CN117965382A - Bacillus intermedium for efficiently degrading formaldehyde and application thereof - Google Patents

Abstract

The invention provides a bacillus intermedius (Ochrobactrum intermedium) BQB-B02 capable of efficiently degrading formaldehyde and application thereof. The pale bacillus (Ochrobactrum intermedium) BQB-B02 is preserved in the China general microbiological culture Collection center (China general microbiological culture Collection center) on the 9 th month of 2021, and has a preservation address of North Star Xili No. 1,3 in the Korean region of Beijing, china, and a preservation number of CGMCC No.23339. The invention provides a new strain with higher formaldehyde degradation capability, which provides a foundation for degrading formaldehyde by a biological method.

Description

A strain of Ochrobacterium intermedia with high efficiency in formaldehyde degradation and its application

Technical Field

The invention belongs to the technical field of microorganisms and relates to a strain of Ochrobacterium intermedia that efficiently degrades formaldehyde and application thereof.

Background technique

Formaldehyde is an organic compound, a colorless gas with a strong irritating and suffocating odor. Commonly used panels, paints, carpets, wallpapers, etc. for interior decoration contain and release formaldehyde. Incomplete combustion of fuel and tobacco leaves also releases formaldehyde. Formaldehyde is highly irritating to the eyes, respiratory tract and skin. Contact with formaldehyde vapor causes conjunctivitis, keratitis, rhinitis, bronchitis, etc. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer published a list of carcinogens, and formaldehyde was listed in the Class 1 carcinogen list.

There are several main measures for removing formaldehyde: (1) Photocatalyst: It uses nano-sized titanium dioxide as the main material, combined with a variety of light absorbers and oxidants. Under specific light conditions, it can efficiently decompose formaldehyde and other VOCs, and even destroy bacteria and viruses. However, for photocatalysts to work, they need ultraviolet light to be stimulated, and ultraviolet light cannot always be present indoors, which limits the effect of photocatalysts. (2) Negative ions: It is equivalent to an active agent, which is conducive to the volatilization of harmful substances such as formaldehyde. However, the treatment time is long, and specific materials must be protected, and the deodorization ability is not strong. (3) Formaldehyde scavengers: One type is formaldehyde scavengers for artificial boards, and the other type is formaldehyde scavengers for air purification. Among them, the most effective is the neutralization reaction type formaldehyde scavenger for artificial boards. However, the deodorization ability is not strong; and it is mainly used in the initial processing stage of the board, which is prone to rebound. (4) Bioenzymes: It uses microbial degradation technology to produce bioenzymes produced by the reproduction of microorganisms themselves. Under the dual action of microorganisms and enzymes, formaldehyde and other indoor pollutants are neutralized. Microbial degradation technology belongs to natural degradation, environmentally friendly and safe. It can quickly neutralize pollutants such as formaldehyde. Therefore, screening functional microorganisms with high efficiency in formaldehyde degradation and developing high efficiency formaldehyde degradation microbial preparations have become an urgent need.

Summary of the invention

The invention aims to provide a strain of Ochrobacterium intermedia for efficiently degrading formaldehyde and application thereof.

To achieve the above object, the present invention adopts the following technical solution:

A strain of Ochrobactrum intermedium BQB-B02 that efficiently degrades formaldehyde. The intermediate Ochrobactrum BQB-B02 is classified and named Ochrobactrum intermedium. It was deposited in the General Microbiology Center of China National Microbiological Culture Collection on September 2, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No. 23339.

The colony characteristics and bacterial morphology of the above-mentioned Ochrobacterium intermedia BQB-B02 are as follows:

The colonies formed by strain BQB-B02 after culturing on NA plates for 24 hours were white, translucent, dull, round, and non-fluid, with a colony diameter of 4-5 mm; the bacterial body morphology was rod-shaped, Gram-positive, without spores and capsules.

The physiological and biochemical characteristics of the above-mentioned Ochrobacterium intermedia BQB-B02 are:

BQB-B02 catalase reaction was negative, VP assay was negative, MR assay was negative, glucose acid production test was negative, glucose gas production test was negative, citrate test was positive, nitrate reduction was positive, starch hydrolysis was negative, indole test was negative, malonic acid assay was positive, and H ₂ S production test was positive.

