CN110628680A - New strain of Paenibacillus and its cultivation method and application - Google Patents

Abstract

The invention relates to a new paenibacillus strain and a culture method and application thereof. The Paenibacillus provided by the invention is separated from soil, and the preservation number is KCTC 43073. According to the heterogeneous taxonomy method, the strain is a new species of Paenibacillus, and the classification status is Paenibacillus sp. The new paenibacillus strain provided by the invention grows at medium temperature (30 ℃), and is easy to culture. The discovery and utilization of the novel paenibacillus enrich the available microbial resources, the novel paenibacillus has the function of degrading organophosphorus pesticide residues, and a novel microbial degradation strain resource can be provided for organophosphorus pesticide residues required by agricultural production.

Description

New strain of Paenibacillus and its cultivation method and application

technical field

The invention belongs to the technical field of microbial engineering, and in particular relates to a new strain of Paenibacillus and its cultivation method and application.

Background technique

Paenibacillus can be isolated from a wide range of sources, such as rhizosphere soil, air, water and food. In 1996, the relevant characteristics of Paenibacillus were revised by Heyndrickx et al. Aerobic or facultative anaerobic growth, spore production, rod-shaped cells, Gram-positive cell wall, movement with perinatal flagella, moderate temperature, the main fatty acid is trans-isosaturated fatty acid C15:0. Paenibacillus G+C content ranges from 39 to 59 mol%.

Paenibacillus has oval spores in enlarged cysts and has no soluble pigment on nutrient agar. Facultatively anaerobic or strictly aerobic. Almost all species were positive for catalase except two subspecies of Paenibacillus larvae. Oxidase reactions are variable. The V-P reaction (acetylmethylmethanol production) is variable, and the pH of the V-P liquid is 4-6. Does not produce hydrogen sulfide. Some species produce indole. Nitrate reduction to nitrite variable. Hydrolysis of casein, starch and urea was variable. Tyrosine breakdown is also variable. The growth at pH 5.6 and 50°C is also variable, the optimum pH is 7.0, and the optimum temperature is 28-30°C. The optimum growth temperature for Paenibacillus macquariensis is 20-23℃. 10% NaCl can inhibit growth. Some species cannot grow in 0.001% lysozyme. The model species is Paenibacillus polymyxa.

Many microorganisms of the Paenibacillus genus can produce a variety of biologically active substances, such as enzymes and antibacterial substances, and have been widely used in the agricultural field as important plant biocontrol bacteria and plant rhizosphere growth-promoting bacteria. Paenibacillus is a bacterium with strong resistance to stress. It can produce highly active flocculation substances, degrade microcystins produced by Microcystis, and degrade oil. It also has broad application prospects in environmental pollution control.

As an indispensable means of production in modern agriculture, pesticides play a huge role in reducing crop pests, increasing the yield of agricultural products, and improving the quality of agricultural products. However, due to the unreasonable and excessive use of chemical pesticides in the process of preventing and controlling crop diseases and insect pests, the pesticide residues in agricultural products are getting higher and higher, causing serious pesticide residues in agricultural products, and even directly polluting food, threatening human health, and seriously polluting soil and water bodies And the atmospheric environment, reducing the diversity of the surrounding biological populations, destroying the balance of the ecosystem, but also affecting the health of the people and the export of agricultural products, destroying the agricultural ecological environment.

With the development of our country's economy and the improvement of people's living standards, food safety issues are increasingly concerned by people. In recent years, food safety problems have emerged one after another, among which food safety problems caused by pesticide residues are also on the rise, seriously threatening people's lives, health and safety, and causing adverse social impacts.

