A phosphorus-removing and phenol-degrading bacterium and its application

Abstract

The present invention discloses a phosphorus-removing and phenol-degrading bacterium and its application. This bacterium is a new strain of Zobellella sp., with the strain number S101 and the preservation number CGMCC No. 29317 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. This bacterium can perform heterotrophic nitrification and phosphorus removal, and can degrade phenol, and will have broad application prospects in aspects such as environmental governance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a phosphorus-removing and phenol-degrading bacterium and its application. Background Art

[0002] Zobellella was established in 2006, and there are currently only 6 effectively described species. Phenol is a raw material or intermediate in the production of industries such as coking, oil refining, medicine, and pesticides. The discharge of phenol-containing wastewater causes water pollution and seriously threatens human health. Developing functional microorganisms that can degrade phenol has important value. At the same time, phosphorus is a key nutrient causing water eutrophication, which will cause excessive reproduction of algae and deteriorate water quality. Biological phosphorus removal is based on the principle that phosphorus-removing bacteria uptake and release phosphorus under aerobic and anaerobic conditions, and realizes phosphorus removal through the alternating operation of aerobic-anaerobic conditions. Phosphorus-removing bacteria are a special type of bacteria in traditional activated sludge processes. Under aerobic conditions, they can absorb phosphorus in sewage into their bodies in excess, making the phosphorus content in their bodies several times higher than that in ordinary bacteria. Such bacteria are widely used for biological phosphorus removal. Generally, phosphorus-removing bacteria are divided into two types, facultative anaerobic denitrifying phosphorus-removing bacteria and aerobic phosphorus-removing bacteria. Among them, denitrifying phosphorus-removing bacteria can use oxygen or nitrate as electron acceptors, while aerobic phosphorus-removing bacteria can only use oxygen as an electron acceptor. Summary of the Invention

[0003] The purpose of the present invention is to provide a phosphorus-removing and phenol-degrading bacterium and its application.

[0004] In the first aspect, the present invention claims to protect a new species of Zobellella.

[0005] The Zobellella sp. S101 claimed to be protected by the present invention has a preservation number of CGMCC No. 29317 at the China General Microbiological Culture Collection Center.

[0006] In the second aspect, the present invention claims to protect a bacterial agent.

[0007] The bacterial agent claimed to be protected by the present invention has the above-mentioned Zobellella sp. S101 as its active ingredient.

[0008] Further, in the above-mentioned microbial agent, the microbial agent may further include a carrier. The carrier can be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material can be at least one of peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the biological material is at least one of straws of various crops, pine shells, rice straws, peanut shells, corn flour, soybean powder, starch, peat and animal feces; the liquid carrier can be water. In the microbial agent, the active ingredient can exist in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture or a mixture of cells and filtrate. The dosage form of the microbial agent can be various dosage forms, such as liquid agent, emulsion, suspension, powder, granule, wettable powder or water dispersible granule. According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the microbial agent.

[0009] In the third aspect, the present invention claims the use of the Zobellella sp. S101 described above or the microbial agent described above in heterotrophic nitrification and / or phosphorus removal.

[0010] Among them, the phosphorus can be total phosphorus.

[0011] In the fourth aspect, the present invention claims the use of the Zobellella sp. S101 described above or the microbial agent described above in degrading phenol.

[0012] The Zobellella sp. S101 provided by the present invention can carry out heterotrophic nitrification and phosphorus removal, and can degrade phenol, and will have broad application prospects in aspects such as environmental governance.

[0013] Depository Description

[0014] Name of the strain: Zobellella

[0015] Latin name: Zobellella sp.

[0016] Strain number: S101

[0017] Depository institution: China General Microbiological Culture Collection Center

[0018] Abbreviation of the depository institution: CGMCC

[0019] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences

[0020] Date of deposit: December 15, 2023

[0021] Accession number in the preservation center: CGMCC No. 29317 Description of the drawings

[0022] Figure 1 It is the phylogenetic tree of S101 and similar valid species.

