CN117402793A - Salt-tolerant alkalophilic surfactant-producing enzyme-producing bacterium and application thereof - Google Patents

Abstract

The object of the present invention is to provide a salt-tolerant, alkali-loving, surfactant- and enzyme-producing bacterium and its application. The name of the strain provided is W16, and the collection number of the General Microbiology Center of China Microbial Culture Collection Committee is CGMCC No. 28376. This bacterium can produce surfactant and β-glucosidase under high salt and high alkali conditions, and can perform heterotrophic nitrification. It is a functional new strain of Nesterenkoia and will have a wide range of applications in environmental management and other aspects. application prospects.

Description

A salt-tolerant and alkali-loving surfactant-producing enzyme-producing bacterium and its application

Technical field

The invention belongs to the field of biotechnology and relates to a salt-tolerant and alkali-resistant surfactant-producing enzyme-producing bacterium and its application.

Background technique

Biosurfactants mainly refer to surface-active secondary metabolites produced by microorganisms. Common biosurfactants include lipopeptides, glycolipids, etc. With the strengthening of the concept of green, environmentally friendly and sustainable development in various fields, biosurfactants have received more and more attention. Cellulase is a type of enzyme that can hydrolyze cellulose into oligoglucose and glucose. Many natural microorganisms contain cellulase. Cellulases can be widely used in textile, paper, food and biofuel industries. However, during the process of cellulase hydrolysis, the large amount of cellobiose and oligosaccharides produced will strongly inhibit the activity of cellulase, resulting in a sharp decrease in cellulose hydrolysis efficiency. β-Glucosidase can efficiently catalyze the conversion of cellobiose and oligosaccharides into glucose, and will relieve product inhibition, thereby ensuring that the entire hydrolysis process is continuous and stable. Ammonia nitrogen can consume a large amount of dissolved oxygen in water bodies, causing hypoxia in water bodies, and may also cause eutrophication of water bodies. Traditional nitrifying bacteria are autotrophic bacteria that require strict growth environments, slow growth rates, and are difficult to cultivate in large quantities, resulting in limited applications. Heterotrophic nitrifying bacteria can directly use organic matter nutrients in the water body to grow while degrading ammonia nitrogen. They have strong adaptability, fast growth rate, and can remove COD and ammonia nitrogen at the same time.

Contents of the invention

One object of the present invention is to provide Nesterenkonia sp. W16.

The deposit number of Nesterenkonia sp. W16 provided by the present invention is CGMCC No. 28376.

The application of the above-mentioned Nesterenkoella or its bacterial suspension or its culture solution or its fermentation product in at least one of the following is also within the scope of protection of the present invention:

1) Application in the production of biosurfactants or bioemulsifiers or application in the preparation of products that produce biosurfactants or bioemulsifiers;

2) Application in the production of β-glucosidase or application in the preparation of products for the production of β-glucosidase;

3) Application in degrading ammonia nitrogen or application in preparing products that degrade ammonia nitrogen.

Another object of the present invention is to provide a product.

The product provided by the invention includes the above-mentioned Nesterenkoella or its bacterial suspension or its culture solution or its fermentation product.

The above products have at least one of the following functions 1)-3):

1) Application of the product in the production of biosurfactants or bioemulsifiers;

2) Application of the product in the production of β-glucosidase;

3) Application of the product in degrading ammonia nitrogen.

The above-mentioned culture of Nesterenkoia W16 also belongs to the protection scope of the present invention. The culture of Nestrenkoella W16 is a substance obtained by culturing Nestrenkoella W16 in a microbial medium (for example, a substance containing Nestrenkoella W16 and secreted into a liquid medium i.e. fermentation broth, or if it contains Nesterenkoia W16 and substances secreted into solid culture media).

The above-mentioned culture of Nesterenkoia W16 has at least one of the following functions 1)-3):

1) Produce biosurfactants or bioemulsifiers;

2) Produce β-glucosidase;

3) Degradation of ammonia nitrogen.

Experiments of the present invention prove that the name of the strain provided by the present invention is W16, and the collection number of the General Microorganism Center of China Microbial Culture Collection Committee is CGMCC No. 28376. This bacterium can produce surfactant and β-glucosidase under high salt and high alkali conditions, and can perform heterotrophic nitrification. It is a new functional strain of Nesterenkoia.

Preservation instructions

Species name: Nesterenkoia

Latin name: Nesterenkonia sp.