Among all kinds of pesticides, organophosphorus pesticides are currently the most widely used in my country, accounting for about 30% of my country's total agricultural output. Because of their high efficiency and high toxicity, they are widely used in the control of crop diseases and insect pests. However, its residual time is long, it is easy to pollute the environment, and it is a potential threat to human health. Its residue in the environment has been widely concerned. At present, there have been many advances in the treatment of organophosphorus pesticide residues, including physical methods, chemical methods, photocatalytic methods, and biological methods: Among them, physical methods generally use various materials that have the ability to adsorb pesticide residues to remove pesticides in soil. After the residue is adsorbed, it is transferred to other places for treatment. For example, Liu Le et al. ^[1] used magnesium-iron nano-layered double hydroxide to adsorb dichlorvos for 75 minutes, and the adsorption rate can reach 88.16%. However, physical methods generally only transfer pollutants. After that, further degradation is required, and the preparation cost of the adsorption material is relatively high, which is not conducive to large-scale promotion; chemical methods generally use oxidants to oxidize organophosphorus pesticide residues and convert them into harmless substances, such as Chen Chunyan et al. ^[2] Using self-made Fe ₂ O ₃ -Ce O ₂ /γ-Al ₂ O ₃ as a catalyst, using catalytic wet hydrogen peroxide oxidation method to pretreat organophosphorus pesticide wastewater can effectively reduce the concentration of organophosphorus pesticide in wastewater, but wastewater often needs to use Diluted with a large amount of water, resulting in large processing equipment, low load, high operating costs, and requires a large amount of oxidant; the principle of photocatalytic method is to use catalysts such as TiO ₂ to convert organophosphorus pesticide residues into harmless substances under the irradiation of ultraviolet light , such as Peng Yanzhi ^[3] showed that the UV-TiO ₂ -Fenton photocatalytic system can make the degradation rate of trichlorfon pesticide reach 92.50%. cost.

Compared with the above methods, microorganisms are widely distributed in the environment, have the characteristics of high degradation efficiency, multiple metabolic pathways, and no secondary pollution, and have become the main means of degrading organophosphorus pesticide residues at present. A large number of studies have proved that there are a variety of microorganisms in nature that can use organic phosphorus as a carbon source and energy source, degrade it into small molecules with low toxicity or non-toxic substances, and finally convert it into CO ₂ , water and minerals to achieve harmless treatment. There are mainly three types of microorganisms in the bioremediation process: indigenous microorganisms, genetically engineered bacteria and alien microorganisms. The reported microorganisms that can effectively degrade pesticides include bacteria, actinomycetes, fungi, and algae. Bacteria occupy a dominant position in bioremediation due to their characteristics such as easy mutagenesis and strong adaptability.

Contents of the invention

One of the objectives of the present invention is to provide a new strain of Paenibacillus.

To achieve the above-mentioned purpose, the present invention adopts following technical scheme:

A Paenibacillus, the preservation number of which is KCTC 43073.

The second object of the present invention is to provide a method for cultivating the new strain. The specific steps of the method are: inoculating the Paenibacillus in the culture medium and cultivating it under the condition of 20-50 DEG C and pH 5.0-12.0.

Further, the above medium is R2A medium.

Further, the above culture temperature is 30°C.

Further, the above pH is 7.0-8.0.

Further, there is 1% NaCl in the above-mentioned culture medium.

Further, the above-mentioned culturing is carried out under aerobic conditions.

The third object of the present invention is to provide the application of the above-mentioned Paenibacillus in the degradation of organophosphorus pesticide residues. Specifically, the above-mentioned Paenibacillus is fermented and cultured to make a degrading bacterial agent for use. Further, the medium components of the fermentation culture are glucose 0.1wt%, NaCl 1.0wt%, peptone 0.5wt%, yeast extract 0.25wt%, and the rest is distilled water.

The new strain of Paenibacillus provided by the present invention grows at medium temperature (30 DEG C) and is easy to cultivate. The discovery and utilization of the new species of bacillus in the present invention has enriched our available microbial resources. The new species of bacillus has the function of degrading organophosphorus pesticide residues and can provide a new microbial degradation method for organophosphorus pesticide residues required for agricultural production. strain resources.

