Kosaconia oryzae HN05 and its uses

Abstract

The present invention relates to the technical field of environmental microorganisms, in particular to Kosakonia oryzae HN05 and its uses. The strain is classified as Kosakonia oryzae HN05 and can degrade diquat under anaerobic conditions. It has the activity of reducing anthraquinone compounds and has a wide spectrum of electron utilization. At the same time, the strain HN05 can synergize with AQDS to significantly promote the anaerobic degradation of diquat, making it suitable for the treatment of diquat-contaminated water and soil remediation, and has good potential for use in the fields of pesticide pollution treatment and soil remediation.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to a Chinese patent application filed with the China Patent Office on September 18, 2021, with application number "202111096588.1" and titled "Kosaconia oryzae HN05 and its use", the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the technical field of environmental microorganisms, in particular to Kosaconia oryzae HN05 and uses thereof. [Background technology]

[0003] Diquat (1,1'-ethylene-2,2'-bipyridine dibromide, diquat) is a non-selective contact type bipyridine herbicide, widely used in agricultural and forestry production such as no-till cultivation, rapid crop rotation, and direct seeding rice. Diquat has persistent and environmental health toxicity. It inhibits the oxidative phosphorylation reaction of zebrafish embryos, causes growth disorders of rainbow trout embryos, chronic death of northwestern salamanders, leads to imbalance of aquatic ecosystems, produces strong toxicity to mammals through groundwater soaking or food chain form, long-term exposure to diquat may cause reproductive toxicity in female mice, farmers are at high risk of Parkinson's disease, acute poisoning of diquat may cause 2128 kinds of diseases, and there is no special antidote after biological accumulation or acute poisoning. Therefore, it is very necessary to accelerate the decomposition of diquat in the environment.

[0004] Diquat is highly soluble in water and is more persistent as surface runoff enters soil, groundwater bodies and deposits in various low-oxygen environments, with a half-life of up to 10 years. Therefore, the key to accelerating the degradation of diquat in the environment is to enhance its anaerobic degradation. It has been reported that methods such as advanced oxidation and anaerobic digestion pool method can effectively accelerate the anaerobic degradation of organic pollutants. However, these methods have the disadvantages of secondary pollution and high cost. Microbial in situ remediation technology is considered to be a good way to enhance the anaerobic degradation of organic pollutants due to its convenient, economical, efficient and green characteristics.

[0005] There are microorganisms in soil that can tolerate diquat, and these bacteria can perform aerobic respiration using diquat as a carbon or nitrogen source, or in a co-metabolic form. The currently reported aerobic decomposition bacteria for diquat include Aspergillus niger and the yeast Lipomyces starkeyi, but no microorganisms related to the decomposition and transformation of diquat under anaerobic conditions have been reported.

[0006] There are currently nine species of Kosakonia strains, and according to reports, except for Kosakonia quasisacchari and Kosakonia cowanii, which are isolated from the human body, the other seven species of Kosakonia sp. are all isolated from plant roots and have the ability to fix nitrogen, making them excellent nitrogen fixers for plants such as rice, corn, wheat, and peanut. At present, there are no literature reports related to the ability of Kosakonia sp. to reduce anthraquinone and decompose diquat. Summary of the Invention [Problem to be solved by the invention]

[0007] In view of this, the present invention provides Kosaconia oryzae HN05 and its use, the strain being isolated from soil and having the characteristics of anaerobic degradation of diquat and reduction of anthraquinone. [Means for solving the problem]

[0008] The technical solution of the present invention is thus realized.

[0009] The present invention provides Kosakonia oryzae HN05, named Kosakonia oryzae HN05, which was deposited at the China Typical Culture Depository Center, Wuhan University, Wuhan, Hubei on July 30, 2021, and the deposit number given to the strain by the depository center is CCTCC NO:M 2021956.

[0010] As further described, the 16S rDNA sequence of Kosakonia oryzae HN05 is the nucleotide sequence shown in SEQ ID NO.1.

[0011] The use of Kosakonia oryzae HN05 in the reduction of anthraquinones compounds as further described.

