Aquamicrobe and application thereof in degradation of atrazine

Abstract

The invention discloses aquatic microbe and an application of the aquatic microbe in degradation of atrazine. The water microbe is named as Aquammicrobium lusiense HR8, and the preservation number of the water microbe is CGMCC (China General Microbiological Culture Collection Center) No. 28916. The invention also discloses a microbial inoculum containing the aquatic microbe, a method for culturing the aquatic microbe and a method for degrading triazine herbicides. The invention discloses that Aquammicrobe is used for degrading atrazine for the first time, the degradation rate of Aquammicrobe HR8 on 200 mg / L atrazine in 48 hours is 100%, the Aquammicrobe HR8 is applied to bioremediation treatment of an atrazine polluted environment, the defect that the bioremediation removal effect on atrazine is poor at the present stage can be overcome, and meanwhile secondary pollution to the environment is reduced.

Description

Technical Field

[0001] The invention relates to the field of biodegradation, and in particular to a water microbe and application thereof in degrading atrazine. Background Art

[0002] Atrazine (2-chloro-4-diethylamino-6-isopropylamino-1,3,5-triazine) is a water-soluble triazine herbicide with a broad spectrum of weed control. It can remove a variety of annual grass and broad-leaved weeds, and also has a certain inhibitory effect on some perennial weeds. It can effectively control crabgrass, barnyard grass, foxtail grass, sedge, foxtail grass, Polygonum, Chenopodium, cruciferous and leguminous weeds, and is suitable for dryland crops such as corn, sorghum, sugarcane, fruit trees, nurseries, and woodlands. It was first patented in 1958 and began commercial use in the United States in 1959. At present, it is one of the most widely used herbicides in the world, with an annual output of more than 36,000 tons.

[0003] The widespread use of atrazine has brought about a series of environmental and ecological problems. Its persistence and mobility in the aquatic environment lead to its high residues in soil and groundwater. Once it enters the watershed, it can spread for miles. In addition to the direct problems of reduced biodiversity, reduced production and food safety, it also has long-term reproductive, endocrine disrupting and hormone interrupting effects, which not only harms crustaceans, insects, mollusks, fish, amphibians and reptiles, but can also cause human birth defects, low birth weight, reproductive tumors, low sperm count in men, and menstruation in women.

[0004] Therefore, removing atrazine from the environment is considered to be a top priority, and exploring effective remediation measures to eliminate environmental atrazine has become a research hotspot. At present, from the perspectives of cost, benefit, quality, degradation efficiency and safety, bioremediation based on microbial enhancement is still the most promising method for pesticide degradation. Researchers have isolated and screened a large number of atrazine-degrading bacteria from a variety of environments, including bacteria, fungi, actinomycetes, etc., but the existing degradation strains still have great limitations. Therefore, screening highly efficient atrazine-degrading strains, processing them into bacterial agents through fermentation, and applying them to the degradation of pesticide residues, thereby achieving the elimination of atrazine residues in soil, water bodies, and agricultural products, is still the primary task of remediation work. Summary of the invention

[0005] In order to solve the defect that there is no bacteria or bacterial agent that can efficiently degrade triazine herbicides such as atrazine in the prior art, the present invention provides an aquamicrobium and its application in degrading atrazine. The Aquamicrobium lusatiense HR8 provided by the present invention has the advantage of efficiently degrading triazine herbicides.

[0006] To achieve the above purpose, this paper provides the following technical solutions:

[0007] One of the technical solutions provided by the present invention is: an isolated bacterium, the bacterium is Aquamicrobium lusatiense, the bacterium is named Aquamicrobium lusatiense HR8, and it is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.28916 and a deposit time of November 8, 2023.

[0008] The biological characteristics of the bacteria are as follows: the colony morphology is regular, round, with smooth edges, light yellow to yellow, opaque, the colony is moist and sticky and easy to pick, and the diameter of the mature colony is 0.5 cm. In the R2A liquid fermentation medium, the logarithmic growth phase is reached within 48 hours of shaking culture on a shaker, and the stable phase can be reached within 5 days of culture. The fermentation liquid is turbid and milky yellow and sticky. The bacteria are Gram-negative aerobic bacteria that can use atrazine as the sole carbon source; they grow in an environment with a temperature of 20 to 40°C and a pH value of 5.0 to 9.0.

