WO2021196735A1 - 一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用 - Google Patents

一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用 Download PDF

Info

Publication number
WO2021196735A1
WO2021196735A1 PCT/CN2020/136200 CN2020136200W WO2021196735A1 WO 2021196735 A1 WO2021196735 A1 WO 2021196735A1 CN 2020136200 W CN2020136200 W CN 2020136200W WO 2021196735 A1 WO2021196735 A1 WO 2021196735A1
Authority
WO
WIPO (PCT)
Prior art keywords
strain
bacillus
culture
tianshenii
medium
Prior art date
Application number
PCT/CN2020/136200
Other languages
English (en)
French (fr)
Inventor
胡著然
王丽荣
单嘉劲
张修
张文娟
樊梅娜
韩广泉
齐素敏
冉新炎
申小冉
Original Assignee
山东碧蓝生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东碧蓝生物科技有限公司 filed Critical 山东碧蓝生物科技有限公司
Priority to KR1020217024585A priority Critical patent/KR20210124232A/ko
Publication of WO2021196735A1 publication Critical patent/WO2021196735A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K17/00—Soil-conditioning materials or soil-stabilising materials
    • C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K17/00—Soil-conditioning materials or soil-stabilising materials
    • C09K17/40—Soil-conditioning materials or soil-stabilising materials containing mixtures of inorganic and organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the invention belongs to the field of microbial technology, and specifically relates to a strain of Bacillus tianshenli strain P46, its bacterial agent and its application in the field of soil heavy metal remediation.
  • Soil is an important resource for human survival in the natural environment and agricultural production.
  • the food, resources and environmental issues facing the world are closely related to soil.
  • the 1920s due to the development of industry, the output of metals has increased significantly, and the resulting heavy metal environmental pollution problems have also appeared.
  • soil pollution seriously hinders agricultural production.
  • the modernization of society and the development of production the problem of soil pollution has become more and more serious, which has attracted widespread attention.
  • the heavy metals that are often paid attention to in environmental science are mercury, cadmium, nickel, chromium, alum, arsenic, selenium, iron, manganese, copper, zinc, lead, etc. Among them, a considerable part of heavy metals are trace elements necessary for organisms, and some heavy metals are shown as environmental pollutants. At present, most researches are on cadmium (Cd), lead (Pb), copper (Cu), and zinc (Zn). And mercury (Hg). In agricultural production, heavy metal pollution not only reduces the fertility of the soil, but also causes the quality of the agricultural products produced does not meet the requirements of ecological safety, and endangers the health of humans and animals, which is very harmful.
  • Bioremediation is an environmentally friendly technology that combines science and technology with natural life processes and uses the life activities of bacteria, fungi, and green plants to repair the contaminated environment.
  • microorganisms have the characteristics of low cost, low energy consumption, no secondary pollution, and high efficiency in the remediation of heavy metal contaminated soil.
  • some microorganisms have good adaptability, cell mechanism, and they can also absorb various types of heavy metals. While shortening the repair time, they can also achieve a good conversion effect of heavy metals in the soil.
  • microorganisms can participate in the regulation of nutrient cycling in the soil and increase the rate of soil heavy metal conversion. Therefore, the use of some fungi, bacteria, actinomycetes, etc. to repair heavy metal contaminated soil has great potential.
  • fungi, bacteria, actinomycetes, etc. to repair heavy metal contaminated soil has great potential.
  • sulfate-reducing bacteria and phosphate solubilizing bacteria have become a hot spot in the research of microbial remediation of heavy metals due to their high soil heavy metal tolerance and potential to repair heavy metals.
  • the present invention screens and obtains a Bacillus tianshenii strain P46 with phosphorus solubilization and heavy metal repair functions from soil contaminated by heavy metals.
  • the Bacillus tenisen has a good ability to solubilize phosphorus, promote plant growth and repair heavy metals in the soil, and has a significant repair effect on the metallic lead and cadmium in the soil.
  • the present invention provides the following technical solutions:
  • the first aspect of the present invention provides a strain of Bacillus tianshenli strain P46, which has been deposited on March 2, 2020 in the General Microbiology Center of the China Microbial Strain Collection Management Committee, referred to as CGMCC, address: Chaoyang District, Beijing No. 3, No. 1, Beichen West Road, its biological preservation number is: CGMCC NO.19444.
  • Bacillus Tenshin was isolated for the first time in 14 years and was identified as a new species. At present, the research on Bacillus cerevisiae is relatively blank in this field, and its biological activity and whether it has probiotic functions are still unclear.
  • the Bacillus tianshenii strain P46 provided by the present invention is a strain with good phosphorus solubilizing ability, which can help improve soil fertility and promote plant growth.
  • the phosphate solubilizing bacteria also have a good passivation ability for heavy metals. When used alone, the degradation rate of heavy metal cadmium in the soil can reach more than 60%, and the degradation rate of metal lead can reach more than 80%.
  • the Bacillus tianshenii strain P46 described in the first aspect has the 16S rDNA sequence shown in SEQ ID NO:1.
  • the strain was isolated from the sewage around the electroplating plant, and its bacterial morphology satisfies the characteristics of Bacillus.
  • the identity of the strain with the known strain Bacillus tianshenii strain YIM M13235 (98%) after genome sequencing comparison can reach 98%, and it is identified as Bacillus spore Bacillus, named Bacillus tianshenii strain P46.
  • Bacillus tianshenii strain P46 The morphological characteristics of Bacillus tianshenii strain P46 are as follows:
  • Bacterial characteristics The cell size of this strain is 0.3 ⁇ m ⁇ 0.5 ⁇ m ⁇ 1.4 ⁇ m ⁇ 3.0 ⁇ m, rod-shaped, gram-positive; the spores are oval, the size is 0.6 ⁇ m ⁇ 0.9 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.5 ⁇ m, there are Sportiness.
  • Colony characteristics The colony is round or irregular, the edges are irregular, the surface is free of wrinkles, roughness, dirty white, and no bumps.
  • Bacillus tianshenii strain P46 The physiological and biochemical characteristics of Bacillus tianshenii strain P46 are as follows:
  • glucose can use glucose, xylose, arabinose, etc., can hydrolyze molasses, and has the function of dissolving phosphorus; it has the highest growth rate when cultured at 30°C, and does not grow under anaerobic conditions.
  • an inoculum comprising the Bacillus tianshenii strain P46 and/or a culture of the bacteria described in the first aspect.