[0012] As further described, the use of Kosakonia oryzae HN05 in pesticide contamination treatment and soil remediation.

[0013] As will be further described, the Kosakonia oryzae HN05 can be used to degrade diquat, achieving the purpose of diquat-contaminated water treatment and soil remediation.

[0014] As will be further described, Kosakonia oryzae HN05 is used to degrade diquat under anaerobic conditions.

[0015] As will be further described, Kosakonia oryzae HN05 is used in combination with sodium anthraquinone-2,6-disulfonate to promote the anaerobic degradation of diquat. Effect of the Invention

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention is Kosakonia oryzae HN05, which is concentrated, separated and purified from the sedimentary soil of Nandu River, Hainan Province, this strain can degrade diquat under anaerobic conditions, has anthraquinone compound reduction activity, and has a wide spectrum of electron utilization; at the same time, Kosakonia oryzae HN05 can synergize with AQDS to significantly promote the anaerobic decomposition of diquat, which can be effectively applied to diquat-polluted water treatment and soil remediation, and has good potential for use in the fields of pesticide pollution treatment and soil remediation. [Brief description of the drawings]

[0017] [Figure 1] This is a transmission electron microscope image of the morphological structure of the Kosaconia oryzae HN05 strain of the present invention. [Diagram 2] FIG. 16S rRNA phylogenetic tree of Kosaconia oryzae HN05 of the present invention. [Diagram 3] FIG. 1 is an electron donor spectrum of anaerobic reduced AQDS from Kosakonia oryzae HN05 of the present invention. [Figure 4] FIG. 2 is a kinetic diagram of the anaerobic decomposition of diquat by Kosaconia oryzae HN05 of the present invention. [Diagram 5] The first level kinetic fitting curve of the present invention's Kosaconia oryzae HN05 and AQDS jointly promoting the anaerobic degradation of diquat.

[0018] Description of biological deposit On July 30, 2021, Kosakonia oryzae HN05 was deposited in the China Typical Culture Depository Center, Wuhan University, Wuhan, Hubei, with the deposit number CCTCC NO:M 2021956. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In order to better understand the technical contents of the present invention, the following provides specific examples to further illustrate the present invention.

[0020] The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.

[0021] Materials and reagents used in the examples of the present invention are obtained from commercial sources unless otherwise specified.

[0022] Example 1 - Concentration and separation of Kosaconia oryzae HN05 (1) Under sterile operating conditions, a 5 g sediment sample was placed in 100 ml of anaerobic liquid medium containing 0.5 mmol / L LAQDS (sodium anthraquinone-2,6-disulfonate, electron acceptor), 2.5 g NaHCO3, 0.25 g NH4Cl, 0.68 g NaH2PO4·2H2O, 0.1 g KCl, 10.0 ml vitamin stock solution, and 10.0 ml trace element stock solution per liter of medium. Here, the vitamin solution contains 5.0 mg folic acid, 0.2 mg vitamin B6, 6.5 mg vitamin B2, 3.0 mg vitamin B1, 10 mg nicotinamide, 1 mg calcium pantothenate, 0.2 mg vitamin B12, and 2.0 mg vitamin H per liter of deionized water, and the trace element solution contains 1.6 g ethylenediaminetetraacetic acid, 3.0 g MgSO4·7H2O, 0.5 g MnSO4·H2O, 1.0 g NaCl, 0.1 g CoCl2·6H2O, 0.1 g CaCl2, 0.01 g CuSO4·5H2O, 0.01 g AlK(SO4)2·12H2O, 0.01 g H3BO3, and 0.025 g Na2MoO4·2H2O per liter of deionized water. A gas mixture (N2 / CO2 = 80 / 20) was blown into the soil-culture medium for 30 minutes to remove oxygen. Once the blowing was complete, the mixture was immediately covered with a rubber cover, and the aluminum cover was pressed to seal it. The mixture was then left to stand at 30°C, away from light, and cultured, and the color change of the culture solution was observed. (2) When the color of the supernatant gradually changed from colorless to orange and tended to stabilize, it was transferred to another fresh enrichment medium with an inoculum amount of 10%, and transferred in this manner three times. (3) Finally, the final culture solution was cultured by dilution coating plate method using NA medium (NaCl 5g / L, beef extract 5g / L, bacterial peptone 10g / L, agar powder 18-20g / L, 1% trace element solution and 1% vitamin solution, at 1 × 10 5 Coating isolation was performed on a surface (sterilized with PA for 20 min), and the surface was aerobically cultured at 30 °C until a single colony was formed on the surface of the medium. A single colony was picked up and single colony isolation and purification were performed to obtain Kosaconia oryzae HN05. (4) A single colony was picked up and re-cultured in fresh enrichment medium. If the color of the system changed from colorless to orange, it indicated that the strain had the property of reducing AQDS.

