterial solutions from the 6 subcultures were then processed according to a 10... -1 -10 -6 After multiple dilution, the culture was spread on plates and incubated in a 30°C incubator for 3 days. Single colonies were picked and streaked for purification to finally obtain pure culture strain YZ-1.
[0043] (2) Identification of fecal alkaloid-producing bacteria YZ-1
[0044] The pure culture strain YZ-1 obtained in step (1) was sent to Sangon Biotech (Shanghai) Co., Ltd. for PCR amplification and sequencing. The 16S rDNA sequencing results are shown in SEQ ID NO. 1. The sequencing results were submitted to the Ezbiocloud.net platform and compared with the standard strains in the database. The phylogenetic tree was constructed using the Neighbor-Joining method with MEGA9.05 software, and the confidence level was tested using the Bootstrap method (repeated 1000 times). The resulting phylogenetic tree is shown in Figure 1.
[0045] The identification results showed that the strain was a fecal alkali-producing bacterium, named Alcaligenes faecalis, and deposited at the China Center for Type Culture Collection (CCTCC) on March 19, 2026, with accession number CCTCC M 2026471, deposited at Wuhan University, Wuhan, China, postal code 430072.
[0046] refer to Figure 2 The morphological characteristics of *Alkali-producing bacteria* YZ-1 are as follows: the bacteria are rod-shaped and non-spore-forming; the colonies are white, round, opaque, smooth, moist, and easily picked up, with the bacterial growth following the streaks. Furthermore, the physiological and biochemical characteristics of strain YZ-1 are: aerobic.
[0047] Example 2: Large-scale culture of alkaloid-producing bacteria YZ-1
[0048] (1) Slant culture: The fecal alkali-producing bacteria YZ-1 strain obtained by screening and identification in the example was inoculated onto R2A solid slant culture medium and cultured in a constant temperature environment of 30℃ for 24 h to obtain slant cells.
[0049] (2) Preparation of bacterial suspension: Pick a loop of colonies from the slant obtained in step (1) with an inoculation loop and inoculate it into an inorganic salt culture medium. At the same time, add succinic acid to the culture medium to an initial concentration of 1.7 g / L. Shake and culture at 30℃ and 160 r / min for 24 h to obtain bacterial suspension.
[0050] Example 3: Degradation characteristics of fecal alkali-producing bacteria YZ-1 on succinic acid
[0051] In this embodiment, succinic acid nitrile was used as the sole carbon and energy source required for the growth of *Alkali-producing Bacteria YZ-1*. The bacterial suspension prepared using the method shown in Example 2 was inoculated into 100 mL of inorganic salt culture medium, and the initial bacterial concentration (expressed as OD₂O₅) was adjusted. 600 Value representation) to OD 600 =0.1, and then succinic acid was added to the culture medium to bring the initial concentration of succinic acid to 1.7 g / L. The culture medium was then placed in a shaker at 30℃ and 160 r / min for incubation. Samples were taken every 12 h during the incubation period to detect and calculate the degradation rate of succinic acid; simultaneously, an appropriate amount of bacterial culture was drawn using a 5 mL syringe to determine the OD of the bacterial cells. 600 Values, related experimental results are as follows Figure 3 As shown.
[0052] To ensure the accuracy and reliability of the experimental results, three parallel experimental samples were set up during the experiment, and a blank control group without inoculation with this strain was added to eliminate interference. The experimental results showed that... Figure 3As the culture time increased, the bacterial cell concentration gradually increased, reaching its peak at 72 h. 600 The value is approximately 0.601, at which point the succinic acid degradation rate can reach 71%. The results of this example confirm that the fecal alkali-producing bacteria YZ-1 can grow and reproduce using succinic acid as the sole carbon and energy source, and possesses stable and efficient succinic acid degradation efficiency.
[0053] Example 4: Large-scale culture of alkaloid-producing bacteria YZ-1
[0054] This example is used for the large-scale culture of fecal alkali-producing bacteria YZ-1. The operation procedure is basically the same as that in Example 2, with only the following differences. The other identical operation steps will not be repeated here:
[0055] (1) Slant culture: The isothermal culture time of slant culture was adjusted to 36 h. The amount of agar added in the R2A solid medium was adjusted to 20 g / L, while the other medium components and concentrations remained unchanged.
[0056] (2) Preparation of bacterial suspension: The duration of the bacterial suspension shaking culture was adjusted to 36 h, and the remaining culture conditions and operation requirements were the same as in Example 2.
