Enterobacter ludwigii strain for degrading formaldehyde and application thereof

Abstract

The application belongs to the technical field of microorganisms, and relates to a strain of Enterobacter ludwigii ZTB-A30 capable of efficiently degrading formaldehyde and application of the strain. Enterobacter ludwigii The strain is classified and named as Enterobacter ludwigii (CGMCC No. 23340) and was preserved in the China General Microbiological Culture Collection Center on September 2, 2021. The address of the preservation center is No. 1, Yihuangyuan 3rd, Beichen West Road, Chaoyang District, Beijing, China. The Enterobacter ludwigii ZTB-A30 can effectively remove formaldehyde in the environment, and can be applied to removal of formaldehyde in air, water and other environments, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a formaldehyde-efficient Ludwig's Enterobacter strain and its applications. Background Technology

[0002] Formaldehyde is an organic compound, a colorless gas with a strong, pungent, and suffocating odor. Commonly used interior decoration materials such as wood-based panels, paints, carpets, and wallpapers often contain and release formaldehyde. Incomplete combustion of fuels and tobacco also releases formaldehyde. Formaldehyde is highly irritating to the eyes, respiratory tract, and skin. Exposure to formaldehyde vapor can cause conjunctivitis, keratitis, rhinitis, bronchitis, and other ailments.

[0003] The main measures for removing formaldehyde are as follows: (1) Photocatalyst: It uses nano-sized titanium dioxide as the main material, combined with various light absorbers and oxidants. Under specific light source conditions, it can efficiently decompose formaldehyde and other VOCs, and even play a role in destroying bacteria and viruses. However, photocatalysts need ultraviolet light to be activated to play a role, but ultraviolet light cannot be present indoors all the time, which limits the role of photocatalysts. (2) Negative ions: They are equivalent to an active agent, which is conducive to the volatilization of harmful substances such as formaldehyde. However, the treatment time is relatively long, and specific materials need to be protected. The deodorizing ability is not strong. (3) Formaldehyde remover: There are two types: one is formaldehyde remover for artificial boards, and the other is formaldehyde remover for air purification. The most effective one is the neutralization reaction formaldehyde remover for artificial boards. However, the deodorizing ability is not strong; and it is mainly used in the initial processing stage of boards, which is prone to rebound. (4) Bio-enzyme: It uses microbial degradation technology. Through the reproduction of microorganisms, bio-enzymes are produced. Under the dual action of microorganisms and enzymes, indoor pollutants such as formaldehyde are neutralized. Microbial degradation technology is a natural degradation, which is environmentally friendly and safe. They can rapidly neutralize pollutants such as formaldehyde. Therefore, screening for highly efficient formaldehyde-degrading microorganisms and developing highly efficient formaldehyde-degrading microbial preparations has become an urgent need. Summary of the Invention

[0004] The purpose of this invention is to provide a formaldehyde-efficient Ludwig C. strain and its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A strain of Enterobacter ludwigii ZTB-A30, classified as Enterobacter ludwigii, was deposited on September 2, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 23340.

[0006] The colony characteristics and cell morphology of the above-mentioned Ludwig's Enterobacter ZTB-A30 strain are as follows: After culturing Ludwig's Enterobacter ZTB-A30 on NA plates for 24 hours, the colonies formed are white, translucent, dull, round, and non-mobile, with a colony diameter of 4-5 mm. The bacterial cells are rod-shaped, Gram-positive, and have no spores or capsules.

[0007] The physiological and biochemical characteristics of the aforementioned Ludwig's Enterobacter ZTB-A30 strain are as follows: Ludwig's Enterobacter ZTB-A30 showed negative results for catalase reaction, negative results for VP assay, positive results for MR assay, positive results for glucose acid production test, negative results for glucose gas production test, positive results for citrate test, positive results for nitrate reduction test, negative results for starch hydrolysis test, negative results for indole test, positive results for malonic acid assay, and positive results for H2S production test.

[0008] The 16S rDNA sequence (SEQ ID NO.1) of Enterobacter ludwigii ZTB-A30 was compared with the sequence in the GenBank database. The results showed that Enterobacter ludwigii ZTB-A30 and Enterobacter ludwigii belong to the same branch, and its 16S rDNA sequence has a similarity of 99.65% with Enterobacter ludwigii (CP024812.1).

[0009] The aforementioned Ludwig's bacterium ZTB-A30 can be used to remove formaldehyde.

[0010] Furthermore, the aforementioned Ludwig's Enterobacter ZTB-A30 strain can be used to remove formaldehyde from industrial wastewater.

[0011] The aforementioned Ludwig's bacterium ZTB-A30 can be used to remove formaldehyde from indoor air.

[0012] A microbial formaldehyde removal product containing the aforementioned Ludwig vulgaris ZTB-A30.

[0013] The aforementioned microbial formaldehyde removal product can be used to remove formaldehyde.