  • the formulation of the bacterial agent is a liquid bacterial agent, powder or granule; further it is a water suspension, a dispersible oil suspension, a wettable powder or a water dispersible granule.
  • the bacterial agent also includes agrochemically acceptable excipients, which are selected from the group consisting of dispersants, wetting agents, disintegrants, binders, defoamers, and antifreeze agents. , One or more of thickeners, fillers and solvents.
  • agrochemically acceptable excipients which are selected from the group consisting of dispersants, wetting agents, disintegrants, binders, defoamers, and antifreeze agents. , One or more of thickeners, fillers and solvents.
  • the present invention does not have special restrictions on the sources of the agrochemical acceptable auxiliary materials, etc., and generally, commercially available products can be used.
  • the third aspect of the present invention provides the application of the strain described in the first aspect and the bacterial agent described in the second aspect in the remediation of heavy metal soils.
  • a composite inoculum composed of Bacillus tianshenli strain P46 and Bacillus subtilis strain P30 or a culture thereof; the Bacillus subtilis strain ( Bacillus subtilis strain) P30, has been deposited in the General Microbiology Center of the China Microbial Strain Collection Management Committee, CGMCC for short, on March 2, 2020.
  • the address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and its biological preservation number It is: CGMCC NO.19443.
  • the Bacillus tenisen has a multi-faceted probiotic effect, including decomposing phosphorus in the soil to provide nutrients for plant growth, promoting plant growth, and inactivating and restoring various metal elements in the soil, and is applied to pollution Farmland restoration has good application prospects.
  • Figure 1 is a graph showing the effect of the culture conditions described in Example 6 on the culture of P46;
  • Figure 1A is a broken line diagram of the influence of temperature and pH on the number of viable bacteria
  • Figure 1B is a broken line diagram of the influence of temperature and pH on the number of spores
  • Figure 1C is a broken line diagram of the influence of rotation speed and time on the number of viable bacteria
  • Figure 1D is a broken line diagram of the influence of rotation speed and time on the number of spores.
  • the present invention proposes a Bacillus deennis, its fungus and its application in the field of heavy metal repair.
  • the first aspect of the present invention provides a strain of Bacillus tianshenli strain P46, which has been deposited on March 2, 2020 in the General Microbiology Center of the China Microbial Strain Collection Management Committee, referred to as CGMCC, address: Chaoyang District, Beijing No. 3, No. 1, Beichen West Road, its biological preservation number is: CGMCC NO.19444.
  • the Bacillus tianshenli strain P46 has the 16S rDNA sequence shown in SEQ ID NO:1.
  • the preparation method of the Bacillus tianshenli strain is as follows: inoculate the Bacillus tianshenli strain P46 on a slant medium for cultivation; take the cultivated slant, and inoculate the strain on the seed under aseptic conditions
  • the liquid medium the first-level seed liquid is obtained by shaking culture; the second-level seed liquid is obtained by expanding the first-level seed liquid; the second-level seed liquid is inoculated into the liquid fermentation medium with an inoculum amount of 0.8 to 1.2%.
  • the rate reaches more than 90%, the fermentation broth is collected, added with light calcium carbonate and spray-dried to obtain bacterial powder.
  • one loop of the bacterial species is inoculated on the solid slant medium, and cultured at 37° C. for 24 to 30 hours.
  • the first-level seed culture is cultured at 37° C. and shaking at 180 rpm for 18-24 hours.
  • the expanded culture is 1% (v/v) inoculum amount
  • the first-level seed liquid is inoculated in 500mL ⁇ 1000mL seed liquid medium
  • the culture is shaken for 12 ⁇ 14h at 37°C to prepare Secondary seed liquid.
  • the seed culture medium formula is as follows: 4.0% molasses, 2.0% soybean meal, 0.7% K 2 HPO 4 , 0.3% KH 2 PO 4 , 0.15% (NH 4 ) 2 SO 4 , 0.05% sodium citrate, 0.01% magnesium sulfate, the balance is water, adjust the pH to 6.8, and sterilize at 121°C for 30 minutes.
  • the solid slant medium is a NB synthetic liquid medium with 1.5-2.0% agar powder added.
  • the liquid fermentation medium formula is: 4.0% molasses, 2.0% soybean meal, 0.7% K 2 HPO 4 , 0.3% KH 2 PO 4 , 0.15% (NH 4 ) 2 SO 4 , 0.05% sodium citrate , 0.01% magnesium sulfate, the balance is water, adjust the pH to 6.8.
  • an inoculum in a second aspect of the present invention, there is provided an inoculum.
  • the inoculum includes the Bacillus tianshenii strain P46 and/or a culture of the bacteria described in the first aspect.
  • the formulation of the bacterial agent in the second aspect is a liquid bacterial agent, powder or granule; further it is a water suspension, a dispersible oil suspension, a wettable powder or a water dispersible granule.
  • the bacterial agent also includes agrochemically acceptable excipients, which are selected from the group consisting of dispersants, wetting agents, disintegrants, binders, defoamers, and antifreeze agents. , One or more of thickeners, fillers and solvents.
  • agrochemically acceptable excipients which are selected from the group consisting of dispersants, wetting agents, disintegrants, binders, defoamers, and antifreeze agents. , One or more of thickeners, fillers and solvents.
  • the present invention does not have special restrictions on the sources of the agrochemical acceptable auxiliary materials, etc., and generally, commercially available products can be used.
  • the third aspect of the present invention provides the application of the strain described in the first aspect and the bacterial agent described in the second aspect in the remediation of heavy metal soils.
  • a composite inoculum composed of Bacillus tianshenli strain P46 and Bacillus subtilis strain P30 or a culture thereof; the Bacillus subtilis strain ( Bacillus subtilis strain) P30, has been deposited in the General Microbiology Center of the China Microbial Strain Collection Management Committee, CGMCC for short, on March 2, 2020.
  • the address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and its biological preservation number It is: CGMCC NO.19443.
  • the Bacillus subtilis strain P30 has a 16S rDNA sequence shown in SEQ ID NO: 2.
  • Strain nutrient medium NB synthetic medium. On the basis of NB medium, 1.5% agar is added as solid medium.
  • Screening medium for phosphate solubilizing bacteria strains (NBRIP): glucose 1.0%, Ca 3 (PO 4 ) 2 0.5%, MgCl 2 0.5%, peptone 0.5%, agar powder 2.0%, pH 7.2 ⁇ 7.4, 121°C, sterile Bacteria 30min.
  • Preliminary screening of phosphate-dissolving bacteria Take 1 gram (1ml) from the soil and water samples obtained from the electroplating plant and mix them into a triangular flask containing 99ml of sterile physiological saline. A constant temperature shaker at 30°C, 180 rpm , Incubate with shaking for 2h.
