WO2023041008A1

WO2023041008A1 - Kosakonia oryzae hn05 and use thereof

Info

Publication number: WO2023041008A1
Application number: PCT/CN2022/119217
Authority: WO
Inventors: 武春媛; 阴文芳; 吴东明
Original assignee: 中国热带农业科学院环境与植物保护研究所
Priority date: 2021-09-18
Filing date: 2022-09-16
Publication date: 2023-03-23
Also published as: CN113913326B; JP2023544961A; CN113913326A

Abstract

Provided are a Kosakonia oryzae HN05 strain and the use thereof. The strain can oxidize and degrade diquat under an anaerobic condition, and has the reduction activity of an anthraquinone compound. The strain can cooperate with AQDS to promote anaerobic degradation of diquat, and has good application prospects in the fields of pesticide pollution treatment and soil remediation when applied to treatment of diquat polluted water and soil remediation.

Description

A strain of Cossackia oryzae HN05 and its application

This application claims the priority of the Chinese patent application submitted to the China Patent Office on September 18, 2021, with the application number "202111096588.1", and the invention title "A Cossackia oryzae HN05 and its application", the entire content of which Incorporated in this application by reference.

technical field

The invention relates to the technical field of environmental microorganisms, in particular to a strain of Cossackia oryzae HN05 and its application.

Background technique

Diquat (1,1'-ethylene-2,2'-bipyridyl dibromide, diquat), is a non-selective contact bipyridyl herbicide, widely used in no-tillage and rapid crop rotation , Live rice and other agricultural and forestry production. Diquat has persistence and environmental health toxicity: Interfering with the oxidative phosphorylation reaction of zebrafish embryos, causing impaired growth of rainbow trout embryos, chronic death of Northwestern salamanders, and resulting in imbalances in aquatic ecosystems; Produces strong toxicity, long-term exposure to diquat can cause reproductive toxicity to female mice, and farmers have an increased risk of Parkinson's disease; acute poisoning of diquat can cause 2,128 diseases, and there is no special effect after bioaccumulation or acute poisoning Antidote. Therefore, it is necessary to accelerate the degradation of diquat in the environment.

Diquat is easily soluble in water. It enters the soil and groundwater with surface runoff, and is deposited in various anoxic environments. It is more persistent, with a half-life of up to ten years. Therefore, the key to accelerating the degradation of diquat in the environment is to strengthen its anaerobic degradation. It has been reported that methods such as advanced oxidation method and anaerobic digester method can effectively accelerate the anaerobic degradation of organic pollutants. However, these methods have disadvantages such as secondary pollution and high cost. Microbial in situ remediation technology is considered to be a good method to enhance the anaerobic degradation of organic pollutants due to its convenience, economy, high efficiency, and green features.

There are diquat-tolerant microorganisms in the soil, which can use diquat as a carbon or nitrogen source, or perform aerobic respiration in the form of co-metabolism. The reported aerobic degradation bacteria of diquat include Aspergillus niger Aspergillus niger, yeast Lipomyces starkeyi, and microorganisms related to the degradation and transformation of diquat under anaerobic conditions have not been reported yet.

There are currently 9 species of Kosakonia sp., and it is reported that except for Kosakonia quasisacchari and Kosakonia cowanii, which are isolated from human body, the other 7 species of Kosakonia sp. are all isolated from the rhizosphere of plants and have the ability to fix nitrogen. , corn, wheat, groundnut and other plants' dominant nitrogen-fixing bacteria, there is no literature report on the ability of Kosakonia sp. to reduce anthraquinone and degrade diquat.

Contents of the invention

In view of this, the present invention proposes a strain of Cossackia oryzae HN05 and its application. The strain is isolated from soil and has the characteristics of anaerobic degradation of diquat and reduction of anthraquinone.

Technical scheme of the present invention is realized like this:

The present invention provides a strain of Kosakonia oryzae HN05, which is classified as Kosakonia oryzae HN05, and was preserved in the Chinese Type Culture Collection Center located at Wuhan University, Wuhan City, Hubei Province on July 30, 2021. The collection center gave the strain The deposit number is CCTCC NO: M 2021956.

