A denitrifying adhesive arrow bacterium and its application

Abstract

This invention discloses a denitrifying adhaerens bacterium and its applications. The denitrifying adhaerens bacterium (Ensifer adhaerens) ST2 has the preservation number CCTCC NO: M20191138. The denitrifying adhaerens bacterium isolated by this invention exhibits excellent nitrous oxide emission reduction effects and can function effectively in the soil for a long period. It can also promote crop growth to a certain extent and can be widely applied to farmland soils such as rice and soybean fields.

Description

Technical Field

[0001] This invention belongs to the fields of agricultural microbiology and environmental protection, and relates to a strain of denitrifying adhesive arrow bacteria and its application. Background Technology

[0002] Nitrous oxide (N₂O) is one of the two most important greenhouse gases besides carbon dioxide and is a primary ozone destroyer. Although nitrous oxide constitutes a small proportion of the atmosphere, its residence time in the atmosphere can reach 114 years, and its global warming potential (GWP) per unit molecular weight is 296 times that of carbon dioxide and 13 times that of methane on a century-scale. The increase in atmospheric nitrous oxide concentration has attracted increasing attention.

[0003] Human activities are a significant source of atmospheric nitrous oxide, with farmland ecosystems accounting for 81% of total anthropogenic emissions. The primary source of nitrous oxide in farmland is applied nitrogen fertilizer, contributing 80% of total agricultural emissions and 20% of global nitrous oxide emissions. Currently, methods for reducing nitrous oxide emissions from farmland soils include field management (including rational fertilization and optimized planting patterns), application of biochar and nitrous oxide emission inhibitors, and inoculation with microbial agents that reduce nitrous oxide emissions. Among these methods, using microbial agents for nitrous oxide reduction offers advantages such as economic efficiency, environmental friendliness, and ease of operation compared to other methods.

[0004] Currently, there are few microbial functional agents developed for soil nitrous oxide emission reduction, and most of them are nitrous oxide reducing bacteria with single functions, which can only reduce nitrous oxide emissions, and their long-term effects in soil are unclear. Summary of the Invention

[0005] To address the problems of current microbial strains used for soil nitrous oxide reduction being limited in species and function, and having unclear long-term effects in soil, this invention provides a denitrifying adhesive sword bacterium that can not only effectively reduce soil nitrous oxide emissions but also efficiently colonize the soil and promote crop growth.

[0006] This invention relates to a denitrifying adhesive arrow bacterium, classified and named as Adhesive Arrow Bacterium ST2 ( Ensifer adhaerens The strain is named ST2 and its preservation number is CCTCC NO: M20191138.

[0007] The technical solution of this invention is as follows: This invention provides the application of this denitrifying adhesive sword bacterium in the preparation of microbial inoculants.

[0008] The first objective of this invention is to provide a microbial inoculant comprising denitrifying adhesive arrow bacteria ST2 ( Ensifer adhaerens ST2 is deposited at the China Center for Type Culture Collection (CCTCC) on December 31, 2019, with accession number CCTCC NO.M20191138.

[0009] Furthermore, the viable count of the microbial agent is 2 × 10⁻⁶. 9 CFU / ml or 2×10 9 CFU / gram or more.

[0010] A second objective of this invention is to provide a method for preparing the aforementioned microbial inoculant, the method comprising inoculating the denitrifying *Strombus aegyptium* into a fermentation medium with a pH of 7.0 and culturing it at 30±1℃ until the viable cell count reaches 2×10⁻⁶. 9 CFU / ml or 2×10 9 CFU / gram or more.

[0011] Furthermore, the fermentation medium comprises 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride, with water as the solvent; or comprises 1.17 g / L sodium chloride, 0.30 g / L potassium chloride, 0.20 g / L potassium dihydrogen phosphate, 0.27 g / L ammonium chloride, 0.40 g / L magnesium chloride hexahydrate, 0.15 g / L calcium chloride dihydrate, 1 g / L yeast extract, and 1 mL / L trace element solution, with water as the solvent.

[0012] The third objective of this invention is to provide a bio-organic fertilizer, wherein the bio-organic fertilizer is organic fertilizer inoculated with the aforementioned microbial agent.

[0013] Furthermore, the bio-organic fertilizer is prepared by inoculating 1 gram or 1 milliliter of microbial agent per gram of organic fertilizer.

[0014] Furthermore, the organic fertilizer is cow manure organic fertilizer.