  • Re-screening of phosphate solubilizing bacteria inoculate the phosphate solubilizing bacteria obtained in the preliminary screening into 250 mL Erlenmeyer flasks (filling volume 50 mL) containing NBRIP liquid medium, rotating at 180 rpm, temperature 37°C, incubating for 24 hours, and observing the growth of the medium The color change and the number of viable bacteria are recorded and the time is recorded.
  • Constant temperature water bath ; incubator; optical microscope.
  • strains P30, P46, S3, and S5 have the best adsorption efficiency for cadmium.
  • microorganisms isolated from the soil and water samples of the electroplating plant were subjected to preliminary screening and re-screening, and a total of 4 strains with high heavy metal degradation performance were screened, and the numbers were P30, P46, S3 and S5.
  • the phytotoxicity test of bacteria is carried out using "pearl" tomato seeds and "crystal” lettuce seeds. Soak the seeds in a single bacterial liquid with a concentration of 2%, a mixed bacterial liquid (1:1:1) and distilled water (control group) for 10 minutes. Then rinse with distilled water, evenly place it in a petri dish covered with wet filter paper, put it into a constant temperature incubator, and set the temperature to 24°C. The budding rate was recorded after 3-4 days, and the weights of the roots and stems of the seedlings after drying in the experimental group and the control group were recorded after 10 days. The phytotoxicity can be judged by observing the delay of seed germination and the growth of seedlings.
  • the morphological identification of the bacteria belongs to the genus Bacillus, the cells are rod-shaped, Gram-positive bacteria, terminal spores, without flagella. It conforms to the characteristics of Bacillus deennis and is preliminarily identified as Bacillus deennis.
  • Table 3 The effects of the selected bacteria on the germination rate and seedling growth of tomatoes and lettuce seeds
  • P30, P46, and the two strains have obvious promoting effects on the germination rate of tomatoes and lettuce seeds, and have a certain growth-promoting effect on tomato and lettuce seedlings.
  • P30 belongs to Bacillus subtilis, which is a well-known strain with plant growth promoting effect.
  • P46 shows a growth promoting effect equivalent to that of P30.
  • Example 3 The shake flask test of each strain on the heavy metal cadmium and lead repairing liquid
  • Treatment 2 Only P46 was inoculated, and 3% nano-adsorbed clay was added at the same time. Replace the inoculation liquid with the same amount of sterile water as the experimental blank.
  • Apparatus and measurement standard Use flame atomic absorption method for measurement, refer to GB15618-1995 for specific operation.
  • the actual concentration of heavy metal cadmium in treatment 1 is 1.9595mg/L, and the actual degradation rate of heavy metal is 60.81%;
  • the actual concentration of heavy metal cadmium in treatment 2 was 1.9755 mg/L, and the actual degradation rate of heavy metal was 60.49%.
  • the actual concentration of heavy metal lead in treatment 1 is 8.1220mg/L, and the actual degradation rate of heavy metal is 83.76%;
  • the actual concentration of heavy metal lead in treatment 2 was 7.9500 mg/L, and the actual degradation rate of heavy metal was 84.10%.
  • Example 1 The horizontal comparison in Example 1 proved that P46 has excellent survival ability and ability to repair cadmium among the phosphorus solubilizing bacteria screened in the same batch.
  • a conventional culture medium was used for the shake flask experiment, and the experimental conditions were closer to actual industrial use conditions. From the above data, it can be seen that the repair efficiency of strain P46 for heavy metal lead can reach more than 83%, and the average removal effect of cadmium can reach 60%.
  • a composite inoculum formed by mixing Bacillus tianshenli strain P46 and Bacillus subtilis strain P30 with the number of viable bacteria in a ratio of 1:1.
  • the determination of the total cadmium and total lead content of heavy metals in the soil uses graphite furnace atomic absorption spectrophotometry, and the specific operation refers to GB/T17141;
  • the determination method of soil heavy metal cadmium and lead in different forms refers to BCR continuous extraction method.
  • the BCR continuous extraction method uses different extractants specified in the international standard to extract soil Cd 2+ and Pb 2+ forms, and quantitatively detects the extracts, and compares the BCR method to extract different forms of Cd 2+ and Pb in the soil. 2+ content, can simply analyze the repair effect of microorganisms and the stability of the final repair,
  • the obtained bacterial cell aggregates were prepared into a bacterial suspension of 3.0-5.0 ⁇ 10 9 CFU/mL.
  • the composite microbial agent composed of the phosphate solubilizing bacteria P30 and P46 can achieve good repair effects on different concentrations of lead and cadmium, and the repair efficiency can reach more than 98% on average.
  • Example 4 For the four groups of soil samples (50 mg/kg CK, 50 mg/kg P) after restoration in Example 4, 2 g was divided into several components and added to a 50 mL polyethylene centrifuge tube. In order to study the effects of different environmental conditions on the composition of the improved soil, the stability of microbial remediation of heavy metal pollution in the soil under different conditions was further explored. The first group is used to judge the effect of pH on repair stability. The specific steps are as follows: adjust 40mL of acid-base solution with different pH 3-9 with NaOH and HCl, add it to the tube, shake at 150rpm, room temperature for 18 ⁇ 2h, centrifuge at 4000g for 15min , Measure the Cd 2+ content in the soil supernatant.
  • the second group is used to judge the influence of different metal cations on the stability of repair.
  • the specific steps are: to analyze pure NaNO 3 , KNO 3 , Ca(NO 3 ) 2 drugs dissolved in deionized water to make 0.01mol/L Na + , K + and Ca 2+ metal cation salt solution 40mL was added to the bottle, shaken at 150rpm for 18 ⁇ 2h, centrifuged at 4000g for 15min, and measured the Cd 2+ content in the soil supernatant.
  • the Ca 2+ solution can extract the most Cd 2+ content. This may be due to the fact that Ca 2+ and Cd 2+ have the same valence.
  • lattice exchange, coordination complexation, etc. activate Cd 2+ from a stable state to an exchangeable state, and the P group has a passivation effect that reduces the activity compared with the CK group.
  • the strain can grow in an environment of 10 ⁇ 40°C, and the strain grows fastest under an environment of 30 ⁇ 40°C; culture it on a shaker at different initial pH values for 48h , Strains can grow in an environment with a pH range of 3.5 to 8.5, and grow fastest in an environment with a pH of 6.5 to 7.5.
  • the best culture conditions for Bacillus tianshen P46 are: pH: 6.8; temperature is 37°C, stirring speed is 180r/min, and culture time is 48h.
  • Table 9 The number of viable bacteria of Bacillus Tenshinii P46 on nutrient media with different components