To further illustrate, the 16S rDNA sequence of rice Kosakonia oryzae HN05 is the nucleotide sequence shown in SEQ ID NO.1.

To further illustrate, the application of Kosakonia oryzae HN05 in the reduction of anthraquinone compounds.

To further illustrate, the application of the rice Kosakonia oryzae HN05 in pesticide pollution treatment and soil remediation.

It is further explained that the rice Kosakonia oryzae HN05 is used to degrade diquat to achieve the purposes of diquat polluted water treatment and soil remediation.

To further illustrate, the rice Kosakonia oryzae HN05 is used to degrade diquat under anaerobic conditions.

It is further illustrated that the rice Kosakonia oryzae HN05 and anthraquinone-2,6-sodium disulfonate act synergistically to promote the application of diquat in anaerobic degradation.

Compared with the prior art, the beneficial effect of the present invention is: the present invention is obtained by the enrichment, isolation and purification of rice Kosakonia oryzae HN05 (Kosakonia oryzae HN05) in the sediment soil of Nandu River, Hainan Province, and the bacterial strain is produced under anaerobic conditions. It can oxidatively degrade diquat, has reducing activity of anthraquinone compounds, and has a wide spectrum of electron utilization; at the same time, Cossackia oryzae HN05 can synergize with AQDS to significantly promote the anaerobic degradation of diquat, which is effectively used in Diquat polluted water treatment and soil remediation have good application prospects in the field of pesticide pollution treatment and soil remediation.

Description of drawings

Fig. 1 is the bacterial strain transmission electron microscope figure of rice cossackia HN05 of the present invention;

Fig. 2 is the 16S rRNA phylogenetic tree of rice cossackia HN05 of the present invention;

Fig. 3 is the electron donor spectrogram of the anaerobic reduction AQDS of Cossackia oryzae HN05 of the present invention;

Fig. 4 is the kinetic diagram of anaerobic degradation of diquat by Cossackia oryzae HN05 of the present invention;

Fig. 5 is a fitting curve of the first-order kinetics of cossackia oryzae HN05 and AQDS synergistically promoting anaerobic degradation of diquat in the present invention.

Biological Deposit Instructions

Kosakonia oryzae HN05 was deposited in the China Center for Type Culture Collection on July 30, 2021. The address is Wuhan University, Wuhan, Hubei, and the deposit number is: CCTCC NO: M2021956.

Detailed ways

In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.

The materials and reagents used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

The enrichment and separation of embodiment 1-rice cossackia HN05

1) Take 5g of sediment samples in 100mL anaerobic liquid medium under aseptic operation conditions, 0.5mmol/LAQDS (anthraquinone-2,6-sodium disulfonate, electron acceptor), 2.5g NaHCO ₃ , 0.25g NH ₄ Cl, 0.68g NaH ₂ PO ₄ ·2H ₂ O, 0.1g KCl, 10.0mL vitamin stock solution, 10.0mL trace element stock solution.

Among them, the vitamin solution contains 5.0mg of folic acid, 0.2mg of vitamin B6, 6.5mg of vitamin B2, 3.0mg of vitamin B1, 10mg of nicotinamide, 1mg of calcium pantothenate, 0.2mg of vitamin B12, and 2.0mg of vitamin H per liter of deionized water; The solution contains 1.6g EDTA, 3.0g MgSO ₄ 7H ₂ O, 0.5g MnSO ₄ H ₂ O, 1.0g NaCl, 0.1g CoCl ₂ 6H ₂ O, 0.1g CaCl per liter of deionized water _2. 0.01g CuSO ₄ ·5H ₂ O, 0.01g AlK(SO ₄ ) ₂ ·12H ₂ O, 0.01g H ₃ BO ₃ , 0.025g Na ₂ MoO ₄ ·2H ₂ O. Inflate the above soil-medium system with mixed gas (N ₂ /CO ₂ =80/20) for 30 minutes to deoxygenate. Immediately cover the rubber cover and pressurize the aluminum cover to seal after the inflation is completed. Keep it at 30°C in the dark for cultivation and observe the cultivation. The color change of the liquid;