Description of drawings

Fig. 1 is the 16S rDNA phylogenetic tree of the strain of the present invention.

The biological material described in the present invention, whose preservation number is KCTC 43073, is classified as Paenibacillus sp., and is named XMF-2, and has been preserved in the Korean Type Culture Collection (KCTC for short, address: South Korea) on May 30, 2019 ).

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions. The room temperature mentioned in the present invention refers to the temperature in the operating room where the test is carried out, which is generally 25°C.

Embodiment 1 Isolation and preparation of new bacterial strain XMF-2 of the present invention

The soil extract from a vegetable field in Wenzhou City, Zhejiang Province was diluted and spread on R2A solid medium, cultured at 30°C for 2-3 days, single colonies were picked, and then purified by streaking.

The composition of R2A medium is: yeast extract powder 0.5g/L; peptone 0.5g/L; casein hydrolyzate 0.5g/L; glucose 0.5g/L; soluble starch 0.5g/L; L; anhydrous magnesium sulfate 0.024g/L; sodium pyruvate 0.3g/L; final pH 7.2±0.2, distilled water to 1000mL. Add agar to solid media.

The obtained purified strain is preserved, and its preservation number is KCTC 43073.

The appearance characteristic of embodiment 2 new bacterial strain XMF-2 of the present invention

1. Colony characteristics

Take a single colony of strain XMF-2, transfer it to R2A solid medium, and culture it in a constant temperature incubator at 30°C for 24h, 36h, and 48h, and observe the size, color, edge, protrusion, smoothness, and viscosity of the colony respectively , transparency and other characteristics. The results showed that the strain XMF-2 formed neat, slightly raised, smooth, transparent, colorless colonies with a diameter of about 2-3mm on the R2A solid medium.

2. Cell Morphological Characteristics

The cells of the strain XMF-2 are Gram-negative bacilli with blunt apex and peripheral flagella; the cell size is 0.9-1.1 μm×2.6-4.7 μm, solitary.

The growth characteristics of embodiment 3 new bacterial strain XMF-2 of the present invention

Pick fresh cultures cultured on R2A solid medium for 24 hours, inoculate them into R2A liquid medium, and cultivate them on a shaker at 30°C for 20-24 hours as seeds.

Growth temperature:

The cultured XMF-2 seeds were transferred to fresh aseptic inoculated R2A liquid medium according to 2% (v/v) inoculum amount, and mixed evenly. They were cultured in water baths at 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, 40°C, 45°C, 50°C and 60°C respectively. The growth was measured at 24h and 48h. The growth temperature range of the obtained strain XMF-2 is 20-50°C, and the optimum temperature is 30°C.

Growth NaCl tolerance:

The cultured XMF-2 seeds were transferred to 0.0%, 2.0%, 5.0%, 7.0%, 9%, 10.0%, 11.0% and 12.0% of the sodium chloride mass concentration by 2% (v/v) inoculum. R2A medium, cultivated at 30°C, and recorded the growth status at 24h and 48h respectively. The results showed that the optimum NaCl concentration for the growth of strain XMF-2 was 1%.

Growth pH range:

Use sterile 1mol/L HCl and 1mol/L NaOH to adjust the sterilized R2A medium to pH values of 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 8.5, 9.0, 10.0, 11.0, At 12.0 and 13.0, the cultured XMF-2 seeds were inoculated at 2% inoculum, cultured at 30°C, and the growth status was recorded at 24h and 48h respectively. The pH range for the growth of strain XMF-2 was 5.0-12.0, and the optimum pH was 7.0-8.0.

Physiological and biochemical characteristics of embodiment 4 new bacterial strain XMF-2 of the present invention

The physiological and biochemical characteristics of strain XMF-2 were identified using API ZYM and API20E identification systems and conventional physiological and biochemical assay methods.