[0009] The bacteria were sequenced by 16S rDNA sequence and compared with the sequences in GenBank by BLAST homology. The results showed that the strain had high homology with Aquamicrobium lusatiense, with a similarity of more than 99%. The strain was named Aquamicrobium lusatiense HR8.

[0010] The second technical solution provided by the present invention is: a bacterial agent, which includes the bacteria as described in one of the technical solutions.

[0011] In a specific embodiment of the present invention, the viable count of the bacteria in the bacterial agent is 1×10 4 CFU / mL or more, 1×10 5 CFU / mL or more, 1×10 6 CFU / mL or more, 1×10 7 CFU / mL or above or 1×10 8 CFU / mL or more, for example 1×10 4 ~1×10 8 CFU / mL.

[0012] In a specific embodiment of the present invention, the bacterial agent further comprises a culture medium for culturing the bacteria and / or a bacterial agent auxiliary material.

[0013] In a specific embodiment of the present invention, the culture medium is LB culture medium or R2A culture medium; and / or the bacterial agent auxiliary material comprises an excipient, a disintegrant, a binder, a thickener, a solubilizer, a preservative, a lubricant and / or a lyoprotectant.

[0014] In the present invention, the bacterial agent is prepared by using an effective amount of the bacteria as the active ingredient, and conventional excipients, disintegrants, adhesives, thickeners, solubilizers, preservatives, lubricants and / or lyophilization protectants and other bacterial agent auxiliary materials can be added according to conventional preparation processes in the art.

[0015] The third technical solution provided by the present invention is: a method for culturing the bacteria as described in one of the technical solutions, the method comprising culturing the bacteria in a culture medium.

[0016] In the present invention, the culture medium can be a conventional culture medium in the art, the culture temperature can be a conventional temperature in the art, the culture time can be a conventional time in the art, the culture pH can be a conventional pH in the art, and the culture speed can be a conventional speed in the art. The initial inoculum size of the culture can be a conventional inoculum size in the art, as long as these culture conditions can grow the Aquamicrobium lusatiense HR8. After the culture is completed, the effective viable count in the culture medium can reach 10 4 More than pcs / mL.

[0017] In a specific embodiment of the present invention, the method satisfies one or more of the following conditions:

[0018] (1) The culture medium is LB culture medium or R2A culture medium;

[0019] (2) The culture temperature is 20 to 40° C., preferably 30° C.;

[0020] (3) The culturing time is 24 to 60 hours, preferably 48 hours;

[0021] (4) The pH of the culture is 5.0 to 9.0, preferably 7.0;

[0022] (5) the culture speed is 150-180 rpm, preferably 160 rpm; and,

[0023] (6) The initial inoculum volume of the culture is 2% (v / v) to 20% (v / v), preferably 15% (v / v).

[0024] The fourth technical solution provided by the present invention is: a method for preparing the bacterial agent as described in the second technical solution, the method comprising culturing the bacteria using the method as described in the third technical solution.

[0025] The fifth technical solution provided by the present invention is: a method for degrading triazine herbicides, the method comprising: applying the bacteria as described in one of the technical solutions or the bacterial agent as described in the second technical solution to an area contaminated by the triazine herbicide.

[0026] Triazine herbicides, also known as triazine herbicides, are widely used for pre-emergence or post-emergence weeding to control the growth of annual herbaceous plants and broad-leaved weeds. There are 36 varieties of triazine herbicides, which are divided into symmetrical triazines (1,3,5-triazines, also known as s-triazines) and asymmetrical triazines (1,2,4-triazines). S-triazines are the main commercial herbicides, which can be further divided into chloro-triazines (such as atrazine (also known as atrazine), simazine, and terbuthylazine), thiomethyl-s-triazines (such as promethazine, amethoxazole, and terbuthylazine), fluoroalkyl-s-triazines (such as triazine indole, triazine fluazifop) and methoxytriazines (such as promethazine and terbuthyl). This type of herbicide is a selective herbicide that targets the D1 protein of photosynthetic system II (PSⅡ) and exerts its weed control effect by inhibiting plant photosynthesis.