2) When the color of the supernatant gradually changes from colorless to orange-yellow and tends to be stable, transfer it to another fresh enrichment medium with a 10% inoculum size, and transfer it 3 times;

3) Finally, the final culture solution is diluted and spread on the NA medium (by NaCl 5g/L, beef extract 5g/L, bacteriological peptone 10g/L, agar powder 18-20g/L, 1% trace element solution and 1% vitamin solution, 1×10 ⁵ Pa sterilized for 20min) surface for coating separation, aerobic culture at 30°C until a single colony was formed on the surface of the culture medium, single colony was picked for single colony isolation and purification, and obtained the Oryzaceae Saxobacteria HN05;

4) Pick a single colony and culture it again in a fresh enrichment medium. When the color of the system changes from colorless to orange-yellow, it indicates that the strain has AQDS reducing properties.

Morphological, physiological, biochemical and molecular biological characteristics of embodiment 2-rice Cossackia HN05

1) Morphological characteristics of bacteria

Observed under an optical microscope, the strain is Gram-negative bacteria, straight rod-shaped, single or in pairs. The perinatal flagella of the strain was observed under a transmission electron microscope (as shown in Figure 1), and it was motile.

After aerobic culture at 30°C for 24 hours on the NA agar solid medium plate with a pH of 7.2, the colony is round, with a moist and smooth surface, a raised middle, translucent, and neat edges, and the diameter of the colony is 1 to 3 mm.

2) Physiological and biochemical characteristics

The strain is fermentative and facultative anaerobic; the growth temperature range is 25-37°C (optimum 30°C), the pH range is 4-9 (optimum pH=6), and the NaCl% range is 0-5 (optimum 0.5%); Other physiological and biochemical characteristics are shown in Table 1.

Table 1 Some physiological and biochemical characteristics of Cossackia oryzae HN05

(Note: "+" means positive reaction, "-" means negative reaction).

3) Molecular biological characteristics

Bacterial DNA was extracted with a bacterial DNA extraction kit (Tiangen Biotechnology Co., Ltd.). Use bacterial 16S rRNA universal primers 27F (5'-AGA GTT TGATCC TGG CTCAG-3', SEQ ID NO.1) and 1492R (5'-TAC GGC TAC CTT GTTACGACT T-3', SEQ ID NO.2) for PCR Amplify. Reaction system 50 μL: template DNA 2 μL, universal primer 27F and 1492R 1 μL each, Taq mix enzyme 25 μL, ddH ₂ O 11 μL. PCR reaction conditions: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing at 52°C for 30s, extension at 72°C for 90s, 35 cycles; extension at 72°C for 10min. The amplified products were detected by 1% agarose gel electrophoresis and sequenced by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

According to the sequencing results, the 16S rRNA gene sequence with high homology was downloaded from http://eztaxon-e.ezbiocloud.net/, and the phylogenetic tree of the strain was constructed by the neighbor-joining method (as shown in Figure 2). Comprehensive morphology, physiology, biochemistry and molecular identification results finally determine that the bacterial strain of the present invention is Cossackia oryzae named Kosakonia oryzae HN05.

Example 3 - Cossackia oryzae HN05 uses different electron donors to reduce AQDS

The present invention uses AQDS as an electron acceptor to investigate the electron utilization spectrum of Cossackia oryzae HN05.

The composition of the reaction system: bacterial suspension, inorganic salts, vitamins, trace elements, the composition and content of each material are the same as the above-mentioned enrichment and separation medium; the electron acceptor is 0.5mmol/LAQDS; the electron donor is selected from acetic acid, glycerol, sucrose, or lactic acid, the added concentration is 5mmol/L; 1mL bacterial suspension, inoculate the strain HN05 in NA liquid medium, culture aerobically at 30℃ for 18h, collect the bacterial cells by centrifugation at 8000r/min, and wash the bacterial cells with inorganic salt medium 2 times, and finally suspend the bacteria in fresh inorganic salt medium to make a bacteria suspension.