The identification results are as follows:

Embodiment 5 PCR amplification and sequence determination of the 16S rDNA gene of the new bacterial strain XMF-2 of the present invention

1. Genomic DNA Extraction

XMF-2 was inoculated in R2A medium, cultured at 30°C for 24 hours, collected by centrifugation, and resuspended with normal saline to remove residual medium. Afterwards, the bacteria were collected by centrifugation again, and the genomic DNA of strain XMF-2 was extracted using a bacterial genomic DNA extraction kit.

2. PCR amplification and sequencing of 16S rDNA gene

The forward primer used for PCR amplification is 5'-AGAGTTTGATCCTGGCTCAG-3'(nt 8-27), and the reverse primer is 5'-AAGGAGGTGATCCAGCC-3'(nt 1541-1557), which correspond to the 16S rDNA of Escherichia coli 8-27 and 1541-1557 bases of the gene.

The reaction system is as follows:

The reaction procedure is as follows:

PCR products were sequenced after agarose gel electrophoresis.

Sequencing results showed that the 16S rDNA sequence (SEQ ID NO: 1) of the strain XMF-2 has a high similarity with Paenibacillus species, and the highest similarity with Paenibacillus Contaminans, so it can be classified as Paenibacillus genus . The phylogenetic tree analysis of 16S rDNA sequence is shown in Figure 1.

Embodiment 6DNA molecular hybridization

Strain XMF-2 was crossed with Paenibacillus Contaminans strainCKOBP-6, which is the most similar strain in genetic relationship.

Proceed as follows:

DNA sample processing: Extract the DNA of the strain to be tested. Before the experiment, it needs to be placed in an ice bath and treated with 40W ultrasonic wave for 24 minutes (set as: play for 3 seconds/stop for 3 seconds; the concentration of the DNA sample is OD260nm2.0, cut the DNA sample Cut into fragments of 2-5 x 10 ⁵ Daltons.

Precisely prepare the DNA samples to be tested with 0.1×SSC so that the OD ₂₆₀ nm is 1.8-2.0, and the OD ₂₆₀ nm values of the two are consistent (accurate to 0.001);

Enter the control program, set the page to set the appropriate measurement parameters. The measurement wavelength was 260 nm, and the total measurement time was set to 30 minutes.

Stabilize the temperature of the cuvette at the optimum refolding temperature.

Take 400 μL each of the DNA samples of the two strains and put them in two centrifuge tubes, and then take 200 μL of the DNA samples of the two strains and put them in the same centrifuge tube as a mixed sample;

Single DNA samples and mixed DNA samples were denatured at 100°C for 15 minutes through the temperature control system before testing, and then cooled to the optimum annealing temperature. Record the OD260nm value, stop reading when the reaction reaches 30 minutes, the temperature of the sample during the whole process should not be lower than TOR, and finally get a straight line with the light absorption value gradually decreasing with time;

According to the experimental results, the homologous hybridization rate was obtained using computer software.

The results showed that the DNA homology between the strain XMF-2 and Paenibacillus Contaminans strain CKOBP-6 was 40%. According to the "Bergey's Bacteria Identification Manual", under the most suitable conditions, the DNA homology of more than 70% belongs to More than 20% of the same species belong to the same genus, so it is judged that the strain XMF-2 belongs to a new species of Paenibacillus.

Embodiment 7 The purposes of novel microorganism of the present invention

Degradation effect of strain XMF-2 on organophosphorus pesticide residues:

Determination of degradation characteristics of organophosphorus pesticides by degrading strain XMF-2 in R2A medium: Pick a single colony of XMF-2 in 50 mL of R2A liquid medium, culture at 30°C and 200 rpm for 24 hours with shaking, and obtain fresh bacterial liquid. Centrifuge 3 mL of fresh cultured bacterial liquid at 6000 rpm for 5 minutes, discard the supernatant, add 10 mL of sterile water to resuspend, and obtain the seed liquid after centrifugation and suspension. The bacterial volume of the seed liquid reaches 6×10 ⁷ CFU/mL above.