[0027] In a specific embodiment of the present invention, the triazine herbicide includes s-triazine herbicides.

[0028] In a specific embodiment of the present invention, the s-triazine herbicide includes atrazine.

[0029] In a specific embodiment of the present invention, in the bacteria or the bacterial agent, the viable cell count of the bacteria is 1×10 4 CFU / g or more, 1×10 5 CFU / g or more, 1×10 6 CFU / g or more, 1×10 7 CFU / g or above or 1×10 8 CFU / g or more, for example 1×10 4 ~1×10 8 CFU / g.

[0030] In a specific embodiment of the present invention, the conditions for the bacteria or the bacterial agent to degrade the triazine herbicide are selected from one or more of the following:

[0031] (1) The temperature is 20 to 40°C, preferably 30°C;

[0032] (2) the period is 2 days or more, 3 days or more, 5 days or more, 7 days or more, 10 days or more, 14 days or more, 20 days or more, or 28 days or more; and,

[0033] (3) pH is 5.0 to 9.0, preferably 7.0.

[0034] In the present invention, the conditions for degrading the triazine herbicide may be the temperature, time and / or pH conventional in the art and suitable for the growth and metabolism of the strain, as long as these conditions can degrade the triazine herbicide.

[0035] The sixth technical solution provided by the present invention is: use of the bacteria described in the first technical solution or the bacterial agent described in the second technical solution in degrading triazine herbicides.

[0036] In a specific embodiment of the present invention, the triazine herbicide includes s-triazine herbicides.

[0037] In a specific embodiment of the present invention, the s-triazine herbicide includes atrazine.

[0038] The reagents and raw materials used in the present invention are commercially available.

[0039] The positive and progressive effects of the present invention are:

[0040] The present invention discloses for the first time the degradation of atrazine by the genus Aquamicrobium. The degradation rate of 200 mg / L atrazine by the genus Aquamicrobium HR8 is 100% for 48 hours. The bioremediation treatment of atrazine-contaminated environments can solve the defect of poor bioremediation and removal effect of atrazine at this stage, and reduce secondary pollution to the environment. In addition, the present invention also provides a bacterial agent for degrading atrazine containing the genus Aquamicrobium HR8. The bacterial agent is easy to use, has low operating cost, good effect, small negative impact on the environment, and has a good prospect for development and utilization. The present invention's genus Aquamicrobium HR8 is of great significance for reducing and eliminating groundwater and soil contaminated by atrazine.

[0041] Biomaterial Deposit Information

[0042] The water microbacterium Aquamicrobium lusatiense HR8 (water microbacterium HR8) of the present invention was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on November 8, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit number is CGMCC No. 28916, the culture name is Aquamicrobium lusatiense HR8, and the classification name is water microbacterium (Aquamicrobium lusatiense). BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0044] Figure 1 The colony morphology of strain HR8 on R2A solid medium.

[0045] Figure 2 This is a picture of the strain HR8 under a thermal field emission scanning electron microscope.

[0046] Figure 3 This is a picture of the colonies forming atrazine dissolution zones after strain HR8 was cultured in MSM-based inorganic salt medium containing 500 mg / L atrazine for 5 days.

[0047] Figure 4 This is the alignment result of strain HR8 on NCBI.

[0048] Figure 5 This is the phylogenetic tree of strain HR8.

[0049] Figure 6 This is the standard curve of atrazine.

[0050] Figure 7 This is the degradation curve of atrazine by strain HR8.

[0051] Figure 8 This is the degradation diagram of atrazine by HR8 under different atrazine concentrations.

[0052] Fig. 9 The degradation diagram of atrazine by HR8 under different inoculation conditions.

[0053] Fig.10 This is the degradation diagram of atrazine by HR8 under different temperature conditions.