[0014] Furthermore, the aforementioned microbial formaldehyde removal product can be used to remove formaldehyde from industrial wastewater.

[0015] Furthermore, the aforementioned microbial formaldehyde removal product can be used to remove formaldehyde from indoor air.

[0016] The advantages of this invention are: The Enterobacter ludwigii ZTB-A30 described in this invention can effectively remove formaldehyde from the environment and can be applied to the removal of formaldehyde in air, water and other environments, showing good application prospects. Detailed Implementation

[0017] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0018] The Ludwig's Enterobacter ZTB-A30 strain described in this invention, classified as Enterobacter ludwigii, was deposited on September 2, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 23340, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0019] The culture media used in the embodiments of the present invention are as follows: Basic inorganic salt culture medium: (NH4)2SO4 1g, K2HPO4 2.16g, MgSO4 0.10g, KH2PO4 0.85g, MnSO4·H2O 0.05g, CaCl2·H2O 0.03g, FeSO4·7H2O 0.01g, agar 18g, water 1000ml, pH=7.0~7.2; sterilize at 121℃ for 20min.

[0020] Basic inorganic salt liquid culture medium: (NH4)2SO4 1g, K2HPO4 2.16g, MgSO4 0.10g, KH2PO4 0.85g, MnSO4·H2O 0.05g, CaCl2·H2O 0.03g, FeSO4·7H2O 0.01g, water 1000ml, pH=7.0~7.2; sterilize at 121℃ for 20min.

[0021] NA medium: 3g beef extract, 5g peptone, 5g NaCl, 18g agar, 1000ml water, pH=7.0~7.2 (for liquid medium, omit agar, i.e., NB medium); sterilize at 121℃ for 20min.

[0022] NA liquid medium: 3g beef extract, 5g peptone, 5g NaCl, 1000ml water, pH=7.0~7.2 (for liquid medium, excluding agar, i.e. NB medium); sterilize at 121℃ for 20min.

[0023] Example 1: (1) Screening process: Thirty-nine isolates were isolated from Fujian mangrove plants using a serial dilution method. The isolated endophytic bacteria were purified using a three-zone streak method, and the purity of the strains was determined by microscopic examination. The purified bacteria were numbered, and single colonies were picked and transferred to NA slants for storage. The strains were screened for formaldehyde degradation activity, initially identifying three strains with strong formaldehyde removal activity. Finally, one strain with good formaldehyde degradation effect was selected and labeled ZTB-A30.

[0024] (2) Colony characteristics and colony morphology: After culturing strain ZTB-A30 on NA plates for 24 hours, the resulting colonies were white, translucent, dull, round, and non-flowing, with a diameter of 4-5 mm. The bacterial cells were rod-shaped, Gram-positive, and had no spores or capsules.

[0025] (3) Physiological and biochemical characteristics: The strain ZTB-A30 was negative for catalase reaction, negative for VP assay, positive for MR assay, positive for glucose acid production test, negative for glucose gas production test, positive for citrate test, positive for nitrate reduction, negative for starch hydrolysis, negative for indole test, positive for malonic acid assay, and positive for H2S production test.

[0026] (4) Initial selection of formaldehyde-degrading active strains Single colonies of the isolated and purified strain were inoculated onto a basic inorganic salt medium containing 300 mg / L formaldehyde and incubated at 30°C for 5 days. Colony growth was then observed, and colony diameters were measured and recorded. Strains that grew normally were preliminarily considered to have the ability to degrade formaldehyde; larger colony diameters indicated stronger formaldehyde-degrading activity.

[0027] Table 1 Preliminary screening of formaldehyde-degrading strains Note: D: Colony diameter.

[0028] Formaldehyde degradation activity: "-": no activity (colon diameter: 0mm); "+": active (colon diameter: 1-5mm); "++": Strong activity (colon diameter: 6-10mm); "+++": Very strong activity (colon diameter: >10mm).

[0029] (5) Rescreening of formaldehyde-degrading strains The strains with formaldehyde degradation activity obtained from the initial screening were inoculated into a liquid culture medium containing 300 mg / L formaldehyde and basic inorganic salts. After incubation at 30°C for 3 days on a shaker at 180 r / min, the changes in the bacterial solution were observed. If the bacterial solution was turbid, it indicated that the strain had formaldehyde degradation activity.

[0030] Table 2. Secondary screening of formaldehyde-degrading strains Note: "-" indicates the bacterial solution is not cloudy; "+" indicates the bacterial solution is cloudy.

[0031] (6) 16S rDNA sequence analysis The 16S rDNA gene sequence of strain ZTB-A30 is shown in SEQ ID NO.1 of the nucleotide sequence listing. Alignment of the obtained 16S rDNA sequence with sequences in the GenBank database showed that strain ZTB-A30 belongs to the same branch as Enterobacter ludwigii, and its 16S rDNA sequence shows 99.65% similarity to Enterobacter ludwigii (CP024812.1). Based on colony morphology, physiological and biochemical characteristics, and 16S rDNA sequence analysis, it was preliminarily identified as Enterobacter ludwigii.