Set up three replicates. The change of AQDS reduction product AH ₂ QDS in the anaerobic system was measured on the 1st, 3rd, 6th, 9th, 12th, and 15th day of culture respectively. A visible-ultraviolet spectrophotometer was used to measure the reduced AH ₂ QDS concentration at a wavelength of 385 nm. The results showed (as shown in Figure 3) that after 15 days of anaerobic cultivation, the AQDS in the anaerobic system was significantly reduced, and AH ₂ QDS in the system was produced in large quantities in the first 9 days, and gradually stabilized in the later period. The ability of Cossackia oryzae HN05 to utilize the four electron donors is obviously different, and the order of utilization ability from large to small is: sucrose > lactic acid > glycerol > acetic acid.

Example 4-Anaerobic degradation of Diquat by Cossackia oryzae HN05

1) Reaction system: In the anaerobic culture medium system, set 7 treatments,

①Sucrose (electron donor) + diquat (control, to investigate whether sucrose and diquat react);

②AQDS (electron acceptor) + diquat (control, to investigate whether AQDS and diquat react);

③HN05+diquat (control, to investigate whether live bacteria directly degrade diquat);

④Sucrose+HN05+diquat (to examine whether diquat can be degraded as an electron acceptor of HN05);

⑤AQDS+HN05+Diquat (to examine whether Diquat can be reduced as an electron donor of HN05);

⑥Sucrose+AQDS+HN05 (inactivated)+diquat (control, to investigate whether the dead bacteria of HN05 degrades diquat);

⑦Sucrose+AQDS+HN05+diquat (to investigate whether the microbial reduction of AQDS promotes the anaerobic degradation of diquat).

The anaerobic medium is composed of inorganic salts, vitamins, and trace elements (the composition and concentration of inorganic salts, microorganisms, and trace elements are the same as the enrichment and separation medium); the concentration of diquat is 50mg/L, AQDS is 0.5mmol/L, sucrose 5mmol/L, 1mL bacterial suspension (the preparation method is the same as the electron donor test). Diquat, AQDS and other components are sterilized separately, sterilized (sterilized at 115°C for 20 minutes) and then mixed, filled with (N ₂ /CO ₂ =80/20) mixed gas for 30 minutes to exhaust oxygen, and immediately cover the rubber cover after inflation And pressurize the aluminum cover to seal, and culture at 30°C in the dark;

2) Diquat detection method: use high performance liquid chromatography (Waters 2695) to determine the content of diquat, the chromatographic conditions are: the detector is PDA, the chromatographic column is Waters C18 column (5μm, 250mm×4.6mm), mobile phase It is 15mmol sodium heptanesulfonate phosphate buffer solution (triethylamine adjusted to pH=2.5): acetonitrile (v:v)=76:24, the flow rate is 1.0mL/min, the injection volume is 10μL, and the detection wavelength is 309nm.

3) Data processing: Diquat degradation rate (%) = (diquat initial concentration C ₀ -diquat concentration C after reaction)/C ₀ ×100%.

Degradation half-life t _1/2 = ln2/k (i.e. t _1/2 = 0.693/k), k is calculated by the formula C _t /C ₀ =e ^-kt , C ₀ is the initial concentration of diquat, and C _t is time t Diquat concentration.

The results are shown in Figure 4, the concentration of diquat in the control group ① and ② in anaerobic culture 0-22d was basically unchanged, indicating that there was no reaction between diquat and sucrose, AQDS, and simple HN05. Diquat did not decrease in treatment ⑤, indicating that diquat could not be degraded as an electron donor of HN05. The concentration of diquat in

treatments

③ and ⑥ decreased by 2.07%, indicating that the initial bacteria had a weak adsorption effect on diquat. The concentration of diquat in treatment ④ decreased by 8.60%, indicating that diquat could be directly degraded by the strain as the electron acceptor of HN05, but the effect was weak. The degradation rate of diquat in treatment ⑦ increased linearly during the incubation time, reaching 41.93% at 22 days, and the degradation effect was remarkable, which indicated that the synergistic effect of HN05 and AQDS could significantly promote the anaerobic degradation of diquat.