[0015] The fourth objective of this invention is to provide the application of the aforementioned denitrifying adhesive sword bacteria ST2, or the aforementioned microbial agents, or the aforementioned bio-organic fertilizers in reducing greenhouse gas nitrous oxide emissions from farmland.

[0016] The fifth objective of this invention is to provide the application of the aforementioned denitrifying adhesive sword bacteria ST2, or the aforementioned microbial inoculants, or the aforementioned bio-organic fertilizer in increasing the root length of soybean plants and / or increasing soybean yield.

[0017] Furthermore, the application involves applying the aforementioned denitrifying adhesive sword bacteria ST2, the aforementioned microbial inoculant, or the aforementioned bio-organic fertilizer to the plant rhizosphere. Preferably, the plant is a soybean plant.

[0018] The beneficial effects of this invention are as follows: This invention isolates a microbial strain from paddy soil in Shantou, Guangdong. The microbial strain is identified as *Arrowobacter fasciatus* by biochemical analysis. This *Arrowobacter fasciatus* has a strong effect on reducing nitrous oxide emissions, can colonize efficiently in the soil, and exert a continuous effect. At the same time, it has a certain promoting effect on crop growth and can be widely used in farmland soils such as rice and soybeans.

[0019] The strain of this invention was deposited on December 31, 2019, with accession number CCTCC NO: M20191138. It is classified and named as: *Arrowheadia spp.* ST2 (… Ensifer adhaerens ST2). The depository is the China Center for Type Culture Collection, located at Wuhan University, China, 430072, China. Attached Figure Description

[0020] Figure 1 The growth and denitrification characteristics of *Streptococcus adipoptera* ST2 when nitrate is used as an electron acceptor include: a represents the growth of denitrifying adhesive brachii ST2 when nitrate is used as an electron acceptor; b represents the changes in nitrate and nitrite content in the bacterial culture of denitrifying adhesive sword bacteria ST2 when nitrate is used as an electron acceptor; c represents the change in nitrous oxide gas content produced by denitrifying adhesive brachii ST2 when nitrate is used as an electron acceptor.

[0021] Figure 2 The growth and denitrification characteristics of *Streptococcus adipoptera* ST2 when using nitrous oxide as an electron acceptor are described, including: a represents the growth of denitrifying adhesive brachii ST2 when nitrous oxide is used as the electron acceptor; b represents the change in nitrous oxide gas content in the culture medium when denitrifying adhesive sword bacteria ST2 uses nitrous oxide as an electron acceptor.

[0022] Figure 3 The phenotypic characteristics of denitrifying adhesive brazil ST2 include: a represents the growth status of denitrifying adhesive brachii ST2 on solid LB medium; b represents the growth status of denitrifying adhesive sword bacteria ST2 in liquid LB medium.

[0023] Figure 4 The physiological and biochemical characteristics of denitrifying adhesive brazil ST2 include: a represents the starch hydrolysis capacity of denitrifying adhesive brass ST2; b represents the oxidase activity of denitrifying adhesive brazier ST2.

[0024] Figure 5 To characterize the growth of denitrifying adhesive brazil ST2 under different pH and salinity conditions, the following was observed: a shows the growth of denitrifying adhesive brachii ST2 at different pH levels; b shows the growth of denitrifying adhesive sword bacteria ST2 under different salinities.

[0025] Figure 6 The effect of mixing denitrifying adhesive spirochete ST2 with organic fertilizer (without adding soil) on nitrous oxide emission flux.

[0026] Figure 7 The effect of denitrifying adhesive bacterium ST2 on nitrous oxide emission flux in soybean soil under pot experiment conditions.

[0027] Figure 8 The effects of denitrifying adhesive bacterium ST2 on soybean agronomic traits under pot experiment conditions, including: a represents the effect of denitrifying adhesive brass ST2 on soybean stem length; b represents the effect of denitrifying adhesive brass ST2 on soybean root length; c represents the effect of denitrifying adhesive brass ST2 on the fresh weight of soybean aboveground parts; d represents the effect of denitrifying adhesive brazier ST2 on the aboveground dry weight of soybean; e represents the effect of denitrifying adhesive brass ST2 on the fresh weight of soybean underground parts; f represents the effect of denitrifying adhesive brass ST2 on the dry weight of soybean underground parts; g represents the effect of denitrifying adhesive brass ST2 on the number of nodules in soybean; h represents the effect of denitrifying adhesive brass ST2 on soybean yield. Detailed Implementation