Abstract

从重金属污染的土壤中筛选得到一株天神芽孢杆菌( Bacillus tianshenli )P46。所述菌株为解磷菌并且对金属镉、铅具有良好的降解作用，应用于作物培养对植物生长无毒，具有一定的促进作用，不影响土壤的稳定性。

Description

一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用 技术领域
本发明属于微生物技术领域，具体涉及一株天神芽孢杆菌(Bacillus tianshenli strain)P46，其菌剂及其在土壤重金属修复领域的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
土壤是人类赖以生存的自然环境和农业生产的重要资源，世界面临的粮食、资源和环境问题与土壤密切相关。自20世纪20年代以来，由于工业的发展，金属的产量明显增加，因而由此产生的重金属环境污染问题也随之出现。在许多发展中和发达国家，都面临着土壤污染严重阻碍农业生产的问题。随着社会现代化和生产的发展，土壤污染问题日益严重，因而已引起人们的广泛关注。
在环境科学中常注意的重金属有汞、镉、镍、铬、矾、砷、硒、铁、锰、铜、锌、铅等。其中有相当一部分重金属是生物所必需的微量元素，另有一些重金属则表现为环境污染物，目前，研究较多的是镉(Cd)、铅(Pb)、铜(Cu)、锌(Zn)和汞(Hg)。在农业生产上，重金属污染不仅使土壤肥力下降，又致使生产出的农产品质量不符合生态学安全的要求，危害人畜健康，因而具有很大的危害性。
同时，土壤污染修复技术也飞速发展，尤其是生物修复技术的发展，为重金属污染修复提供了更为高效、绿色的手段。生物修复是一种环境友好型技术，它将科学技术与自然生命过程相结合，利用细菌、真菌、绿色植物等的生命活动，来修复受污染环境。其中，微生物在重金属污染土壤修复中具有成本低、能耗低、无二次污染、效率高等特点。同时部分微生物自身良好适应能力、细胞机制，也可以吸收多种类型重金属在缩短修复时间的同时，也可以达到良好的重金属土壤转化效果。同时微生物可以参与调控土壤中养分循环，提高土壤重金属转化速度。所以利用一些真菌、细菌、放线菌等修复重金属污染土壤具有很大的潜力。在众多种类的微生物中，硫酸盐还原菌和解磷菌以较高的土壤重金属耐受性和修复重金属的潜力而成为现今研究微生物修复土壤重金属的热点。
发明内容
本发明从重金属污染的土壤中筛选得到一株具有解磷及重金属修复功能的天神芽孢杆菌(Bacillus tianshenii strain)P46。所述天神芽孢杆菌具有良好的解磷、促进植物生长及土壤中重金属修复能力，对土壤中的金属铅和镉具有显著的修复效果。
针对以上技术效果，本发明提供以下技术方案：
本发明第一方面，提供一株天神芽孢杆菌(Bacillus tianshenli strain)P46，已于2020年3月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，其生物保藏号为：CGMCC NO.19444。
天神芽孢杆菌于14年首次分离，并被认定为一个新的种。目前本领域内针对天神芽孢杆菌的研究相对空白，对其生物活性和是否具有益生功能尚不明确。本发明提供的所述天神芽孢杆菌(Bacillus tianshenii strain)P46是一株具有良好解磷能力的菌株，能够帮助提升土壤肥力，促进植物的生长。另外，解磷菌还对于重金属具有良好的钝化能力，单独使用的情况下，对土壤中重金属镉的降解率达到60％以上，金属铅降解率达到80％以上。
第一方面所述的天神芽孢杆菌(Bacillus tianshenii strain)P46具有SEQ ID NO:1所示的16S rDNA序列。
所述菌株分离自电镀厂周围的污水中，其细菌形态满足芽孢杆菌特征，经基因组测序比对与已知菌株Bacillus tianshenii strain YIM M13235(98％)的同一性可达98％，认定为天神芽孢杆菌，定名为天神芽孢杆菌(Bacillus tianshenii strain)P46。
所述天神芽孢杆菌(Bacillus tianshenii strain)P46的形态学特征如下：
菌体特征：该菌种细胞大小为0.3μm～0.5μm×1.4μm～3.0μm，呈杆状，革兰氏阳性；芽孢椭圆形，大小为0.6μm～0.9μm×1.0μm～1.5μm，有运动性。
菌落特征：菌落呈圆形或不规则性，边缘不整齐，表面无皱褶、粗糙，污白色，无隆起。
所述天神芽孢杆菌(Bacillus tianshenii strain)P46的生理生化特征如下：
能利用葡萄糖、木糖及阿拉伯糖等，能水解糖蜜，具有解磷功能；在30℃下培养生长速率最高，在厌氧条件下不生长。
本发明第二方面，提供一种菌剂，所述菌剂包括第一方面所述的天神芽孢杆菌(Bacillus tianshenii strain)P46和/或菌的培养物。
所述菌剂剂型为液体菌剂、粉剂或颗粒剂；进一步的为水悬浮剂、可分散油悬浮剂、可湿性粉剂或水分散颗粒剂。
作为优选，所述菌剂中还包括农药学上可接受的辅料，所述农药学上可接受的辅料选自分散剂、润湿剂、崩解剂、粘结剂、消泡剂、抗冻剂、增稠剂、填料和溶剂中的一种或多种。本发明对所述农药学上可接受的辅料的来源等没有特殊限制，一般采用市售产品即可。
本发明第三方面，提供上述第一方面所述菌株、第二方面所述菌剂在重金属土壤修复方面的应用。
本发明第四方面，提供一种复合菌剂，所述复合菌剂由天神芽孢杆菌(Bacillus tianshenli strain)P46与枯草芽孢杆菌(Bacillus subtilis strain)P30或其培养物组成；所述枯草芽孢杆菌(Bacillus subtilis strain)P30，已于2020年3月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，其生物保藏号为：CGMCC NO.19443。
以上一个或多个技术方案的有益效果如下：
1.天神芽孢杆菌作为一个新的属，相关研究几乎处于空白状态，上述研究结果为天神芽孢杆菌的发掘和应用提供了可供借鉴的经验。
2.所述天神芽孢杆菌具有多方面的益生作用，包括分解土壤中的磷元素为植物生长提供养分，促进植株的生长，以及对土壤中多种金属元素的钝化、修复效果，应用于污染农田修复具有良好的应用前景。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为实施例6中所述培养条件对P46培养的影响结果图；
其中，图1A为温度、pH值对活菌数量的影响折线图；
图1B为温度、pH值对芽孢数量的影响折线图；
图1C为转速、时间对活菌数量的影响折线图；
图1D为转速、时间对芽孢数量的影响折线图。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的，针对现有技术中存在的不足，本发明提出了一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用。
本发明第一方面，提供一株天神芽孢杆菌(Bacillus tianshenli strain)P46，已于2020年3月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，其生物保藏号为：CGMCC NO.19444。
所述天神芽孢杆菌(Bacillus tianshenli strain)P46具有SEQ ID NO:1所示的16S rDNA序列。
优选的，所述天神芽孢杆菌菌粉制备方法如下：将所述天神芽孢杆菌(Bacillus tianshenli strain)P46接种于斜面培养基进行培养；取培养好的斜面，在无菌条件下取菌株接种于种子液体培养基中，振荡培养获得一级种子液；对一级种子液进行扩大培养获得二级种子液；以0.8～1.2％的接种量取二级种子液接种于液体发酵培养基中，当芽孢率达到90％以上时，收集发酵液，加入轻质碳酸钙后喷雾干燥得到菌粉。
进一步优选的，所述斜面培养中，将菌种一环接种于固体斜面培养基上，在37℃下培养24～30h。
进一步优选的，所述一级种子培养在37℃条件下，180rpm震荡培养18～24h。
进一步优选的，所述扩大培养以1％(v/v)的接种量，将一级种子液接于500mL～1000mL种子液体培养基中，在37℃条件下，震荡培养12～14h，制得二级种子液。
进一步优选的，所述喷雾干燥的步骤中，向发酵液中加入0.67～1.00％(质量百分数)轻质碳酸钙。
进一步优选的，所述种子培养基配方如下：4.0％糖蜜，2.0％豆粕、0.7％K 2HPO 4、0.3％KH 2PO 4，0.15％(NH 4) 2SO 4，0.05％柠檬酸钠，0.01％硫酸镁，余量为水，调pH至6.8，121℃条件下灭菌30min。
进一步优选的，所述固体斜面培养基为NB合成液体培养基中添加1.5～2.0％的琼脂粉。