Further, in the system of HN05 and AQDS synergistically promoting the degradation of diquat, the degradation rate was fitted with first-order kinetics, and the results are shown in Figure 5. The coefficient of determination R ² =0.9091, k=0.0210±0.0030, conforming to the first-order Kinetic equations. On this basis, the half-life of strain HN05 to diquat degradation in this system was calculated to be (33.7±4.6)d.

Example 5 - Cossackia oryzae HN05 uses sucrose as an electron donor to reduce anthraquinone compounds

Using sucrose (5mmol/L) as the electron donor, the reduction ability of Cossackia oryzae HN05 to four anthraquinone compounds was investigated and compared.

Inoculate 1mL of bacterial suspension into basic anaerobic medium, add 0.5mmol/L anthraquinone-1-sodium sulfonate (α-AQS), anthraquinone-2-sodium sulfonate (AQS), anthraquinone-2, Four kinds of anthraquinone compounds such as 6-disodium disulfonate (AQDS) or anthraquinone-1,5-sodium disulfonate (1,5-AQDS) were used as potential electron acceptors, without adding HN05 and without adding sucrose The system was used as a control, and the culture conditions were the same as those for enrichment and separation. Set up three replicates. The changes of anthraquinone, the reduction product of anthraquinone in the anaerobic system were measured on the 1st, 3rd, 6th, 9th, 12th, and 15th day of culture respectively. Visible-ultraviolet spectrophotometry was used to measure the concentration of anthrahydroquinone. The ultraviolet absorption wavelengths of α-AQS, AQS, AQDS, and 1,5-AQDS reduction products were 380nm, 382nm, 385nm, and 385nm, respectively.

The results are shown in Table 2 below. After 15 days of anaerobic culture, in the "HN05+sucrose+anthraquinone" anaerobic system, the four anthraquinones were significantly reduced, while the control of "HN05+anthraquinone" and "sucrose+anthraquinone" In the system, no anthrahydroquinone was detected, indicating that Cossackia oryzae HN05 can use sucrose as an electron donor to anaerobically reduce four anthraquinone compounds, and the order of utilization ability from large to small is: AQDS>α-AQS> AQS>1,5-AQDS.

Table 2 Concentration of the reduction product anthrahydroquinone in the "HN05+sucrose+anthraquinone" system at 15 days

Note: AH ₂ QDS, α-AH ₂ QS, AH ₂ QS, and 1,5-AH ₂ QDS are the reduction products of AQDS, α-AQS, AQS, and 1,5-AQDS, respectively.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims

A strain of Kosakonia oryzae HN05 is characterized in that it is named Kosakonia oryzae HN05, and is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 2021956HN05.
A strain of Kosakonia oryzae HN05 is characterized in that: the 16S rDNA sequence of Kosakonia oryzae HN05 is the nucleotide sequence shown in SEQ ID NO.1.
The application of Cossackia oryzae HN05 described in claim 1 in the reduction of anthraquinone compounds.
The application of Cossackia oryzae HN05 combined with sucrose in the anaerobic reduction of anthraquinone compounds according to claim 1.
The application according to claim 4, characterized in that the concentration of the sucrose used is 5mmol/L.
The application according to any one of claims 3-5, characterized in that the anthraquinone compound includes one of AH 2 QDS, α-AH 2 QS, AH 2 QS and 1,5-AH 2 QDS or several.
The application of Cossackia oryzae HN05 described in claim 1 in pesticide pollution treatment and soil remediation.
The application according to claim 7, characterized in that: the Cossackia oryzae HN05 is used to degrade diquat to achieve the purposes of diquat polluted water treatment and soil remediation.
The application according to claim 8, characterized in that the Cossackia oryzae HN05 is used to degrade diquat under anaerobic conditions.
The application of cossackia oryzae HN05 combined with anthraquinone-2,6-sodium disulfonate in anaerobic degradation of diquat according to claim 1.
The application according to claim 10, characterized in that the concentration of the sodium anthraquinone-2,6-disulfonate is 0.5mmol/L.