[0028] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0029] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0030] Example 1: Denitrification characteristics of *Strombus fasciatus* ST2 1. Using nitrates as electron acceptors: Single colonies of ST2 strain were picked from the plate and inoculated into 30 mL of SRM liquid medium (containing 1.17 g NaCl, 0.3 g KCl, 0.20 g KH2PO4, 0.27 g NH4Cl, 0.40 g MgCl2•6H2O, 0.15 g CaCl2•2H2O, 1 g yeast extract, 1 mL trace element solution, and water per liter). The medium was then anaerobically incubated at 30 °C until OD200 reached. 600The value is around 1.0, and the viable count is 2 × 10⁻⁶. 9 CFU / ml or higher, then 300 μL of bacterial culture was inoculated into an anaerobic serum bottle containing 30 mL of SRM liquid medium and 10 mmol / L potassium nitrate and cultured. SRM liquid medium without strain ST2 but with potassium nitrate was used as a control (CK). Each treatment was repeated in triplicate. OD values ​​of the medium were measured at 0, 12, 24, 36, 48, 60, and 72 hours. 600 The levels of nitrate and nitrite were determined by collecting 1 mL of headspace gas at 0, 24, 48, and 72 hours and measuring the nitrite content using gas chromatography with an electron capture detector (ECD-GC). The results are shown in [Figure number missing]. Figure 1 .

[0031] Figure 1 The results showed that strain ST2 was in the logarithmic growth phase from 0 to 36 hours and entered the stationary phase after 48 hours.

[0032] Figure 1 b indicates that from 0 to 36 hours, strain ST2 reduced nitrate to nitrite. After 36 hours, the nitrite content also decreased significantly, indicating that the strain further reduced nitrite and produced nitrous oxide or nitrogen gas.

[0033] Figure 1 c shows that strain ST2 continuously produces nitrous oxide when using nitrate as an electron acceptor, reaching its highest concentration after 48 hours.

[0034] 2. Using nitrous oxide as an electron acceptor: The bacterial culture was conducted in the same manner as described above. 10,000 ppm nitrous oxide gas was added to the SRM medium as an electron acceptor, with SRM medium without added nitrous oxide gas serving as a control (CK). The OD values ​​of the fermentation broth were measured at 0, 12, 24, 36, 48, 60, and 72 hours. 600 The nitrous oxide content of the culture system was determined at 0, 24, 48, and 72 hours.

[0035] The results are as follows Figure 2 As shown, when nitrous oxide is used as the electron acceptor, strain ST2 grows relatively slowly, reaching its maximum OD value after 48 hours, and almost completely reducing nitrous oxide after 48 hours.

[0036] Example 2: Identification of denitrifying adhesive brachii ST2 When cultured on LB medium, ST2 colonies are round, slightly raised, whitish-yellow, moist, opaque, with neat and smooth edges; no biofilm forms when cultured in liquid medium. Figure 3Figure a shows the morphological characteristics of the strain on the plate, and figure b shows the state of the fermentation broth in liquid culture. The strain is Gram-negative, rod-shaped, and non-spore-forming. It exhibits starch hydrolysis and oxidase activity, but is negative for indole reaction, gelatin liquefaction, phenylalanine deaminase, citrate utilization, H2S production, and lactose fermentation. The strain possesses good denitrification ability and can reduce nitrates, nitrites, and nitrous oxide.

[0037] The strain was identified as *Cyclophorus fasciatus* by whole-genome sequencing (see nucleotide sequence SEQ ID No: 1, GenBank accession number: GCA_002940685.1); physiological and biochemical characteristics are shown in Table 1 and... Figure 4 .

[0038] Table 1 Physiological and biochemical characteristics of denitrifying adhesive brazier ST2 Indole reaction - Starch hydrolysis + Gelatin liquefaction - Phenylalanine deaminase test - Citrate utilization - Oxidase + <![CDATA[H2S]]> - lactose fermentation - Figure 4 The results show that denitrifying adhesive brass ST2 can hydrolyze starch and has oxidase activity.

[0039] Example 3: Determination of fermentation parameters of denitrifying adhesive sword bacteria ST2 in culture medium Absorb 90 microliters of well-adjusted OD 600 The value of denitrifying adhesive sword bacteria ST2 (viable count 2×10⁻⁶) 9 (CFU / ml or higher) were inoculated into 30 mL of LB medium (3:1000 volume ratio) at pH 4.0, 6.0, 8.0, 10.0 and salinity 0%, 2%, 3%, and 4%, respectively, and cultured at 30°C and 190 rpm / min for 6, 12, 24, and 36 hours. LB liquid medium without added bacterial culture was used as a control. The OD of the fermentation broth was measured. 600 Growth curves of the strains in LB broth were plotted. Each treatment was performed in triplicate, and the results are shown below. Figure 5 As shown.