Add cyhalofop-ethyl with a final concentration of 100mg/L in the R2A medium, and insert the seed solution of the bacterial strain XMF-2 at an inoculum size of 3% by volume; at the same time, add a final concentration of 100mg/L in the inorganic salt medium cyhalofop-ethyl, insert the inactivated bacterial strain XMF-2 seed liquid as a contrast by the inoculation amount of 3% volume ratio, cultivate 24h in 30 ℃ constant temperature shaker, adopt high performance liquid chromatography to detect bacterial strain XMF-2 to B The degradation of acifluorfen and the calculation of the degradation rate showed that the degradation rate of acifluorfen by strain XMF-2 could reach 90% within 24 hours.

Using the same method to test the degradation of fenoxyprop-p-ethyl, fluazifop-p-ethyl, quizalofop-p-p, clodinafop-propargyl and other aryloxyphenoxycarboxylate pesticides, after 48 hours of reaction , fluazifop-p-p, quizalofop-p-p, and clodinafop-propargyl have good degradation effects.

The degradation effect of strain XMF-2 on organophosphorus pesticides is shown in the table below.

Table 1 Degradation effect of strain XMF-2 on organophosphorus pesticides

The preparation of embodiment 8 bacterial strain XMF-2 degrading bacterial agent

1) The degradation strain XMF-2 isolated and screened in Example 1 was inoculated into a test tube containing 3 mL of R2A medium and cultivated to the logarithmic phase, and the test tube liquid was inoculated in 100 mL of fermentation medium with an inoculum size of 0.5% by volume, 30°C, 150rpm shaking culture to the logarithmic phase, to obtain fermentation strains;

2) Inoculate 500 liters of seeds with 70% liquid content (based on the volume of the fermenter, the same below) with an inoculation amount of 10% (v/v, based on the volume of the culture medium) of the fermented strains obtained above. Cultivate in the culture medium of the tank (the culture medium has been sterilized by high pressure damp heat at 121°C and cooled), and cultivate to the logarithmic growth phase. Ratio), the stirring speed is 150rpm, and the cultivation temperature is 30°C; the seed solution is obtained;

3) the inoculum amount of the seed liquid is inserted into the medium of 5000 liters of production tanks with a liquid volume of 70% by 10% (v/v, taking the medium volume as a basis) to cultivate and ferment (the production tank medium has been used at 1.1 kg/cm ² pressure, 121 ℃ high pressure damp heat sterilization, cooling), the ventilation rate of sterile air per minute during the cultivation and fermentation process is 1:1 (volume ratio of medium to sterile air), and the stirring speed is 180rpm, The culture temperature is 30°C, and the culture time is 30 hours. After the fermentation, the number of bacteria reaches more than 1 billion/mL. After the fermentation is completed, the culture solution is directly packed into liquid dosage forms in plastic barrels or bottles, which are degrading bacteria. agent.

Among them, the fermentation medium, the medium of the seed tank, and the medium formula of the production tank are the same, all of which are glucose 0.1wt%, NaCl 1.0wt%, peptone 0.5wt%, yeast extract 0.25wt%, solvent is distilled water, pH7.2 -7.5.

The method of using the degrading bacterial agent is as follows: when the temperature is favorable for the growth of microorganisms, the bacterial agent is evenly sprayed on the soil surface in an amount of 100mL/m ² and tilled to mix the bacterial agent and the soil to be treated evenly. After the microorganisms grow and degrade the pesticide residues for 3 to 4 days, the degradation effect can be detected.

The degradation effect of the prepared bacterial agent on the soil containing organophosphorus pesticides is shown in Table 2 below.