[0023] Figure 2 It is the phylogenetic tree of S101 and similar strains. Detailed implementation manners

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0026] Example 1: Isolation and identification of Zobellella sp. S101

[0027] I. Isolation of Zobellella sp. S101

[0028] The pure strain S101 was isolated from the sediment sample of the river channel in Beijing in July 2022.

[0029] II. Identification

[0030] 1. Morphological, physiological and biochemical identification

[0031] The above-isolated S101 strain formed cyan, metallic luster colonies on the LB solid medium (formula: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7 - 8, agar powder 1.5%). The activated S101 was inoculated into the LB liquid medium (formula: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7 - 8), and cultured with shaking at 30 °C and 150 rpm for 24 h as the subsequent fresh seed bacterial solution. The pH value of the peptone 10 g / L and yeast extract 5 g / L liquid medium was adjusted to 7 - 8, and sodium chloride with final concentrations of 0, 5, 10, 11, 12, 13, 14, 15% was added respectively. The fresh seed bacterial solution of S101 was inoculated at 1% (v / v) respectively, and cultured with shaking at 30 °C and 150 rpm. S101 grew in 0 - 10% sodium chloride. According to the "Handbook of Common Bacterial System Identification", glucose fermentation and starch hydrolysis were measured. S101 was positive for glucose fermentation and negative for starch hydrolysis.

[0032] 2. 16S rDNA gene identification

[0033] The activated S101 was inoculated in LB liquid medium and cultured with shaking at 30 °C and 150 rpm for 48 h. 1 ml of the freshly cultured bacterial liquid was centrifuged at 8000 rpm for 5 min at 4 °C, and the cells were collected in a 2 ml centrifuge tube. DNA was extracted using a DNA extraction kit. After electrophoresis detection, PCR amplification was performed using the universal primers 8F / 1492R. After electrophoresis detection of the PCR products, the 16S rDNA gene sequence was determined, and the specific sequence is shown as Sequence 1 in the sequence listing.

[0034] After aligning the 16S rDNA gene sequence of the above S101 strain using the Ezbiocloud database, it was found that this strain belongs to the genus Zobellella, but has a low similarity with the valid species strains, and the highest similarity with Zobellella denitrificans is only 98.67%. The related sequences of the genus Zobellella in the Ezbiocloud database were used to construct a phylogenetic tree using the software MEGA as Figure 1 , and S101 is in an independent branch on the phylogenetic tree. There are differences in colony morphology, sodium chloride growth concentration, and starch hydrolysis between S101 and Zobellella denitrificans, as shown in Table 1.

[0035] Table 1 Differences between S101 and Zobellella denitrificans

[0036] S101 Zobellella denitrificans Colony morphology Cyan Greyish white Sodium chloride growth concentration 0-10％ 0-12％ Hydrolyzed starch - +

[0037] The related sequences of the genus Zobellella in the Ezbiocloud database, the most similar sequences MT368026, CP012621, MK100779 in NCBI, and the sequences in patents CN114231439A, CN113800652A, CN113913336A, CN113308410A, CN109897804A, CN109609419A, CN104845920A, CN103232954A were used to construct a phylogenetic tree using the software MEGA as Figure 2 , and S101 is still in an independent branch on the phylogenetic tree.

[0038] Therefore, the S101 strain is a new species of a newly discovered Zobellella sp.

[0039] Zobellella sp. S101 was deposited at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on December 15, 2023, with the deposit registration number of CGMCC No. 29317.