Strain number: W16

Collection institution: General Microbiology Center of China Committee for the Collection of Microbial Cultures

Abbreviation of depository institution: CGMCC

Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing

Storage date: September 7, 2023

Collection center registration number: CGMCC No.28376

Description of the drawings

Figure 1 shows the NCBI comparison results.

Figure 2 shows the W16 phylogenetic tree.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1. Isolation and identification of Nesterenkonia sp.W16

1. Isolation of Nesterenkonia sp.W16

In September 2020, pure strain W16 was isolated from Zhangjiakou saline-alkali soil samples.

2. Identification

1. Morphological, physiological and biochemical identification

The W16 strain 2216E isolated above formed fine green-yellow colonies on the solid medium. The activated W16 was inoculated into a liquid culture medium containing 5g/L peptone, 1g/L yeast powder, 25g/L sodium chloride, and a pH value adjusted to 9-10, and cultured with shaking at 30°C and 150rpm for 24 hours as the subsequent fresh seed bacterial liquid. . Adjust the pH values of peptone 5g/L, yeast powder 1g/L, and sodium chloride 25g/L liquid culture medium to 7, 8, 9, 10, 11, and 12 respectively, and connect them at 1% (v/v) respectively. W16 fresh bacterial liquid is cultured with shaking at 30°C and 150rpm. W16 grows at a pH value of 9-11, and the optimal pH is 10. Adjust the pH values of peptone 5g/L and yeast powder 1g/L liquid culture medium to 9 respectively, add sodium chloride with final concentrations of 0, 2.5, 5, 7.5, 10, 15, and 20%, respectively. (v/v) Add fresh bacterial liquid of W16, culture with shaking at 30°C and 150rpm, and grow W16 in 0-10% sodium chloride.

2. Identification of 16S rDNA gene

The activated W16 was inoculated into a liquid medium containing 5g/L peptone, 1g/L yeast powder, 25g/L sodium chloride, and a pH value adjusted to 9-10, and cultured with shaking at 30°C and 150rpm for 24 hours. Take 1 ml of fresh bacterial culture and centrifuge it at 4°C and 8000 rpm for 5 min, collect the cells in a 2 ml centrifuge tube, and use a DNA extraction kit to extract DNA. After electrophoresis detection, universal primer 8F/1492R was used for PCR amplification. After electrophoresis detection of the PCR product, the 16S rDNA gene sequence was determined. The specific sequence is shown in Sequence Listing 1.

After comparing the 16S rDNA gene of the above-mentioned W16 strain using the NCBI database and the Ezbiocloud database, it was found that the strain belongs to the genus Nesterenkonia, but its similarity to existing model strains is low. The most similar sequence to NCBI, KJ417902, and the most similar effective species, Nesterenkonia haasae strain Hz 6-5 (serial number MG279104, this strain is also the patented CN109439562A strain), are only 99.00% similar, as shown in Figure 1. There are differences in sodium chloride utilization concentration and optimal pH value between W16 and the most similar effective species Nesterenkonia haasae, as shown in Table 1.

Table 1Differences between W16 and Nesterenkonia haasae

project W16 Nesterenkonia haasae Sodium chloride concentration range 0-15% 0-25% Optimum pH value 10 9-9.5

The related sequences of the genus Nesterenkonia in the Ezbiocloud database, as well as the sequences of suspected new species disclosed in patents CN108239612A and CN109439562A, and the NCBI most similar sequence KJ417902, were used to construct a phylogenetic tree using the software MEGA as shown in Figure 2. W16 is in an independent group on the phylogenetic tree. branch.

Therefore, strain W16 is a newly discovered species of the genus Nesterenkonia.

Nesterenkonia sp. W16 has been deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures (CGMCC for short) on September 7, 2023. The address is: No. 1 Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is CGMCC No. 28376.

Example 2. Testing of surfactant-producing function of W16 CGMCC No. 28376 strain

The W16 CGMCC No. 28376 strain obtained in Example 1 was inoculated into a liquid medium containing 5g/L peptone, 1g/L yeast powder, 25g/L sodium chloride, and a pH value adjusted to 9-10, and shaken at 30°C and 150rpm. After culturing for 72 hours, W16 bacterial liquid was obtained. Centrifuge the bacterial solution at 4°C and 8000 rpm for 5 min, take the supernatant, and obtain the supernatant culture liquid.

The blank culture medium without W16 bacterial liquid was used as a control to obtain a control culture medium.

The emulsification index (E24) was determined according to the method in the literature "Study on the Properties of Biosurfactant Rhamnolipids".