Table 2 Bacteria to the degradation effect of organophosphorus pesticides in soil

sequence listing

<110> Nanjing University of Technology

<120> New species of Paenibacillus and its cultivation method and application

<130>xb19101101

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1455

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tcggcggctg gctccttgcg gttacctcac cgacttcggg tgttgtaaac tctcgtggtg 60

tgacgggcgg tgtgtacaag acccgggaac gtattcaccg cggcatgctg atccgcgatt 120

actagcaatt ccgacttcat gcaggcgagt tgcagcctgc aatccgaact gagaccggct 180

ttttaggatt ggctccgcct cgcggcttcg cggcccgttg taccggccat tgtagtacgt 240

gtgtagccca ggtcataagg ggcatgatga tttgacgtca tccccacctt cctccggttt 300

gtcaccggca gtcatcctag agtgcccaac tcaatgctgg caactaagat caagggttgc 360

gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac catgcaccac 420

ctgtcttgga tgtcccgaag gagggcccca tctctaatgc ttgcatccag atgtcaagac 480

ctggtaaggt tcttcgcgtt gcttcgaatt aaaccacata ctccactgct tgtgcgggtc 540

cccgtcaatt cctttgagtt tcactcttgc gagcgtactc cccaggcggc atgcttaatg 600

tgtttacttc ggcaccaagg gtatcgaaac ccctaacacc tagcatgcat cgtttacggc 660

gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgcgcct cagcgtcagt 720

tataggccag aaagtcgcct tcgccactgg tgttcctcca catctctacg catttcaccg 780

ctacacgtgg aattccactt tcctctccta cactccagtc tcccagtttc cagtgcgacc 840

caaggttgag ccctgggttt aaacaccgaa cttaaaagac cgcctgcgcg cgctttacgc 900

ccaataattc cggacaacgc ttgcccccta cgtattaccg cggctgctgg cacgtagtta 960

gccggggctt tcttctcagg taccgtcaaa tgaagagcag ttactctcct catccttctt 1020

ccctggcaac agagttttac gatccgaaaa ccttcatcac tcacgcggcg ttgctcggtc 1080

agactttcgt ccattgccga agattcccta ctgctgcctc ccgtaggagt ctgggccgtg 1140

tctcagtccc agtgtggccg ttcaccctct caggtcggct acgcatcgtc gccttggtga 1200

gccgttacct caccaactag ctaatgcgcc gcaggcccat ctgtaagtga cagcttgcgc 1260

cgtctttcct gatctctcca ggaggagaaa ccacctatcc ggttttagca cacgtttccg 1320

tgagttatcc cgatcttaca ggcaggttgc ctacatgtta ctcacccgtc cgccgctaac 1380

ctttcccgaa ggaaagatcc gctcgacttg catgtattag gcacgccgcc agcgatcgtc 1440

ctgagccagg atcaa 1455

Claims (10)

1. A Paenibacillus, which is characterized by having a deposit number of KCTC 43073.

2. A method of culturing the Paenibacillus of claim 1, comprising the steps of: inoculating Paenibacillus into culture medium, and culturing at 20-50 deg.C and pH of 5.0-12.0.

3. The method of claim 2, wherein the culture medium is R2A culture medium.

4. The method according to claim 2, wherein the temperature of the culture is 30 ℃.

5. The method according to claim 2, wherein the pH of the culture is 7.0 to 8.0.

6. The method according to claim 2 or 3, wherein the culture medium further comprises 1% by mass of NaCl.

7. The method of claim 2, wherein the culturing is carried out under aerobic conditions.

8. The use of the Paenibacillus of claim 1 for degrading organophosphorus pesticide residues.

9. The use of claim 8, wherein the paenibacillus strain of claim 1 is fermented and cultured to obtain the degrading bacteria agent.

10. The use of claim 9, wherein the medium composition of the fermentation culture is glucose 0.1 wt%, naci 1.0 wt%, peptone 0.5 wt%, yeast extract 0.25 wt%, and the balance distilled water.