进一步优选的，所述液体发酵培养基配方为：4.0％糖蜜，2.0％豆粕、0.7％K 2HPO 4、0.3％KH 2PO 4，0.15％(NH 4) 2SO 4，0.05％柠檬酸钠，0.01％硫酸镁，余量为水，调pH至6.8。
本发明第二方面，提供一种菌剂，所述菌剂，所述菌剂包括第一方面所述的天神芽孢杆菌(Bacillus tianshenii strain)P46和/或菌的培养物。
优选的，第二方面所述菌剂剂型为液体菌剂、粉剂或颗粒剂；进一步的为水悬浮剂、可分散油悬浮剂、可湿性粉剂或水分散颗粒剂。
作为优选，所述菌剂中还包括农药学上可接受的辅料，所述农药学上可接受的辅料选自分散剂、润湿剂、崩解剂、粘结剂、消泡剂、抗冻剂、增稠剂、填料和溶剂中的一种或多种。本发明对所述农药学上可接受的辅料的来源等没有特殊限制，一般采用市售产品即可。
本发明第三方面，提供上述第一方面所述菌株、第二方面所述菌剂在重金属土壤修复方面的应用。
本发明第四方面，提供一种复合菌剂，所述复合菌剂由天神芽孢杆菌(Bacillus tianshenli strain)P46与枯草芽孢杆菌(Bacillus subtilis strain)P30或其培养物组成；所述枯草芽孢杆菌(Bacillus subtilis strain)P30，已于2020年3月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，其生物保藏号为：CGMCC NO.19443。
优选的，所述枯草芽孢杆菌(Bacillus subtilis strain)P30具有SEQ ID NO:2所示的16S rDNA序列。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。
实施例1 菌种的筛选
1、材料与方法
1.1样品
电镀厂周边土壤及水样等11份样品材料。
1.2筛选培养基
菌株营养培养基：NB合成培养基，NB培养基的基础上加入1.5％的琼脂即为固体培养基。
解磷菌菌株的筛选培养基(NBRIP)：葡萄糖1.0％、Ca 3(PO 4) 2 0.5％、MgCl 2 0.5％、蛋白胨0.5％、琼脂粉2.0％，pH7.2～7.4，121℃，灭菌30min。
1.3样品初筛
解磷菌的初筛：从电镀厂取得的土样及水样中取样1克(1ml)，混合放入装有99ml无菌生理盐水的三角瓶中，恒温摇床30℃，180转/分，震荡培养2h。获取悬浮液后做连续稀释，取1ml做10 -3-10 -5稀释，取10 -3-10 -5稀释液各0.1ml加入到NBRIP培养基平板上进行稀释涂布分离单菌落，挑取有解磷圈的单菌落进行分离纯化并斜面保存于冰箱中备用。
1.4菌种复筛
解磷菌的复筛：将初筛得到的解磷菌分别接种到装有NBRIP液体培养基的250mL三角瓶内(装液量50mL)，转速180rpm，温度37℃，培养24h，观察培养基的颜色变化及活菌数并记录时间。
1.5仪器
恒温水浴锅；培养箱；光学显微镜。
2、结果
2.1初筛结果
如表1所示，本实施例从11个样品中，共分离了78个菌株，通过解磷透明圈直径比与选择培养扩增速度的筛选，共初步筛选了17株解磷菌。
表1 初筛结果
Figure PCTCN2020136200-appb-000001
2.2复筛结果
如表2所示，P30、P46、S3、S5菌株对镉的吸附效率最好。
表2 复筛结果
Figure PCTCN2020136200-appb-000002
注：1、—：表示测不到；2、P7：为从土壤中筛选的脱氟不动杆菌；3、P10：为从土壤中筛选的克雷伯氏杆菌；4、P30：为从污水中筛选出的枯草芽孢杆菌；5、P46：为从土壤中筛选的天神芽孢杆菌；6、P51：为从污水中筛选出的枯草芽孢杆菌。
上述17株解磷菌中，去除了病害菌及真菌后，筛选解磷效果达到50％以上的菌株进行摇瓶实验，其中镉去除效果较好的四株菌如表2所示，其中，P46菌株对镉去除效果最好。
实施例2 菌种的鉴定及安全试验
1、材料与方法
1.1菌种
从电镀厂土壤及水样等样品中分离得到的微生物经初筛、复筛后，共筛选出4株重金属降解性能较高的菌株，编号分别是P30,P46,S3和S5。
1.2细菌的形态鉴定及分子鉴定
除特别说明外，一般形态及分子试验均参照《伯杰氏细菌鉴定手册》(第9版)和《微生物学实验手册》进行。
1.3安全试验
细菌的植物毒性检测是利用“珍珠”西红柿种子与“水晶”生菜种子进行的。将种子在2％浓度的单一菌液、混合菌液(1:1:1)以及蒸馏水(控制组)中浸泡10分钟。然后用蒸馏水冲洗，均匀放置于铺好湿滤纸的培养皿中，放入恒温培养箱，温度设置为24℃。3-4天后记录出芽率，10天后分别记录实验组与对照组烘干后幼苗根和茎的重量。通过观察种子发芽的延迟程度和幼苗的生长情况，判断植物毒性。
2、结果
2.1菌种的鉴定
经细菌的形态鉴定，属于芽孢杆菌属，细胞呈杆状，革兰氏阳性菌，端生芽孢，无鞭毛。符合天神芽孢杆菌特征，初步认定为天神芽孢杆菌。
挑取单菌落接种至NB液体培养基中，37℃，180rpm震荡培养24h，取1-5ml菌液利用细菌基因组DNA提取试剂盒提取菌株基因组DNA。16S rRNA通用引物8f(5'-agagtttgatcctggctcag-3')和1492r(5'-ggttaccttgttacgactt-3')对该菌株基因组DNA扩增并送至上海生工生物公司测序。BLAST软件比对分析该菌株与以下菌株的同一性可达98％：Bacillus tianshenii strain YIM M13235(98％)。
综合测序与形态鉴定结果，确定该菌株属于天神芽孢杆菌。
2.2安全试验
表3 所筛选细菌对西红柿和生菜种子的发芽率及幼苗生长影响
Figure PCTCN2020136200-appb-000003
Figure PCTCN2020136200-appb-000004
由表3可知，P30、P46、两株菌对西红柿及生菜种子的发芽率均具有明显的促进作用，且对西红柿及生菜幼苗由一定的促生长作用。P30属于枯草芽孢杆菌，是一种公知具有植物促生作用的菌种，P46显示出与P30相当的促生效果。
实施例3 各菌株对重金属镉、铅修复液体摇瓶试验
1、材料和方法
1.1菌种
P30、P46
1.2培养基
NB液体合成培养基，121℃，灭菌30min。
1.3重金属镉含量测定方法及仪器
测定方法：
(1)配制NB液体培养基，加入重金属镉(使培养基的重金属镉含量为5mg/L)灭菌备用。
(2)设置2个处理，分别接种对应菌株菌液1ml，2个处理分别为：
处理1：只接种P30；
处理2：只接种P46，同时加入3％纳米吸附粘土。以等量的无菌水替代接菌液作为实验空白。
(3)对接种后的摇瓶37℃，180rpm震荡培养3天。
(4)对震荡培养后的各处理进行离心(转速5000转)，取上清测定重金属镉含量。
仪器及测定标准：使用火焰原子吸收法测定，具体操作参考GB15618-1995。
2试验内容与结果
1)初始重金属镉的浓度为5mg/L
结论：
处理1的重金属镉实际浓度为1.9595mg/L，重金属实际降解率为60.81％；
处理2的重金属镉实际浓度为1.9755mg/L，重金属实际降解率为60.49％。
2)初始重金属铅的浓度为50mg/L
结果：
处理1的重金属铅实际浓度为8.1220mg/L，重金属实际降解率为83.76％；
处理2的重金属铅实际浓度为7.9500mg/L，重金属实际降解率为84.10％。
结论：
实施例1中通过横向对比证实了P46在同一批次筛选获得的解磷菌中具有优良的生存能力和修复镉的能力。本实施例中采用常规培养基进行摇瓶实验，实验条件更加贴近工业实际使用状况。由上述数据可以看出，菌株P46对于重金属铅的修复效率可达到83％以上，对镉的去除效果平均能够达到60％。
实施例四 重金属修复盆栽试验
本实施例中，提供一种由天神芽孢杆菌(Bacillus tianshenli strain)P46与枯草芽孢杆菌(Bacillus subtilis strain)P30以活菌数量1:1混合而成的复合菌剂。
1、土壤重金属镉、铅含量测定方法：
土壤重金属总镉、总铅含量测定使用石墨炉原子吸收分光光度法，具体操作参考GB/T17141；
土壤重金属镉、铅不同形态含量的测定方法参考BCR连续提取法。
BCR连续提取法是利用国际标准中规定的不同提取剂对土壤Cd 2+、Pb 2+形态进行提取，并对提取液进行定量检测，通过对比BCR法提取土壤中的不同形态Cd 2+、Pb 2+含量，可简单分析微生物的修复效果和最终修复的稳定性，
2、重金属修复盆栽试验方法