[0023] Example 2 – Morphological, physiological, biochemical and molecular biological characteristics of Kosaconia oryzae HN05 (1) Morphological characteristics of the bacterial cell Under optical microscopy, the strain was Gram-negative, straight rod shaped, single or paired. Under transmission electron microscopy, the peritrichous tissue of the strain was observed (shown in Figure 1) and was motile. After aerobic incubation at 30°C for 24 h on a pH 7.2 NA agar solid medium plate, the colonies were round, had a moist and smooth surface, a protruding center, translucent, neat edges, and a colony diameter of 1 to 3 mm. (2)Physiological and biochemical properties The strain is fermentative and facultatively anaerobic, with a growth temperature range of 25-37°C (most suitable 30°C), pH range of 4-9 (most suitable pH=6), NaCl% range of 0-5 (most suitable 0.5%), and other physiological and biochemical characteristics as shown in Table 1.

[0024] [Table 1]

[0025] (3) Characteristics of molecular biology Bacterial total DNA was extracted with a bacterial DNA extraction kit (Tiangen Biotechnology Co., Ltd.). The bacterial 16S rRNA universal primer 27F (5'-AGA GTT TGA TCC TGG CTC AG-3', SEQ ID NO. 2 ) and 1492R (5'-TAC GGC TAC CTT GTT ACG ACT T-3', SEQ ID NO. 3 ) was used to perform PCR amplification. The reaction system was 50μL: 2μL template DNA, 1μL each of universal primers 27F and 1492R, 25μL Taq mix enzyme, and 11μL ddH2O. PCR reaction conditions: 5min initial denaturation at 95℃, 30s denaturation at 95℃, 30s annealing at 52℃, 90s extension at 72℃, 35 cycles, 10min extension at 72℃. The amplified products were detected by 1% agarose gel electrophoresis and the sequencing was completed by Shanghai Biotechnology Engineering Technology Services Co., Ltd.

[0026] According to the sequencing results, the 16S rRNA gene sequences with higher homology were downloaded from http: / / eztaxon-e.ezbiocloud.net / , and a phylogenetic evolutionary tree of the strains was constructed by the neighbor-joining method (shown in Figure 2). Combining the results of morphology, physiological biochemical and molecular identification, the strain of the present invention was finally determined to be Kosakonia oryzae, named Kosakonia oryzae HN05.

[0027] Example 3 – Kosaconia oryzae HN05 reduced AQDS using different electron donors

[0028] In this study, AQDS was used as the electron acceptor and the electron utilization spectrum of Kosaconia oryzae HN05 was investigated.

[0029] 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 concentrated isolation medium, the electron acceptor is 0.5 mmol / LAQDS, the electron donor is selected from acetic acid, glycerin, sucrose, or lactic acid, and the added concentration is 5 mmol / L. 1 mL of bacterial suspension. HN05 strain was inoculated into NA liquid medium and aerobic cultured at 30°C for 18 h. The bacterial cells were collected by centrifugation at 8000 r / min, then the bacterial cells were washed twice with inorganic salt medium, and finally the bacterial cells were suspended in fresh inorganic salt medium to make a bacterial suspension.