[0040] The results showed that the optimal growth pH for denitrifying adhesive sword bacteria ST2 was neutral (pH 6-8), but it could not grow normally under excessively acidic or alkaline conditions; the optimal salinity for the strain was 0%-2%, and the strain's growth was limited at a salinity of 3%, and it could not grow normally at a salinity of 4%.

[0041] Example 4: Analysis of the effect of denitrifying adhesive bacterium ST2 on nitrous oxide emission reduction when mixed with organic fertilizer (without soil). Preparation of bacterial suspension: Single colonies of *Streptococcus adiponitrile* were picked and inoculated into LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0-7.2, solvent: water) and incubated at 30°C and 190 rpm for at least 10 hours. The OD was adjusted using LB liquid medium. 600 =1.0.

[0042] 20 grams of cow dung organic fertilizer (in this example, the organic fertilizer used was cow dung organic fertilizer produced by Jiangsu Enweibo Biotechnology Co., Ltd.) were mixed with 20 ml of denitrifying adhesive sword bacterium ST2 bacterial solution with an OD value of 1.0 and placed in a culture bottle. The organic fertilizer was kept submerged. The organic fertilizer-bacterial solution mixture was placed in a 100 ml serum bottle and incubated at 28°C. To determine the nitrous oxide flux, after sealing the bottle with a rubber stopper and aluminum cap for 3 hours, 1.0 ml of headspace gas was extracted from the bottle using a glass syringe and subsequently analyzed using a gas chromatography-electron capture detector (ECD-GC). A comparison was made with sterile LB liquid medium (CK group) without bacterial inoculation. Results are shown below. Figure 6 .

[0043] Depend on Figure 6 It can be seen that inoculating organic fertilizer with denitrifying adhesive sword bacteria ST2 (ST2 group) significantly reduced the cumulative emission flux of organic fertilizer nitrous oxide by 32.1%, and significantly reduced nitrous oxide emissions by 46.5% on the fourth day after inoculation.

[0044] Example 5: Analysis of the reduction of nitrous oxide emissions from soybean soil by denitrifying adhesive brassicactus ST2 under greenhouse pot cultivation conditions. Soil for testing: Soil was collected from 0-20 cm soil of construction land in Chengwu County, Heze City, Shandong Province. After being air-dried, it was sieved through a 2 mm sieve and set aside for use.

[0045] The soil physicochemical properties are as follows: pH: 8.11; electrical conductivity: 0.41 ms / cm; organic carbon: 5.75 g / kg (per kilogram of air-dried soil); total potassium: 19.80 g / kg (per kilogram of air-dried soil); total phosphorus: 1.19 g / kg (per kilogram of air-dried soil); total nitrogen: 0.07% (per 100 grams of air-dried soil).

[0046] Pre-cultivation of soybean seedlings: Select healthy soybean seeds, sterilize their surface, soak them in sterile water for 10 minutes, then wash them with a 2% sodium hypochlorite solution for 5 minutes, and rinse them gently and repeatedly with sterile water until clean. Sow the seeds evenly on a 5 cm layer of vermiculite (pre-watered with a small amount of water to keep it moist). Cover with newspaper before germination to keep it in the dark; remove the newspaper after germination. Water 2-3 times daily with a spray bottle to maintain sufficient humidity. Once the soybeans have developed two cotyledons, select seedlings of uniform growth and transplant them into pots.

[0047] Preparation of bacterial suspension: Single colonies of *Streptococcus adiposa* were picked and inoculated into LB broth. The culture was incubated at 30°C on a shaker at 180 rpm / min for at least 12 hours. The resulting bacterial suspension was centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were collected. The suspension was resuspended in sterile deionized water and centrifuged again at 6000 rpm for 5 min to remove residual culture medium. After removing the supernatant, the OD value of the bacterial suspension was adjusted using sterile deionized water. 600 The value was adjusted to 1.0 (10 7 -10 8 (CFU / mL), to be used.

[0048] Pot inoculation experiment: Weigh 5 kg of air-dried soil into a 5-liter sealed container, add an appropriate amount of sterile water to ensure the soil moisture reaches 60% of maximum field capacity. Carefully remove soybean seedlings from the vermiculite, wash away the vermiculite adhering to the root surface with sterile water, and carefully transplant them into pots, taking care not to damage the roots. Transplant 4 soybean seedlings into each pot. After the soybeans have adapted to growth in the pots for one week, use a 20 mL syringe to draw the prepared inoculum, and then inoculate the inoculum at a rate of 4% (4 mL of inoculum per 100 g of soil) into the rhizosphere of the soybean plants, 50 mL per soybean plant, for a total of 200 mL. The control group was inoculated with an equal amount of sterile deionized water. Each group had 4 replicates, and all were placed in a constant temperature greenhouse for cultivation.