[0040] Example 2: Detection of the heterotrophic nitrification and phosphorus removal functions of the S101 CGMCC No. 29317 strain

[0041] The activated S101 CGMCC No. 29317 was inoculated into LB liquid medium and cultured with shaking at 30 °C and 150 rpm for 48 h. Freshly cultured bacterial liquid was centrifuged at 4 °C and 8000 rpm for 5 min, and the cells were collected and then suspended in an equal volume of sterile water to form a bacterial suspension. The S101 bacterial suspension was inoculated into the heterotrophic nitrification medium (formula: ammonium chloride 0.382 g / L, sodium acetate 2 g / L, magnesium sulfate 0.2 g / L, dipotassium hydrogen phosphate 0.2 g / L, sodium chloride 0.12 g / L, manganese sulfate 0.01 g / L, ferrous sulfate 0.01 g / L) at 2% (v / v), and there was an uninoculated control. It was cultured with shaking at 30 °C and 150 rpm for 5 d. The ammonia nitrogen content of the inoculated culture solution and the uninoculated control was determined using the standard salicylic acid spectrophotometric method for the determination of ammonia nitrogen in water quality HJ536 - 2009. The ammonia nitrogen degradation rate was calculated as (ammonia nitrogen concentration of the uninoculated control - ammonia nitrogen concentration of the inoculated culture solution) / ammonia nitrogen concentration of the uninoculated control × 100%, and the ammonia nitrogen degradation rate was 64.15%, indicating that the strain S101 has heterotrophic nitrification function. At the same time, the total phosphorus content of the inoculated culture solution and the uninoculated control was further determined using the ammonium molybdate spectrophotometric method for the determination of total phosphorus in water quality GB / T 11893 - 89. The total phosphorus degradation rate was calculated as (total phosphorus concentration of the uninoculated control - total phosphorus concentration of the inoculated culture solution) / total phosphorus concentration of the uninoculated control × 100%, and the total phosphorus degradation rate was 46.87%, indicating that the strain S101 has phosphorus removal function.

[0042] Example 3: Detection of the phenol degradation function of the S101 CGMCC No. 29317 strain

[0043] The activated S101 CGMCC No.29317 was inoculated into LB liquid medium and cultured with shaking at 30 °C and 150 rpm for 48 h. Freshly cultured bacterial liquid was centrifuged at 4 °C and 8000 rpm for 5 min, and the cells were collected and then suspended in an equal volume of sterile water to form a cell suspension. Fresh bacterial liquid was taken at 2% (V / V) and inoculated into phenol solution A (formula: dipotassium hydrogen phosphate 1 g / L, ammonium dihydrogen phosphate 1 g / L, ammonium sulfate 1 g / L, potassium nitrate 3 g / L, magnesium sulfate 0.2 g / L, phenol 0.1 g / L) and phenol solution B (formula: dipotassium hydrogen phosphate 1 g / L, ammonium dihydrogen phosphate 1 g / L, ammonium sulfate 1 g / L, potassium nitrate 3 g / L, magnesium sulfate 0.2 g / L, phenol 0.1 g / L, glucose 5 g / L) respectively, and cultured aerobically with shaking at 30 °C and 150 rpm for 3 d. Since phenol has a stable maximum absorption peak at a wavelength of 270 nm, and there is a good linear relationship between the concentration and OD270nm. Therefore, the ultraviolet spectrophotometry was used to select 270 nm to measure the initial phenol and the phenol concentration after the action of S-101. The phenol degradation rates of S-101 were calculated according to (initial phenol concentration - phenol concentration after action) / initial phenol concentration, which were: the phenol degradation rate in phenol solution A was 57.49%; the phenol degradation rate in phenol solution B was 92.19%.

Claims

1. Zobellella sp. S101, whose accession number at the General Microbiological Center of China National Microbiological Culture Collection Administration is CGMCC No.29317.

2. A bacterial agent, the active ingredient of which is the Zobellella sp. S101 described in claim 1.

3. Use of the Zobellella sp. S101 described in claim 1 or the bacterial agent described in claim 2 in heterotrophic nitrification and / or phosphorus removal.

4. Use of the Zobellella sp. S101 described in claim 1 or the bacterial agent described in claim 2 in degrading phenol.

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