The 16S rDNA sequence (SEQ ID NO.1) of the above-mentioned Ochrobactrum intermedium BQB-B02 was compared with the sequence in the GenBank database. The results showed that BQB-B02 and Ochrobactrum intermedium were on the same branch, and the similarity of its 16S rDNA sequence with Ochrobactrum intermedium (MK249656.1) was 99.85%. Combined with the colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, it was preliminarily identified as Ochrobactrum intermedium.

The advantages of the present invention are:

The Ochrobactrum intermedium BQB-B02 of the present invention can effectively remove formaldehyde in the environment, can be applied to the removal of formaldehyde in the air, water and other environments, and has good application prospects.

Detailed ways

In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

The intermediary Ochrobactrum BQB-B02 described in the present invention is classified and named as intermediary Ochrobactrum (Ochrobactrumintermedium) BQB-B02, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 2, 2021, with the deposit number: CGMCC No.23339, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

The culture medium used in the following examples is as follows:

Basic inorganic salt culture medium: (NH ₄ ) ₂ SO ₄ 1g, K ₂ HPO ₄ 2.16g, MgSO ₄ 0.10g, KH ₂ PO4 0.85g, MnSO ₄ ·H ₂ O 0.05g, CaCl ₂ ·H ₂ O 0.03g, FeSO ₄ ·7H ₂ O 0.01g, water 1000ml, pH 7.0-7.2 (no agar is added for liquid culture medium), sterilize at 121°C for 20min.

NA medium: 3g beef extract, 5g peptone, 5g NaCl, 18g agar, 1000ml water, pH 7.0-7.2 (no agar is added for liquid culture medium, i.e. NB medium), sterilize at 121℃ for 20min.

Example 1 Separation, screening and identification

(1) Screening process:

319 isolates were isolated from Fujian mangrove plants by gradient dilution method. The isolated endophytic bacteria were purified by three-zone line method, and the purification of the strains was determined by microscopy. The purified bacteria were numbered, single colonies were picked, and transferred to NA slant for storage. The formaldehyde degradation activity was screened by strains, and 3 strains with strong formaldehyde removal activity were initially screened out. Finally, a bacterium with good formaldehyde removal effect was screened out and marked as BQB-B02.

(2) Colony characteristics and colony morphology:

The colonies formed after BQB-B02 was cultured on NA plates for 24 hours were white, translucent, dull, round, and non-fluid, with a colony diameter of 4-5 mm; the bacterial body morphology was rod-shaped, Gram-staining was positive, and there were no spores or capsules.

(3) Physiological and biochemical characteristics:

BQB-B02 catalase reaction was negative, VP assay was negative, MR assay was negative, glucose acid production test was negative, glucose gas production test was negative, citrate test was positive, nitrate reduction was positive, starch hydrolysis was negative, indole test was negative, malonic acid assay was positive, and H ₂ S production test was positive.

(4) Preliminary selection of active strains for formaldehyde degradation

The single colony of the isolated and purified bacterial strain was inoculated onto a basic inorganic salt medium containing 300 mg/L formaldehyde, and then cultured at 30°C for 5 days to observe the growth of the colony, measure the colony diameter and record it. The strains that can grow normally are preliminarily considered to have the ability to degrade formaldehyde, and the larger the colony diameter, the stronger the activity of the strain in degrading formaldehyde.

Table 1 Preliminary screening of formaldehyde-degrading strains

Note: D: colony diameter.

Ammonia degradation activity: “-”: inactive (colony diameter: 0 mm); “+”: active (colony diameter: 1-5 mm); “++”: relatively strong activity (colony diameter: 6-10 mm); “+++”: very strong activity (colony diameter: >10 mm).

(5) Rescreening of formaldehyde-degrading strains

The strain with formaldehyde degradation activity obtained from the above preliminary screening was inoculated into a basic inorganic salt liquid culture medium containing 300 mg/L formaldehyde, and cultured at 30°C at 180 r/min on a shaker for 3 days to observe the changes in the bacterial solution. If the bacterial solution is turbid, it indicates that the strain has formaldehyde degradation activity.

Table 2 Rescreening of formaldehyde-degrading active strains

Note: The bacterial solution is not turbid: “-”; the bacterial solution is turbid: “+”.

(6) 16S rDNA sequence analysis

The 16S rDNA gene sequence of strain BQB-B02 is shown in nucleotide sequence table SEQ ID NO.1. The measured 16S rDNA sequence was compared with the sequence in GenBank database, and the results showed that BQB-B02 and Ochrobactrum intermedium were on the same branch, and the similarity of its 16S rDNA sequence with Ochrobactrum intermedium (MK249656.1) was 99.85%. Combined with colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, it was preliminarily identified as Ochrobactrum intermedium.