以接种解磷菌P30、P46于LB培养基中，以160rpm，37℃恒温培养2天后，将得到的细菌菌体团聚物制备成3.0-5.0×10 9CFU/mL细菌悬液。将不同浓度梯度的Cd污染土壤以200g/盆分装于各个花盆中，设置两组实验组，分别为未加菌组(CK组)，单独加复合菌液(P组，其中P46、P30的活菌数量比例为1:1)。 CK组不加细菌，P组加入1mL复合菌液，向各组中加水至土壤含水率为60％-70％，然后将所有花盆中的土样以木铲搅拌均匀，保证加入的细菌在土壤中分布均匀，每组三个平行。待细菌加入土壤一个月后，测量土壤可交换态、可氧化态、可还原态及残渣态重金属镉、铅的含量。土壤重金属初始可交换态含量设置为2ml/kg、5ml/kg、10ml/kg、20ml/kg、50ml/kg。
3、试验内容与结果
表4 盆栽试验一月后取样4种价态重金属镉含量测定结果
Figure PCTCN2020136200-appb-000005
表5 盆栽试验一月后取样4种价态重金属铅含量测定结果
Figure PCTCN2020136200-appb-000006
Figure PCTCN2020136200-appb-000007
结论：
通过表4和表5的数据可以看出，所述解磷菌P30和P46组成的复合菌剂对于不同浓度的铅和镉均能够实现良好的修复作用，修复效率平均可达98％以上。
实施例5 重金属修复菌剂稳定性试验
针对实施例4中修复后的四组土壤样品(50mg/kg CK、50mg/kg P)均以2g为一组分成若干组份加入50mL聚乙烯离心管中。为了研究不同环境条件对已改良土壤成分的影响，进一步探讨微生物修复土壤重金属污染对不同条件的稳定性。第一组用以判断pH对修复稳定性的影响，具体步骤为：以NaOH和HCl调节不同pH 3-9的酸碱溶液40mL加入管中，以150rpm，室温摇18±2h后，4000g离心15min，测量土壤上清液中的Cd 2+含量。第二组用以判断不同金属阳离子对修复稳定性的影响，具体步骤为：以分析纯NaNO 3，KNO 3，Ca(NO 3) 2药品溶于去离子水中制成0.01mol/L的Na +，K +和Ca 2+金属阳离子盐溶液40mL加入瓶中，以150rpm，摇18±2h后，4000g离心15min，测量土壤上清液中的Cd 2+含量。
表7 PH对修复后土壤稳定性的影响
Figure PCTCN2020136200-appb-000008
表8 重金属阳离子对修复后土壤稳定性的影响
Figure PCTCN2020136200-appb-000009
表7结果说明，土壤酸碱性会一定程度上改变已形成的稳定性Cd形态，尤其在H +大量存在时，CdS或Cd 2(PO 4) 3等生成产物可能会被氧化或分解，导致微生物修复结果的稳定性下降，二次释放回环境中。在碱性环境中，微生物修复产 物仍能保持稳定，不易被洗脱出释放至环境中。而P组相对CK组表现出降低活性的钝化效果。
从对比三种金属阳离子对土壤稳定性的结果来看，Ca 2+溶液能够提取的Cd 2+含量最多，这可能是由于Ca 2+与Cd 2+拥有相同的化合价，在进行离子交换时，可能发生晶格交换，配位络合等将Cd 2+从稳定态向可交换态活化的现象，而P组相对CK组均表现出降低活性的钝化效果。
土壤酸碱及金属阳离子均可导致微生物修复结果的稳定性下降，但对稳定性的影响不大，在实际应用中可忽略不计。
实施例6 培养基及培养条件的优化
将P46菌株置于不同温度下摇床培养48h，在10～40℃环境中的菌株能够生长，且30～40℃环境下的菌株生长最快；置于不同初始pH值环境下摇床培养48h，在pH值范围为3.5～8.5的环境中菌株能够生长，且在pH值为6.5～7.5的环境中生长最快。置于37℃、pH值为6.8的条件下摇床培养，转速分别设置120rpm、150rpm、180rpm，于第12、24、36、48、60取培养液计数。天神芽孢杆菌P46最佳培养条件为：pH：6.8；温度为37℃，搅拌速度180r/min，培养时间48h。
表9 天神芽孢杆菌P46在不同组分的营养培养基上的活菌数
Figure PCTCN2020136200-appb-000010
表注：
1、甘蔗糖蜜4.0％；豆粕2.0％；KH 2PO 4 0.3％；(NH) 2SO 4 0.15％；柠檬酸钠0.05％；MgSO 4·7H 2O 0.01％，MnSO 4 0.05％；余量为水；pH 7.0-7.2。
2、玉米粉6.0％；豆粕3.5％；硫酸铵0.3％；碳酸氢钠0.6％；碳酸钙0.1％；pH＝7.0。
根据活菌数与芽孢数综合分析，最终确定培养基成分为：玉米粉6.0％；豆粕3.5％；硫酸铵0.3％；碳酸氢钠0.6％；碳酸钙0.1％；其余为水；pH＝7.0。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一株天神芽孢杆菌(Bacillus tianshenli strain)P46，已于2020年3月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，其生物保藏号为：CGMCC NO.19444。
  2. 如权利要求1所述天神芽孢杆菌(Bacillus tianshenii strain)P46，其特征在于，具有SEQ ID NO:1所示的16S rDNA序列。
  3. 如权利要求1所述天神芽孢杆菌(Bacillus tianshenii strain)P46，其特征在于，所述天神芽孢杆菌菌粉制备方法如下：将所述天神芽孢杆菌(Bacillus tianshenli strain)P46接种于斜面培养基进行培养；取培养好的斜面，在无菌条件下取菌株接种于种子液体培养基中，振荡培养获得一级种子液；对一级种子液进行扩大培养获得二级种子液；以0.8～1.2％的接种量取二级种子液接种于液体发酵培养基中，当芽孢率达到90％以上时，收集发酵液，加入轻质碳酸钙后喷雾干燥得到菌粉。
  4. 如权利要求3所述天神芽孢杆菌(Bacillus tianshenii strain)P46，其特征在于，所述斜面培养中，将菌种一环接种于固体斜面培养基上，在37℃下培养24～30h；
    或所述一级种子培养在在37℃条件下，180rpm震荡培养18～24h。
  5. 如权利要求3所述天神芽孢杆菌(Bacillus tianshenii strain)P46，其特征在于，所述扩大培养以1％(v/v)的接种量，将一级种子液接于500mL～1000mL种子液体培养基中，在37℃条件下，震荡培养12～14h，制得二级种子液；
    或所述喷雾干燥的步骤中，向发酵液中加入0.67～1.00％(质量百分数)轻质碳酸钙。
  6. 如权利要求3所述天神芽孢杆菌(Bacillus tianshenii strain)P46，其特征在于，所述种子培养基配方如下：4.0％糖蜜，2.0％豆粕、0.7％K 2HPO 4、0.3％KH 2PO 4，0.15％(NH 4) 2SO 4，0.05％柠檬酸钠，0.01％硫酸镁，余量为水，调pH至6.8，121℃条件下灭菌30min；
    或所述固体斜面培养基为NB合成液体培养基中添加1.5～2.0％的琼脂粉。
  7. 如权利要求3所述天神芽孢杆菌(Bacillus tianshenii strain)P46，其特征在于，所述液体发酵培养基配方为：玉米粉6.0％；豆粕3.5％；硫酸铵0.3％；碳酸氢钠0.6％；碳酸钙0.1％；pH＝7.0。
  8. 一种菌剂，其特征在于，所述菌剂，所述菌剂包括权利要求1-7任一项所述的天神芽孢杆菌(Bacillus tianshenii strain)P46和/或菌的培养物；优选的，所述菌剂剂型为液体菌剂、粉剂或颗粒剂；进一步的为水悬浮剂、可分散油悬浮剂、可湿性粉剂或水分散颗粒剂；优选的，所述菌剂中还包括农药学上可接受的辅料，所述农药学上可接受的辅料选自分散剂、润湿剂、崩解剂、粘结剂、消泡剂、抗冻剂、增稠剂、填料和溶剂中的一种或多种。
  9. 权利要求1-7任一项所述天神芽孢杆菌(Bacillus tianshenii strain)P46、权利要求8所述菌剂在重金属土壤修复方面的应用。
  10. 一种复合菌剂，其特征在于，所述复合菌剂由天神芽孢杆菌(Bacillus tianshenli strain)P46与枯草芽孢杆菌(Bacillus subtilis strain)P30或其培养物组成；所述枯草芽孢杆菌(Bacillus subtilis strain)P30，已于2020年3月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，其生物保藏号为：CGMCC NO.19443。
PCT/CN2020/136200 2020-03-31 2020-12-14 一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用 WO2021196735A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020217024585A KR20210124232A (ko) 2020-03-31 2020-12-14 바실러스 톈션니 균주, 이의 미생물 제제 및 중금속 복구 분야의 응용