[0030] Three replicates were set up. The change status of AH2QDS, the product of AQDS reduction in the anaerobic system, was measured after 1, 3, 6, 9, 12, and 15 d of cultivation, respectively. A visible-ultraviolet spectrometer was adopted to measure the concentration of reduced AH2QDS at a wavelength of 385 nm. The results showed that after 15 d of anaerobic cultivation (as shown in Figure 3), there was an obvious reduction phenomenon in AQDS in the anaerobic system, and a large amount of AH2QDS was produced in the system in the first 9 days, and gradually stabilized in the later period. The utilization abilities of the four electron donors in Kosaconia oryzae HN05 were obviously different, and the utilization abilities were sequentially from large to small: sucrose > lactic acid > glycerin > acetic acid.

[0031] Example 4 - Kosaconia oryzae HN05 anaerobic decomposition of diquat 1. Reaction system: In the anaerobic medium system, seven treatments were set up. (1) Sucrose (electron donor) + diquat (control, to examine whether sucrose and diquat react). (2) AQDS (electron acceptor) + diquat (control, to examine whether AQDS and diquat react). (3) HN05 + diquat (control, to examine whether living bacteria could directly degrade diquat). (4) Sucrose + HN05 + diquat (we considered whether diquat would be decomposed as an electron acceptor for HN05). (5) AQDS + HN05 + diquat (we considered whether diquat was reduced as an electron donor for HN05). (6) Sucrose + AQDS + HN05 (inactivated) + diquat (control, to examine whether HN05 dead bacteria could decompose diquat). (7) Sucrose + AQDS + HN05 + diquat (to examine whether microbial reduction of AQDS promotes the anaerobic degradation of diquat).

[0032] The anaerobic medium is composed of inorganic salts, vitamins, and trace elements (the components and concentrations of inorganic salts, microorganisms, and trace elements are the same as those of the concentrated isolation medium), with diquat concentrations of 50 mg / L, AQDS of 0.5 mmol / L, and sucrose of 5 mmol / L, and 1 mL of bacterial suspension (the manufacturing method is the same as that of the electron donor test). Diquat and AQDS were sterilized separately from other components, and after sterilization (sterilization at 115 ° C for 20 min), they were mixed again, and oxygen was discharged by blowing in a mixed gas (N2 / CO2 = 80 / 20) for 30 min. After the blowing was completed, the rubber cover was immediately placed over the medium, the aluminum cover was pressed to seal it, and the medium was left to stand at 30 ° C and cultured away from light. 2. Detection method of diquat: Use high-efficiency liquid chromatography (Waters 2695) to determine the content of diquat. The chromatography conditions are: detector is PDA, chromatography column is Waters C18 column (5μm, 250mm×4.6mm), mobile phase is 15mmol sodium heptanesulfonate phosphate buffer solution (adjusted to pH=2.5 with triethylamine):acetonitrile (v:v)=76:24, flow rate is 1.0mL / min, sample injection amount is 10μL, detection wavelength is 309nm. 3. Data processing: diquat decomposition rate (%) = (initial concentration of diquat C0 - concentration of diquat after reaction C) / C0 × 100%. Decomposition half-life t 1 / 2 = ln2 / k (i.e. t 1 / 2 =0.693 / k), where K is the formula C t / C0=e -kt where C is the initial concentration of diquat and C t was the concentration of diquat at time t.

[0033] The results are shown in Figure 4. The concentration of diquat in the control groups (1) and (2) cultured anaerobic for 0-22d was basically unchanged, indicating that there was no reaction between diquat and sucrose, AQDS, and simple HN05. The diquat in treatment (5) also did not decrease, indicating that diquat was not decomposed as an electron donor for HN05. The diquat concentration in treatments (3) and (6) decreased by 2.07%, indicating that the initial bacterial cells had a weak adsorption effect on diquat. The concentration of diquat in treatment (4) decreased by 8.60%, indicating that diquat was directly decomposed by the strain as an electron acceptor for HN05, but the effect was weak. The diquat degradation rate in treatment (7) increased linearly during the culture time, reaching 41.93% at 22d, with a significant degradation effect, which explained that the synergistic action of HN05 and AQDS could significantly promote the anaerobic degradation of diquat.