[0054] Fig.11 The degradation diagram of atrazine by HR8 under different pH conditions. DETAILED DESCRIPTION

[0055] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0056] The culture medium used in the embodiment includes:

[0057] LB medium: peptone 10g, yeast powder 5g, NaCl 10g, adjust pH to 7.2-7.4, sterilize at 121℃ for 30min.

[0058] R2A medium: KH2PO4 0.3 g, MgSO4·7H2O 0.05 g, sodium pyruvate 0.3 g, soluble starch 0.5 g, glucose 0.5 g, casein hydrolysate 0.5 g, peptone 0.5 g, yeast extract powder 0.5 g, adjust pH to 7.2, and sterilize at 121°C for 30 min.

[0059] Mineral salt medium (MSM): 0.5g NaNO3, 1.0g (NH4)2SO4, 2.5g Na2HPO4, 1.0g KH2PO4, 1mL trace element storage solution pH 7.0, sterilized at 121℃ for 30min. After cooling to room temperature, add 1mL vitamin solution and 1mL calcium and magnesium solution. Add different concentrations of atrazine as the only nitrogen source according to the experimental design requirements.

[0060] Trace element storage solution: 108mg Al2(SO4) 3· 18H2O, 56mg CoSO4·7H2O, 56mg CuSO4·5H2O, 3.0g FeSO4·7H2O, 611mg H3BO3, 28mg KBr, 56mg KI, 28mg LiCl, 389mg MnCl2·4H2O, 28mgNa2MoO4·2H2O, 28mg Na2WO4·2H2O, 58mg NiCl2·6H2O, 28mg SnCl2·2H2O, 34mg ZnSO4·H2O, filtered through 0.2μm filter membrane, stored at room temperature away from light.

[0061] Vitamin solution: 200mg para-aminobenzoic acid, 200mg biotin, 200mg folic acid, 200mg niacin, 100mg calcium pantothenate, 100mg pyridoxine hydrochloride, 100mg riboflavin, 100mg thiamine, 1mg vitamin B 12 , filtered through a 0.2 μm filter and stored at room temperature away from light.

[0062] Calcium-magnesium solution: 30 g CaCl2, 20 g MgCl2. Sterilize at 121°C for 20 min and store at room temperature away from light.

[0063] The corresponding solid separation and purification medium can be obtained by adding 20 g / L agar to the above liquid medium.

[0064] Experimental equipment: Agilent-1100 high performance liquid chromatography, chromatographic column: Ultimate XB-C18 column (150mm×4.6mm, 5μm), mobile phase: methanol-water (80:20v / v), flow rate: 1.0mL·min -1 , column temperature: 25℃, wavelength: 254nm, injection volume: 5μL.

[0065] The atrazine herbicide used in the examples was purchased from SIGMA (Shanghai, China).

[0066] The above implementation is described in more detail below in conjunction with specific examples.

[0067] Example 1: Acquisition and identification of atrazine-degrading bacteria

[0068] 1. Isolation and screening of degradation bacteria FH-1

[0069] Weigh 10g of soil that has been contaminated with atrazine for a long time (sludge from the sewage treatment pool of Nanjing Pesticide Factory) and add it to a 250mL triangular shake flask containing 100mL of inorganic salt culture medium containing 100mg / L atrazine. Place it on a shaker (30℃, 150rpm) and culture it for 7 days. Take 10mL of the culture solution and inoculate it into 100mL of fresh inorganic salt culture medium containing 200mg / L atrazine to continue culturing. Continue to transfer until the final concentration of atrazine is 1000mg / L. Take 1mL of the bacterial solution and dilute it step by step into 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 5 concentration gradients, each dilution was separated by coating method on solid inorganic salt medium containing 1000mg / L atrazine, cultured at 30℃ for two days, single colonies were picked according to their different appearance and characteristics, repeatedly streaked and purified, and the same strains were merged according to the appearance and morphological characteristics of the colonies and the bacterial morphological characteristics observed by microscopic morphology, and the obtained strains were inoculated on the R2A solid medium slant, stored and used. Finally, a strain that can grow on the separation medium with an atrazine concentration of 1000mg / L was isolated and named HR8. Its colony morphology on R2A solid medium is as follows Figure 1 As shown in the figure, the bacterial image under the thermal field emission scanning electron microscope is as follows Figure 2 shown.