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010243541.2 2020-03-31
CN202010243541.2A CN111235076B (zh) 2020-03-31 2020-03-31 一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用

Publications (1)

Publication Number Publication Date
WO2021196735A1 true WO2021196735A1 (zh) 2021-10-07

Family

ID=70880671

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/136200 WO2021196735A1 (zh) 2020-03-31 2020-12-14 一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用

Country Status (3)

Country Link
KR (1) KR20210124232A (zh)
CN (1) CN111235076B (zh)
WO (1) WO2021196735A1 (zh)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113755401A (zh) * 2021-10-11 2021-12-07 中国热带农业科学院热带作物品种资源研究所 一种新鞘氨醇杆菌Novosphingobium capsulatum及其应用
CN114107130A (zh) * 2021-12-16 2022-03-01 天津科技大学 一种复合微生物菌肥及其制备方法和应用
CN114231459A (zh) * 2021-12-27 2022-03-25 中南大学 一种大肠埃希氏菌、微生物菌剂、组合物及应用
CN114621904A (zh) * 2022-05-13 2022-06-14 中国科学院地理科学与资源研究所 一种微球菌及其应用
CN114990024A (zh) * 2022-06-24 2022-09-02 浙江省农业科学院 一种耐高温抗生素降解混合菌剂及其制备方法和应用
CN115141770A (zh) * 2022-06-27 2022-10-04 安阳工学院 一种能够降解畜禽废水中cod的特基拉芽孢杆菌h1及其应用
CN115197708A (zh) * 2022-06-15 2022-10-18 江苏天象生物科技有限公司 一种土壤修复剂、制备装置及制备方法
CN115216428A (zh) * 2022-07-15 2022-10-21 厦门大学附属第一医院 一种抗汞细菌及其在汞污染治理方面的应用
CN115353997A (zh) * 2022-08-29 2022-11-18 山西农业大学高粱研究所 一种复合菌剂及其在降解土壤中烟嘧磺隆的应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111235076B (zh) * 2020-03-31 2020-10-30 山东碧蓝生物科技有限公司 一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用
CN112592855B (zh) * 2020-12-17 2022-05-20 有研资源环境技术研究院(北京)有限公司 一株枯草芽孢杆菌及其处理铀、镉污染水体的方法
CN113106083B (zh) * 2021-04-15 2022-06-21 浙江大学 降镉生物改性陶粒及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102168045A (zh) * 2010-12-24 2011-08-31 北京科为博生物科技有限公司 一种枯草芽孢杆菌制剂及其制备方法
CN107151639A (zh) * 2017-06-06 2017-09-12 广东植物龙生物技术股份有限公司 一株来源于蚕沙的菌株Bacillus subtilis SEM‑9及其应用
CN110205273A (zh) * 2019-06-11 2019-09-06 山东碧蓝生物科技有限公司 一种具有促生长及抗病作用的解淀粉芽孢杆菌及其应用
CN111235076A (zh) * 2020-03-31 2020-06-05 山东碧蓝生物科技有限公司 一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用
CN111423996A (zh) * 2020-03-31 2020-07-17 山东碧蓝生物科技有限公司 一种复合菌剂、其制备方法及在土壤修复领域的应用