Example 2 Determination of the Formaldehyde Degradation Effect of BQB-B02

After strain BQB-B02 is fully activated, it is inoculated in a shake flask filled with NA liquid culture medium to obtain seed liquid. Then the seed liquid is inoculated in a NA liquid culture medium containing 600 mg/L formaldehyde at a ratio of 10% (V/V), and placed on a 180r/min shaker at 30°C for 3 days. Considering the volatility of formaldehyde, two groups of blank controls are set up (blank control 1 is a NA culture medium that is exactly the same as the test group without inoculation of bacteria; blank control 2 is the same volume of sterile water with the same formaldehyde content as the test group). The formaldehyde content is determined by subtracting the volatility value to reduce the error. The formaldehyde content is determined by the acetylacetone method, and the formaldehyde removal rate is calculated.

Table 3 Determination of the effect of BQB-B02 on formaldehyde degradation

As can be seen from Table 3, the formaldehyde content was significantly reduced after treatment with BQB-B02, and the formaldehyde removal rate was 83.71%. It can be seen that BQB-B02 has a significant effect on formaldehyde removal.

Example 3: Determination of the Formaldehyde Removal Effect of BQB-B02

In a wastewater biochemical treatment pool of a chemical plant (formaldehyde content was 150 mg/L, COD value was 1000 mg/L, pH value was 7), BQB-B02 bacterial solution (bacterial solution concentration was 5×10 ⁸ cfu/mL) was added to the wastewater at a dosage of 5% (v/v), the temperature of the treatment pool was controlled at 25℃-28℃, the aeration volume was 5-10m ³ /min, samples were taken after 6 hours and 12 hours, the formaldehyde content was determined by the acetylacetone method, and the formaldehyde removal rate was calculated.

Table 4 Determination of the removal effect of BQB-B02 on formaldehyde

As can be seen from Table 4, after 6 hours of treatment with strain BQB-B02, the formaldehyde content in the chemical wastewater decreased by 83.42%, and after 12 hours of treatment, the formaldehyde content in the chemical wastewater decreased by 90.11%. Ochrobacterium intermedia BQB-B02 has a significant effect on formaldehyde removal.

Example 4: Determination of indoor formaldehyde removal effect of BQB-B02

Select a ^60m3 sealed room, control the indoor temperature at 25℃-28℃, put 2500mL of BQB-B02 bacterial solution (bacterial solution concentration is 5× ¹⁰⁸ cfu/mL) into the spray device (humidifier), turn on the spray device to spray the bacterial solution evenly, and then add formaldehyde at one time, with a formaldehyde concentration of 0.25mg/ ^m3 . Take samples after 2 days and 3 days, determine the formaldehyde content by acetylacetone method, and calculate the formaldehyde removal rate.

Table 5 Determination of the removal effect of BQB-B02 on formaldehyde

The results show that after 2 days, the formaldehyde concentration has dropped to 0.04mg/m ³ , and the formaldehyde removal rate has reached 84%; after 3 days, it has reached 0.01mg/m ³ , and the formaldehyde removal rate has reached 96%. The formaldehyde concentration has fully met the national standard requirements. It can be seen that BQB-B02 bacterial solution can quickly degrade formaldehyde and has a significant effect on removing formaldehyde content in the air.

The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims (6)

1. A formaldehyde-degrading strain, characterized in that: the strain is Ochrobactrum intermedium BQB-B02, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 2, 2021, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No. 23339. 2. The use of the formaldehyde-degrading strain according to claim 1, characterized in that it is used to degrade formaldehyde. 3. The use according to claim 2, characterized in that: Ochrobactrum intermedium BQB-B02 is brought into contact with an environment containing formaldehyde to degrade the formaldehyde. 4. A formaldehyde-degrading bacterial agent, characterized in that the bacterial agent is a liquid bacterial agent, which contains the Ochrobactrum intermedium BQB-B02 described in claim 1. 5. The use of the formaldehyde degrading bacterial agent according to claim 4, characterized in that it is used to degrade formaldehyde. 6. The use according to claim 5, characterized in that a formaldehyde-degrading bacterial agent containing Ochrobactrum intermedium BQB-B02 is brought into contact with an environment containing formaldehyde to degrade formaldehyde.