[0049] Nitrous oxide collection and determination: Nitrous oxide gas samples were collected and analyzed using a static dark chamber-gas chromatography method. Gas was collected from the top of the static chamber at 0, 12, 24, and 36 minutes after sealing. The nitrous oxide concentration was determined using a gas chromatograph with an electron trap (ECD), and the nitrous oxide emission flux and cumulative emission were calculated.

[0050] The results are as follows Figure 7 As shown, this experiment illustrates the effect of denitrifying adhesive bacteria on the nitrogen oxide emission flux in soybean soil under the conditions of a soybean pot experiment.

[0051] Depend on Figure 7 It was found that inoculation with denitrifying clove-like bacteria ST2 significantly reduced the emission flux of nitrous oxide from soybean soil. N2O emissions decreased by 85.7% the day after inoculation, and ST2 effectively colonized the soybean soil, continuously exerting its emission reduction effect. Throughout the soybean life cycle, inoculation with ST2 effectively reduced nitrous oxide emissions by 52.9%.

[0052] Example 6: Analysis of the effect of denitrifying adhesive sword fungus on soybean growth under greenhouse pot cultivation conditions. According to Example 5, after soybeans reached full maturity, soybean plants were collected, and agronomical indicators for each treatment were measured, including plant height, root length, aboveground fresh and dry weight, underground fresh and dry weight, and yield. Specific results can be found in [link to example]. Figure 8 .

[0053] Depend on Figure 8 It can be seen that, compared with the control, inoculation with denitrifying adhesiomycosis ST2 significantly increased the root length of soybean plants, resulting in an increase in average soybean yield, but had no significant effect on soybean plant height, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, and nodule number.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microbial inoculant, characterized in that, The microbial agent includes denitrifying adhesive ribbon bacteria ST2 ( Ensifer adhaerens ST2 is deposited at the China Center for Type Culture Collection (CCTCC) on December 31, 2019, with accession number CCTCC NO.M20191138.

2. The microbial agent according to claim 1, characterized in that, The microbial agent contains more than 200 million live bacteria per milliliter or 200 million per gram.

3. The method for preparing the microbial inoculant according to claim 1, characterized in that, The method includes inoculating the denitrifying adhesive sword bacteria into a fermentation medium with a pH of 7.0 and culturing it at 30±1℃ until the viable count reaches 2×10⁻⁶. 9 CFU / ml or 2×10 9 CFU / gram or higher.

4. The method for preparing the microbial inoculant according to claim 4, characterized in that: The fermentation medium comprises 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride, in water; or comprises 1.17 g / L sodium chloride, 0.30 g / L potassium chloride, 0.20 g / L potassium dihydrogen phosphate, 0.27 g / L ammonium chloride, 0.40 g / L magnesium chloride hexahydrate, 0.15 g / L calcium chloride dihydrate, 1 g / L yeast extract, and 1 mL / L trace element solution, in water.

5. A bio-organic fertilizer, characterized in that, The bio-organic fertilizer is organic fertilizer inoculated with the microbial agent described in claim 1.

6. The bio-organic fertilizer according to claim 5, characterized in that, The bio-organic fertilizer is prepared by inoculating 1 gram or 1 milliliter of microbial agent per gram of organic fertilizer.

7. The bio-organic fertilizer according to claim 5, characterized in that, The organic fertilizer is cow manure organic fertilizer.

8. The application of the denitrifying adhesive sword bacteria ST2 as described in claim 1, or the microbial agent as described in claim 1, or the bio-organic fertilizer as described in claim 5, in reducing greenhouse gas nitrous oxide emissions from farmland.

9. The application of the denitrifying adhesive sword bacteria ST2 as described in claim 1, or the microbial agent as described in claim 1, or the bio-organic fertilizer as described in claim 5 in increasing the root length of soybean plants and / or increasing soybean yield.

10. The application according to claim 8 or 9, characterized in that, The denitrifying adhesive sword bacteria ST2 as described in claim 1, or the microbial agent as described in claim 1, or the bio-organic fertilizer as described in claim 5, is applied to the rhizosphere of the plant; preferably, the plant is a soybean plant.

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