[0070] Degradation zone experiment: Take 1mL of bacterial solution and inoculate it into a test tube containing 10mL of R2A medium. Place it on a shaker (30℃, 150rpm) and culture it for 24h. Use an inoculation loop to dip the HR8 bacterial solution in the R2A medium and streak it on a plate of MSM basic inorganic salt medium containing 500mg / L atrazine. After 5 days of culture, an atrazine dissolution zone is formed at the bottom of the colony. Figure 3 shown.

[0071] 2. Identification of degradation bacteria HR8

[0072] (1) Morphological identification of the degrading bacteria HR8

[0073] The degrading bacteria HR8 isolated and purified as above was inoculated into solid LB culture medium and placed in a 30°C incubator for static culture. Single colonies in the logarithmic growth phase and with stable colony size were selected for morphological characteristics description: the degrading bacteria HR8 grew very fast on the LB solid plate culture medium, with regular, round shape, smooth edges, light yellow to yellow, opaque, moist and sticky colonies that were easy to pick up, and the diameter of mature colonies was 0.5 cm.

[0074] (2) Homology analysis of 16S rDNA of degrading bacteria

[0075] The 16S rDNA of strain HR8 was extracted, and the universal primers (27F (5'-AGAGTTTGATCCTGGCTCAG-3'), SEQ ID NO: 1; 1492R (5'-GGTTACCTTGTTACGACTT-3'), SEQ ID NO: 2) were used to amplify the 16S rDNA sequence. The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 1.5 min; final extension at 72°C for 10 min; cycle number 35. 50 μL amplification system: 2×Taq Master Mix 25 μL, genomic DNA 1 μL, 1 μL each of 1492R and 27F primers, and sterile water was used to make up to 50 μL. After the reaction, the product was detected by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Engineering Co., Ltd. for sequencing. The 16S rDNA sequence of strain HR8 (SEQ ID NO: 3) was obtained by sequencing, with a total of 1390 base sequences. The comparison results were compared with the NCBI database. Figure 4 As shown, the species with the greatest sequence similarity to the species to be tested is Aquamicrobium lusatiense, with a sequence similarity of 99.78%.

[0076] Aquamicrobium HR8 16S rDNA (SEQ ID NO: 3):

[0077]

[0078] Construction of phylogenetic tree of strain HR8: Seven strains with high sequence similarity to strain HR8 were selected, and the phylogenetic tree of strain HR8 was constructed using MEGA5 software. The constructed phylogenetic tree is Figure 5 The strain with the highest similarity to strain HR8 was Aquamicrobium lusatiense, which was named Aquamicrobium lusatiense HR8.

[0079] Example 2: Study on the degradation characteristics of the strain (different atrazine concentrations, inoculation amounts, temperatures, pH)

[0080] 1. Preparation of atrazine standard curve

[0081] The atrazine standard curve was prepared using a high performance liquid chromatograph, specifically: the herbicide standard was graded diluted with methanol to obtain atrazine samples with concentrations of 1000 mg / L, 500 mg / L, 250 mg / L, 125 mg / L, 62.5 mg / L, and 31.25 mg / L, respectively. The diluted atrazine standard was filtered using a 0.22 μm microporous filter membrane and the bubbles were removed by ultrasound before sampling and testing. The standard curve was prepared using a plane rectangular coordinate system, with the ordinate being the peak area and the abscissa being the atrazine sample concentration. The concentration and peak area are shown in Tables 1 and Figure 6 .

[0082] Table 1 Atrazine sample concentration and peak area

[0083] Concentration mg / L Peak area 1000 4184.989 500 2108.286 250 1092.813 125 562.2705 62.5 288.3488 31.25 146.2462

[0084] From Table 1 and Figure 6 It can be seen that the standard curve of atrazine is y=4.155x+33.789, R2=0.9999.