[0032] Example 2: Determination of the formaldehyde degradation effect of ZTB-A30 After fully activating *Enterobacter ludwigschneidera* ZTB-A30, it was inoculated into a shake flask containing NA liquid medium to obtain a seed culture. The seed culture was then inoculated at a ratio of 10% (v / v) into NA liquid medium containing 600 mg / L formaldehyde and incubated at 30°C for 3 days on a shaker at 180 rpm. Considering the volatility of formaldehyde, two blank controls were set up (blank control 1 was an identical formaldehyde-containing NA liquid medium without bacterial inoculation as the experimental group; blank control 2 was the same volume of sterile water with the same formaldehyde content as the experimental group). The volatility value was subtracted when measuring formaldehyde content to reduce error. The formaldehyde content in the culture medium was determined using the acetylacetone method, and the formaldehyde removal rate was calculated.

[0033] Table 3. Determination of formaldehyde degradation effect of ZTB-A30 As shown in Table 3, the formaldehyde content in the culture medium of *Enterobacter ludwigschneidera sinensis* ZTB-A30 was significantly reduced after treatment, with a formaldehyde removal rate of 85.34%. This demonstrates that *Enterobacter ludwigschneidera sinensis* ZTB-A30 has a significant effect on formaldehyde removal.

[0034] Example 3: Formaldehyde Removal Efficiency Test of ZTB-A30 A chemical plant's wastewater biochemical treatment pond (formaldehyde content 150 mg / L, COD value 1000 mg / L, pH value 7) was treated with Ludwig's Enterobacter ZTB-A30 bacterial solution (bacterial solution concentration 5 × 10⁻⁶). 8Add cfu / mL to the wastewater at a dosage of 5% (v / v), and control the treatment tank temperature at 25-28℃ and the aeration rate at 5-10 m³ / h. 3 The formaldehyde content in the water was determined by sampling at 6 hours and 12 hours, and the formaldehyde removal rate was calculated using the acetylacetone method.

[0035] Table 4. Determination of formaldehyde removal efficiency of ZTB-A30 As shown in Table 4, after 6 hours of treatment with Ludwig's Enterobacter ZTB-A30, the formaldehyde content in the chemical wastewater decreased by 83.76%; after 12 hours of treatment, the formaldehyde content in the chemical wastewater decreased by 90.98%. Ludwig's Enterobacter ZTB-A30 has a significant effect on formaldehyde removal.

[0036] Example 4: Determination of the formaldehyde removal effect of ZTB-A30 on indoor air Choose 60m 3 In a sealed room with the temperature controlled at 25-28℃, 2500mL of Ludwig's Enterobacter ZTB-A30 bacterial suspension (concentration of 5×10⁻⁶) was placed. 8 Add the bacterial solution (cfu / mL) to the spray device (humidifier), turn on the spray device to spray the bacterial solution evenly, and then add formaldehyde all at once, with a formaldehyde concentration of 0.25 mg / mL. 3 Samples were taken after 2 and 3 days, and the formaldehyde content in the room air was determined using the acetylacetone method to calculate the formaldehyde removal rate.

[0037] Table 5. Determination of formaldehyde removal efficiency of ZTB-A30 The results showed that the formaldehyde concentration had decreased to 0.03 mg / m³ after 2 days. 3 The formaldehyde removal rate reached 88%; after 3 days, the formaldehyde concentration dropped to 0.01 mg / m³. 3 The formaldehyde removal rate reached 96%, and the formaldehyde concentration fully met the national standards. This demonstrates that Ludwig's Enterobacter ZTB-A30 bacterial solution can rapidly degrade formaldehyde and has a significant effect on removing formaldehyde from the air.

[0038] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A strain of Enterobacter ludwig's bacterium ZTB-A30, characterized in that: Classified and named Ludwig's Enterobacter ( Enterobacter ludwigii It was deposited on September 2, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 23340.

2. The application of Ludwig's Enterobacter ZTB-A30 as described in claim 1, characterized in that: It is used to remove formaldehyde.

3. The application according to claim 2, characterized in that: It is used to remove formaldehyde from industrial wastewater.

4. The application according to claim 2, characterized in that: It is used to remove formaldehyde from indoor air.

5. A microbial formaldehyde removal product, characterized in that: Contains Ludwig's Enterobacter ZTB-A30 as described in claim 1.

6. The application of the microbial formaldehyde removal product as described in claim 5, characterized in that: It is used to remove formaldehyde.

7. The application according to claim 6, characterized in that: It is used to remove formaldehyde from industrial wastewater.

8. The application according to claim 6, characterized in that: It is used to remove formaldehyde from indoor air.

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