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002192186A (ja) * 2000-12-22 2002-07-10 Fuji Photo Film Co Ltd 重金属捕集方法及び重金属回収方法
PE20080325A1 (es) * 2006-07-04 2008-04-10 Univ Chile Cepa bacteriana aislada y proceso de biosorcion de metales con un inoculante basado en la cepa
CN101255071B (zh) * 2007-03-01 2010-08-11 上海绿乐生物科技有限公司 一种利用城市污泥生产磷细菌生物肥料的方法
CN102757275A (zh) * 2012-07-25 2012-10-31 天津生机集团股份有限公司 一种用于水产养殖池的生物肥料及其制备方法
KR101540861B1 (ko) * 2013-12-16 2015-08-07 재단법인 전주생물소재연구소 양이온성 염료 및 금속에 흡착 효능을 갖는 바실러스 서브틸리스 jb-016 균주 및 이를 이용한 바이오매스
CN104193546B (zh) * 2014-08-06 2016-04-06 湖南省微生物研究院 一种降低镉、铅含量的复合土壤钝化剂及其制备和使用方法
CN105925502B (zh) * 2016-05-09 2019-06-18 广东植物龙生物技术股份有限公司 来源于蚕沙的菌株Bacillus megaterium OP6及其应用
CN106167776A (zh) * 2016-07-18 2016-11-30 暨南大学 一种可活化土壤中重金属镉的蜡样芽孢杆菌（Bacillus cereus）TH‑35及其应用
KR20180031159A (ko) * 2016-09-19 2018-03-28 경상대학교산학협력단 휴믹산에서 분리된 휘발성 유기물질을 생산하는 바실러스 메가테리움 균주, 이를 함유하는 식물환경복원용 조성물 및 이를 이용한 식물환경복원 방법
CN106734159A (zh) * 2016-12-02 2017-05-31 青岛蔚蓝生物集团有限公司 一种重金属污染土壤的修复方法
US10179744B2 (en) * 2017-01-12 2019-01-15 Cisbay Global Inc. Method for water remediation and desalination via selectively breeding and cultivating generations of microbes
US20180194697A1 (en) * 2017-01-12 2018-07-12 Khanh Le Microbial soil enhancements
US10221107B1 (en) * 2017-10-17 2019-03-05 Cisbay Global Inc. Microbial humic soil enhancements
CN108821908B (zh) * 2018-06-15 2021-03-02 湖南省微生物研究院 一种包含巴达维亚芽胞杆菌hj-1的菌剂及其应用
CN108795808B (zh) * 2018-06-15 2020-05-05 湖南省微生物研究院 一株巴达维亚芽胞杆菌及其应用
CN110628659B (zh) * 2018-06-21 2021-10-01 上海交通大学 巨大芽孢杆菌及其菌剂制备及其土壤重金属修复应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102168045A (zh) * 2010-12-24 2011-08-31 北京科为博生物科技有限公司 一种枯草芽孢杆菌制剂及其制备方法
CN107151639A (zh) * 2017-06-06 2017-09-12 广东植物龙生物技术股份有限公司 一株来源于蚕沙的菌株Bacillus subtilis SEM‑9及其应用
CN110205273A (zh) * 2019-06-11 2019-09-06 山东碧蓝生物科技有限公司 一种具有促生长及抗病作用的解淀粉芽孢杆菌及其应用
CN111235076A (zh) * 2020-03-31 2020-06-05 山东碧蓝生物科技有限公司 一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用
CN111423996A (zh) * 2020-03-31 2020-07-17 山东碧蓝生物科技有限公司 一种复合菌剂、其制备方法及在土壤修复领域的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIANG ZHAO, ZHANG DAO-FENG, KHIEU THI-NHAN, SON CHU KY, ZHANG XIAO-MEI, CHENG JUAN, TIAN XIN-PENG, ZHANG SI, LI WEN-JUN: "Bacillus tianshenii sp. nov., isolated from a marine sediment sample", INTERNATIONAL JOURNAL OF SYSTEMATIC AND REVOLUTIONARY MICROBIOLOGY, vol. 64, no. Pt 6, 1 June 2014 (2014-06-01), GB, pages 1998 - 2002, XP055854946, ISSN: 1466-5026, DOI: 10.1099/ijs.0.062224-0 *
LIU JUN-SHENG, XIE XIU-CHAO, LUO YANG-LAN, DENG BAI-WAN, BAI QIU-YUE, YAN MENG-CHEN, BAI XING: "Isolation, Identification, and Biological Characteristics of a Cadmium-Resistant Endophytic Bacteria Bacillus aryabhattai", BIOTECHNOLOGY BULLETIN, vol. 35, no. 2, 27 November 2018 (2018-11-27), pages 64 - 72, XP055854941, DOI: 10.13560/j.cnki.biotech.bull.1985.2018-0803 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113755401B (zh) * 2021-10-11 2023-04-07 中国热带农业科学院热带作物品种资源研究所 一种新鞘氨醇杆菌Novosphingobium capsulatum及其应用
CN113755401A (zh) * 2021-10-11 2021-12-07 中国热带农业科学院热带作物品种资源研究所 一种新鞘氨醇杆菌Novosphingobium capsulatum及其应用
CN114107130A (zh) * 2021-12-16 2022-03-01 天津科技大学 一种复合微生物菌肥及其制备方法和应用
CN114107130B (zh) * 2021-12-16 2024-03-15 天津北洋百川生物技术有限公司 一种复合微生物菌肥及其制备方法和应用
CN114231459A (zh) * 2021-12-27 2022-03-25 中南大学 一种大肠埃希氏菌、微生物菌剂、组合物及应用
CN114231459B (zh) * 2021-12-27 2023-05-26 中南大学 一种大肠埃希氏菌、微生物菌剂、组合物及应用
CN114621904A (zh) * 2022-05-13 2022-06-14 中国科学院地理科学与资源研究所 一种微球菌及其应用
CN115197708A (zh) * 2022-06-15 2022-10-18 江苏天象生物科技有限公司 一种土壤修复剂、制备装置及制备方法
CN114990024A (zh) * 2022-06-24 2022-09-02 浙江省农业科学院 一种耐高温抗生素降解混合菌剂及其制备方法和应用
CN115141770A (zh) * 2022-06-27 2022-10-04 安阳工学院 一种能够降解畜禽废水中cod的特基拉芽孢杆菌h1及其应用
CN115141770B (zh) * 2022-06-27 2023-08-29 安阳工学院 一种能够降解畜禽废水中cod的特基拉芽孢杆菌h1及其应用
CN115216428A (zh) * 2022-07-15 2022-10-21 厦门大学附属第一医院 一种抗汞细菌及其在汞污染治理方面的应用
CN115216428B (zh) * 2022-07-15 2023-09-19 厦门大学附属第一医院 一种抗汞细菌及其在汞污染治理方面的应用
CN115353997A (zh) * 2022-08-29 2022-11-18 山西农业大学高粱研究所 一种复合菌剂及其在降解土壤中烟嘧磺隆的应用
CN115353997B (zh) * 2022-08-29 2023-08-22 山西农业大学高粱研究所 一种复合菌剂及其在降解土壤中烟嘧磺隆的应用

Also Published As

Publication number Publication date
CN111235076B (zh) 2020-10-30
CN111235076A (zh) 2020-06-05
KR20210124232A (ko) 2021-10-14

Similar Documents

Publication Publication Date Title
WO2021196735A1 (zh) 一种天神芽孢杆菌、其菌剂及在重金属修复领域的应用
CN111423996B (zh) 一种复合菌剂、其制备方法及在土壤修复领域的应用
CN110106126B (zh) 一株胶质芽孢杆菌及其在制备盐碱土壤改良剂中的应用
WO2021248974A1 (zh) 具有降低蔬菜重金属含量、提高蔬菜质量的功能菌株及应用
CN104450552A (zh) 一种硫酸盐还原菌-解磷菌及其在联合修复镉污染土壤中的应用
CN111849815A (zh) 一种植物根际促生菌株Gxun-20及其在植物促生中的应用
CN105255794A (zh) 一种假单胞菌及其应用
CN110699303A (zh) 一株高效解磷假单胞菌及其微生物菌剂和应用
CN108587947B (zh) 一株溶磷菌及其与dehp降解菌复合菌剂与在土壤改良中的应用
CN108821908B (zh) 一种包含巴达维亚芽胞杆菌hj-1的菌剂及其应用
WO2024051151A1 (zh) 释硅链霉菌 cs13-6及其应用
CN109055258B (zh) 一种贪铜菌及贪铜菌制剂和贪铜菌制剂在重金属污染土壤修复中的应用
Upadhyay et al. Isolation, characterization and morphological study of Azotobacter isolates
CN108795808B (zh) 一株巴达维亚芽胞杆菌及其应用
CN116676201B (zh) 一种波兰青霉cfp-2及其应用
CN110846250B (zh) 一株高产γ-PGA的枯草芽孢杆菌及其应用
CN115505536A (zh) 一种黑曲霉ca-5及其应用
CN109868242B (zh) 一株耐盐产乙偶姻的枯草芽孢杆菌及其应用
CN109576177B (zh) 一种中华微杆菌株sm8及其在耐盐促生中的应用
CN111334457A (zh) 一株具有土壤重金属修复作用的天神芽孢杆菌及其应用
CN114317373B (zh) 一种鞘氨醇单胞菌pah02、微生物制剂及其作为农作物降镉富硒功能调理剂的应用
CN112779187B (zh) 一株五大连池芽孢杆菌及其应用
CN115960766A (zh) 一种防治青枯病的微生物及其应用
CN108330085B (zh) 一种适用于低温环境的解钾菌株及基于该菌株制备微生态制剂的发酵方法
CN107164245B (zh) 一株撕裂蜡孔菌的促生作用及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20929507

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20929507

Country of ref document: EP

Kind code of ref document: A1