[0085] The degradation rate is calculated as follows:

[0086]

[0087] 2. Study on the degradation characteristics of water microbe HR8

[0088] (1) Degradation curve of the degradation bacteria HR8

[0089] The degradation strain HR8 was inoculated into an inorganic salt medium containing 200 mg / L atrazine, and cultured in a light-proof shaking incubator at 160 rpm and 30°C for 48 hours. Another treatment with the same culture conditions but without inoculation of the degradation strain HR8 was set up, which was recorded as the control group (CK). The culture fluid was collected every 6 hours, and the residual atrazine in the two groups of culture media was determined by high performance liquid chromatography.

[0090] like Figure 7 As shown, strain HR8 had no lag phase after inoculation and could completely degrade 200 mg / L atrazine within 48 h.

[0091] (2) Effect of culture conditions on the degradation ability of HR8

[0092] The effects of different initial atrazine concentrations, initial inoculation amounts, temperatures and pH on the degradation ability of the degrading bacteria HR8 were studied respectively.

[0093] Different initial atrazine concentrations: The initial atrazine concentrations in the culture medium were set to 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L and 800 mg / L, respectively. The inoculation amount was 10%, and the culture was carried out at 30°C for 48 hours. The degradation rate of the strain under each condition was detected after 24 hours and 48 hours. After 24 hours of culture, the degradation rates of the five treatments were 65.54%, 59.57%, 84.80%, 38.62%, and 10.14%, respectively. After 48 hours of culture, the degradation rates of the degrading bacteria for atrazine with initial concentrations of 50 mg / L, 100 mg / L, and 200 mg / L can all reach 100%. Figure 8 shown.

[0094] Initial inoculation amount: The initial inoculation ratio of the modified strain was set to 2%, 5%, 10%, 15% and 20% (v / v), the atrazine concentration was 400 mg / L, and the culture was carried out at 30°C for 48 hours. The degradation rate of the strain under each condition was tested after 24 hours and 48 hours. After 24 hours of culture, the degradation rate of the treatment with an inoculation amount greater than 10% exceeded 50%. After 48 hours of culture, the degradation rate of the strain under all inoculation amounts exceeded 50%, among which the degradation rate of the treatment with an inoculation amount of 15% and 20% could reach 99%. Fig. 9 shown.

[0095] Culture temperature: Set the shaking incubator for culturing HR8 at 20°C, 25°C, 30°C, 35°C, and 40°C, with an inoculation rate of 10%, an atrazine concentration of 400 mg / L, and culture for 48 hours. Detect the degradation rate of the strain under each condition after 24 hours and 48 hours. After culturing for 24 hours at 25°C to 35°C, the degradation rate of the strain can exceed 70%. After culturing for 48 hours at 25°C to 35°C, the degradation rate of the strain is greater than 98%, the degradation rate at 20°C is 77.75%, and the degradation rate at 40°C is 26.41%. Fig.10 shown.

[0096] pH value: The pH of the inorganic salt culture medium was adjusted to 5, 6, 7, 8, and 9. According to the inoculation amount of 10%, the atrazine concentration was 400 mg / L, and cultured at 30°C for 48 hours. The degradation rate of the strain under each condition was detected after 24 hours and 48 hours. After 24 hours of culture, the degradation rate of the strain under acidic and alkaline conditions did not exceed 50%. After 48 hours of culture, the degradation rate of the strain under pH 7 conditions could reach 100%, and the degradation rate under pH 8 conditions could reach 55.82%. This result shows that pH has a great influence on the degradation of atrazine. Fig.11 shown.

[0097] Example 3: Preparation of atrazine bioremediation agent

[0098] (1) The strain of water microbe HR8 was inoculated into a medium containing 200 mg / L of inorganic salts, and cultured at 30°C and 160 rpm with shaking until the logarithmic growth phase, and the degradation rate was measured to obtain the degrading bacteria.

[0099] (2) The cultured bacteria were inoculated into R2A medium at a volume ratio of 15%, and cultured at 30° C. and 160 rpm with shaking until the logarithmic growth phase to obtain seed solution.