Emulsification index (E24) = emulsion height/total liquid height × 100%

Results: The emulsification index (E24) of the W16 supernatant culture medium was 35.7%, and the emulsification index (E24) of the control culture medium was 0, indicating that strain W16 has the function of producing surfactant.

Example 3. β-glucosidase activity of W16 CGMCC No. 28376 strain

Solid culture medium: In a liquid culture medium with peptone 5g/L, yeast powder 1g/L, sodium chloride 25g/L, and a pH value adjusted to 9-10, add 0.05% esculin and 0.25% ferric ammonium citrate. and 1.5% agar, sterilized and poured onto solid plates.

The W16 CGMCC No. 28376 strain obtained in Example 1 was inoculated into a liquid medium containing 5g/L peptone, 1g/L yeast powder, 25g/L sodium chloride, and a pH value adjusted to 9-10, and shaken at 30°C and 150rpm. After culturing for 24 hours, W16 bacterial liquid (CFU content: 10 ⁹ /ml) was obtained.

Take 5 μl of the above-mentioned fresh W16 bacterial liquid and place it on the above-mentioned solid medium, and incubate at 30°C for 48 hours.

The results showed that an obvious black precipitation circle appeared around the W16 colony, and the diameter ratio of the black precipitation circle to the colony was 17/8 = 2.12, indicating that strain W16 had good β-glucosidase activity.

Example 4. Heterotrophic nitrification function detection of W16 CGMCC No. 28376 strain

High-salt and high-alkali liquid heterotrophic nitrification medium formula: (NH ₄ ) ₂ SO ₄ 0.5g/L, sodium acetate 2g/L, K ₂ HPO ₄ ·3H ₂ O3.27g/L, KH ₂ PO ₄ 0.3g/ L, NaCI 50g/L, pH 9.

The W16 CGMCC No. 28376 strain obtained in Example 1 was inoculated into a liquid medium containing 5g/L peptone, 1g/L yeast powder, 25g/L sodium chloride, and a pH value adjusted to 9-10, and shaken at 30°C and 150rpm. After culturing for 24 hours, W16 bacterial liquid (CFU content: 10 ⁹ /ml) was obtained.

Take 1000 μl of the above-mentioned fresh W16 bacterial liquid and centrifuge it at 4°C and 8000 rpm for 5 min, collect the bacterial cells, and then suspend them with an equal volume of sterile water to form a W16 bacterial suspension (CFU content is 10 ⁹ /ml).

Then, the W16 bacterial suspension was inoculated into the liquid heterotrophic nitrification medium at 1% (volume percentage), cultured with shaking at 30°C and 150 rpm for 7 days, and the W16 culture liquid was collected.

The W16 bacterial suspension without inoculation was used as a control to obtain a control culture medium.

The ammonia nitrogen content in W16 culture medium and control culture medium was measured using the standard "HJ536-2009 Determination of Ammonia Nitrogen in Water Quality by Salicylic Acid Spectrophotometry Detection", which was 8.47 mg/L and 101.68 mg/L respectively.

Ammonia nitrogen removal rate = (ammonia nitrogen concentration in the culture medium without bacteria - ammonia nitrogen concentration in culture medium with bacteria W16) / ammonia nitrogen concentration in the culture medium without bacteria × 100%

Results: The ammonia nitrogen removal rate in W16 culture solution was 91.67%, indicating that strain W16 has heterotrophic nitrification function.

Claims (5)

1. Nestrenkoella W16, its deposit number is CGMCC No. 28376. 2. Any of the following applications of Nesterenkoia as claimed in claim 1: 1) Application of Nesterenkoia W16 in the production of biosurfactants or bioemulsifiers or in the preparation of products for the production of biosurfactants or bioemulsifiers; 2) Application of Nesterichia W16 in the production of β-glucosidase or in the preparation of products for the production of β-glucosidase; 3) Application of Nesterenkoia W16 in degrading ammonia nitrogen or in preparing products that degrade ammonia nitrogen. 3. A product, characterized in that it contains Nestrenkoella W16 according to claim 1. 4. Any of the following applications of the product according to claim 3: 1) Application of the product in the production of biosurfactants or bioemulsifiers; 2) Application of the product in the production of β-glucosidase; 3) Application of the product in degrading ammonia nitrogen. 5. The culture of Nesterenkoia according to claim 1, characterized in that: the culture has the following functions 1) or 2) or 3): 1) Produce biosurfactants or bioemulsifiers; 2) Produce β-glucosidase; 3) Degradation of ammonia nitrogen.