[0034] Furthermore, in the system where HN05 and AQDS jointly promote the degradation of diquat, a first-level kinetic fitting was performed for the degradation rate, and the results are shown in Figure 5. The coefficient of determination R 2 = 0.9091, k = 0.0210 ± 0.0030, which follows the first level kinetic equation. Based on this, the half-life range for degradation of diquat by strain HN05 in this system was calculated to be (33.7 ± 4.6) d.

[0035] Example 5 - Kosaconia oryzae HN05 utilizes sucrose as an electron donor to reduce anthraquinone compounds The reduction ability of Kosaconia oryzae HN05 to four anthraquinone compounds using sucrose (5 mmol / l) as the electron donor was examined and compared. 1 mL of the bacterial suspension was inoculated into basic anaerobic medium, and four anthraquinone compounds, such as 0.5 mmol / L anthraquinone-1-sodium sulfonate (α-AQS), anthraquinone-2-sodium sulfonate (AQS), anthraquinone-2,6-disodium disulfonate (AQDS), or anthraquinone-1,5-sodium disulfonate (1,5-AQDS), were added as potential electron acceptors. The culture conditions were the same as those for enrichment and separation, with a system without HN05 and a system without sucrose added as controls. Three replicates were set up. The changes in anthrahydroquinone, the reduction product of anthraquinone, in the anaerobic system were measured when the medium was cultured for 1, 3, 6, 9, 12, and 15 d, respectively. The concentrations of anthrahydroquinones were measured using UV-vis spectrophotometry, and the UV absorption wavelengths of the reduction products of α-AQS, AQS, AQDS, and 1,5-AQDS were 380 nm, 382 nm, 385 nm, and 385 nm, respectively.

[0036] The results are shown in Table 2 below. After 15 days of anaerobic cultivation, in the anaerobic system of “HN05 + sucrose + anthraquinone”, all four kinds of anthraquinones had obvious reduction phenomenon, while in the control systems of “HN05 + anthraquinone” and “sucrose + anthraquinone”, no anthrahydroquinone was detected, indicating that Kosaconia oryzae HN05 can use sucrose as an electron donor to anaerobicly reduce four kinds of anthraquinone compounds, and the utilization ability was in the order from largest to smallest: AQDS>α-AQS>AQS>1,5-AQDS.

[0037] [Table 2]

[0038] The above are only preferred embodiments of the present invention, and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Kosakonia oryzae HN05, characterized in that its classification is named Kosakonia oryzae HN05, deposited in China Typical Culture Depository Center, and has the deposit number CCTCC NO:M 2021956.

2. 2. Kosaconia oryzae HN05 according to claim 1, characterized in that the 16S rDNA sequence of Kosaconia oryzae HN05 is the nucleotide sequence shown in SEQ ID NO.

3. Use of the Kosakonia oryzae HN05 according to claim 1 in reducing anthraquinone compounds.

4. Use of a combination of Kosaconia oryzae HN05 according to claim 1 and sucrose in the anaerobic reduction of anthraquinone compounds.

5. The use according to claim 4, characterized in that the concentration of sucrose used is 5 mmol / L.

6. The reduction product of the anthraquinone compound is AH ２ QDS, α-AH ２ Q.S., A.H. ２ QS and 1,5-AH ２ The use according to any one of claims 3 to 5, characterized in that it comprises one or more of the following: QDS.

7. 2. The use of the Kosaconia oryzae HN05 according to claim 1 in treating and remediating soil polluted with pesticides, wherein the pesticide pollutant is diquat.

8. The use according to claim 7, characterized in that the Kosaconia oryzae HN05 is used for treating diquat-contaminated water, decomposing diquat, and remediating soil.

9. The use described in claim 8, characterized in that the Kosaconia oryzae HN05 is used to decompose diquat under anaerobic conditions.

10. The use according to claim 9, further comprising sodium anthraquinone-2,6-disulfonate, the concentration of which is 0.5 mmol / L.

Images