[0100] (3) Adding 20 mL of the obtained seed solution (OD600 = 2) into LB liquid fermentation medium can promote the high-density growth of the water microbe HR8, reaching a viable bacterial count of 1×10 4 ~1×10 8 CFU / mL and degrade atrazine.

[0101] Example 4: Effect of HR8 bacterial agent on soil remediation

[0102] 1000 g of soil was weighed, and the atrazine aqueous solution was added and mixed evenly to make the atrazine concentration in the soil 20 mg / kg (i.e., the soil was prepared to be contaminated with atrazine). The soil bioremediation agent containing the HR8 strain (obtained in Example 3) was added at 1×10 8 CFU / g was applied to the soil, mixed thoroughly, and cultured in a dark incubator at 30°C. Samples were taken regularly to determine the residue of atrazine in the soil.

[0103] The results showed that the degradation rate of atrazine was significantly improved by using soil bioremediation agents containing the HR8 strain. The degradation rate of atrazine reached 65.92% after adding HR8 agents to the soil for 14 days, while the degradation rate of atrazine without adding agents was only 38.94% after 14 days.

[0104] The above description is only a preferred embodiment of the present invention and does not constitute a limitation on the scope of the claims. Other substantially equivalent substitutes that can be thought of by those skilled in the art are all within the protection scope of the present invention.

Claims

1. An isolated bacterium, wherein the bacterium is Aquamicrobium lusatiense, characterized in that: The bacterium is named Aquamicrobium lusatiense HR8, and its deposit number is CGMCC No.28916.

2. A bacterial agent, characterized in that The bacterial agent comprises the bacteria as claimed in claim 1; Preferably, the viable count of the bacteria in the bacterial agent is 1×10 4 CFU / mL or more, preferably 1×10 4 ~1×10 8 CFU / mL.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent also includes a culture medium for culturing the bacteria and / or a bacterial agent auxiliary material; preferably: The culture medium is LB culture medium or R2A culture medium; and / or, The bacterial agent auxiliary materials include excipients, disintegrants, binders, thickeners, solubilizers, preservatives, lubricants and / or freeze-drying protective agents.

4. A method for culturing the bacterium according to claim 1, characterized in that: The method comprises culturing the bacteria in a culture medium.

5. The method according to claim 4, characterized in that The method satisfies one or more of the following conditions: (1) The culture medium is LB culture medium or R2A culture medium; (2) The culture temperature is 20 to 40° C., preferably 30° C.; (3) The culturing time is 24 to 60 hours, preferably 48 hours; (4) The pH of the culture is 5.0 to 9.0, preferably 7.0; (5) the culture speed is 150-180 rpm, preferably 160 rpm; and, (6) The initial inoculum volume of the culture is 2% (v / v) to 20% (v / v), preferably 15% (v / v).

6. A method for preparing the bacterial agent as claimed in claim 2 or 3, characterized in that: The method comprises culturing the bacteria using the method of claim 4 or 5.

7. A method for degrading triazine herbicides, characterized in that: The method comprises: applying the bacterium according to claim 1 or the bacterial agent according to claim 2 or 3 to an area contaminated by the triazine herbicide; Preferably, the triazine herbicide includes s-triazine herbicide; More preferably, the s-triazine herbicide includes atrazine.

8. The method according to claim 7, characterized in that In the bacteria or the bacterial agent, the viable count of the bacteria is 1×10 4 CFU / g or more, preferably 1×10 4 ~1×10 8 CFU / g.

9. The method according to claim 7 or 8, characterized in that The conditions for the bacteria or the bacterial agent to degrade the triazine herbicide are selected from one or more of the following: (1) The temperature is 20 to 40°C, preferably 30°C; (2) a period of 2 days or more, preferably 14 days or more; and, (3) pH is 5.0 to 9.0, preferably 7.

0.

10. Use of the bacterium according to claim 1 or the bacterial agent according to claim 2 or 3 in degrading triazine herbicides; Preferably, the triazine herbicide includes s-triazine herbicide; More preferably, the s-triazine herbicide includes atrazine.

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