[0057] Example 5: Effects of different pH environments on the degradation of succinic acid bacteria YZ-1 by alkali-producing bacteria
[0058] The inorganic salt culture medium was adjusted to different pH values (5.0, 6.0, 7.0, 8.0, and 9.0) using 1 mol / L sodium hydroxide aqueous solution or 1 mol / L sulfuric acid aqueous solution. The bacterial suspension prepared in Example 4 was inoculated into 100 mL of the pH-adjusted inorganic salt culture medium to control the initial bacterial concentration (expressed as OD). 600 The initial concentration (calculated as 0.1) of succinate was adjusted to 1.7 g / L by adding succinate to the culture medium. The inoculated medium was then placed in a shaker at 30℃ and 160 r / min for incubation. Three parallel samples and one blank control group (without inoculation) were included to ensure the reliability of the results. Samples were taken after 60 h of incubation to detect the succinate degradation rate and OD of the bacterial culture. 600 Values and CO2 production, experimental results are as follows: Figure 4 As shown.
[0059] Depend on Figure 4 It can be seen that when the pH value is in the range of 5 to 9, the degradation rate of succinate by this strain exceeds 75%, and the degradation effect is optimal when the pH value is 8 to 9, with a degradation rate of over 85%. Moreover, the growth of the strain, the amount of CO2 generated and the degradation rate of succinate show the same trend.
[0060] Example 6: Effect of different substrate concentrations on the degradation of succinate by fecal alkali-producing bacteria YZ-1
[0061] The bacterial suspension prepared in Example 4 was inoculated into 100 mL of inorganic salt culture medium, and the initial bacterial concentration was adjusted (in terms of OD). 600 The initial concentration of succinate (calculated as 0.1) was 0.1, and succinate was added to achieve initial concentrations of 1, 1.3, 1.7, 2.0, and 2.5 g / L, respectively. The inoculated culture medium was then placed in a shaker at 30℃ and 160 r / min for incubation. Three parallel samples and one blank control group without inoculation were included in the experiment. Samples were taken periodically during incubation to detect the concentration of succinate in the reaction solution and the OD of the *Alcaligenes faecalis* YZ-1 bacterial culture. 600 Values, experimental results as follows Figure 5 As shown.
[0062] Depend on Figure 5 It can be seen that increasing the initial concentration of the substrate has no significant effect on the degradation of succinate by the fecal alkali-producing bacterium YZ-1. Therefore, it can be inferred that succinate has low toxicity to this strain and that the strain has a strong tolerance to succinate concentration.
[0063] Example 7: Effect of different initial bacterial counts on the degradation of succinic acid bacteria YZ-1 by fecal alkali-producing bacteria
[0064] Take the bacterial suspension prepared by the method in Example 4 and inoculate it into 100 mL of inorganic salt culture medium to achieve an initial bacterial concentration (expressed as OD). 600 The solution was prepared in batches of 0.05, 0.1, 0.15, 0.20, and 0.25 g / L, and then succinate (1.7 g / L) was added. The mixture was then incubated in a shaker at 30 ℃ and 160 r / min. Three parallel samples and one blank control group without inoculation were included in the experiment. Sampling was performed periodically to determine the succinate concentration and OD of the *Alcaligenes faecalis* YZ-1 bacterial culture. 600 Values, results are shown below Figure 6 As shown.
[0065] Through observation Figure 6 It can be seen that, in the initial bacterial count OD 600 Within the range of 0.05 to 0.20, the fecal alkali-producing bacteria YZ-1 exhibited a high degradation rate for succinic acid.
[0066] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.
[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.
Claims
1. A fecal alkaloid-producing bacterium (Alcaligenes faecalis) YZ-1, characterized in that: It is deposited at the China Center for Type Culture Collection, accession number CCTCC M 2026471, deposited on March 19, 2026, at Wuhan University, Wuhan, China, 430072, China.
2. The application of the fecal alkali-producing bacterium YZ-1 as described in claim 1 in the degradation of succinic acid.
3. The application as described in claim 1, characterized in that: Add fecal alkali-producing bacteria YZ-1 to the sample to be degraded succinate to carry out the degradation reaction.
4. The method for degrading succinic acid oxidase YZ-1 based on claim 1, characterized in that: Includes the following steps: (1) Provide a sample containing succinic anion; (2) Add fecal alkali-producing bacteria YZ-1 to the sample and ferment it.
5. The method as described in claim 4, characterized in that: The fermentation is carried out in an environment with a pH of 5 to 9.
6. The method as described in claim 4 or 5, characterized in that: The fermentation is carried out in an environment with a pH of 8 to 9.
7. The method as described in claim 4, characterized in that: The concentration of succinic acid in the sample was below 2.5 g / L.
8. The method as described in claim 4, characterized in that: The amount of fecal alkali-producing bacteria YZ-1 added is based on the OD of the fermentation system. 600 The amount added is to make OD 600 The value is 0.05~0.
25.
9. The method as described in claim 4, characterized in that: The fecal alkali-producing bacteria YZ-1 is added in the form of a bacterial suspension, and the preparation method of the bacterial suspension includes the following steps: The fecal alkali-producing bacteria YZ-1 was inoculated into the culture medium, and succinic acid was added as a carbon source. The culture was then carried out at 25-35℃ and 100-200 r / min for 12-36 h.
10. The method as described in claim 9, characterized in that: The culture medium is an inorganic salt culture medium, and the concentration of succinate added is 1~3g / L.