Phage composition and its use in strengthening soil carbon fixation

Abstract

A phage composition and an use thereof in strengthening soil carbon fixation, said phage composition including three phages, namely, a polyvalent phage YSZKP that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa; a Bacillus cereus phage YSZBA1; and a Bacillus cereus phage YSZBA2. In the present invention, the phage composition is injected into the target soil, and an approach of strengthening soil carbon fixation by means of cooperation with the host bacteria through injection of AMGs from the phage is employed; at the same time, the phage has a function of regulating the soil microbial communities from bottom to top; a network of symbiosis with indigenous bacteria is further optimized, and the soil carbon fixation potential is improved. This technique is an environmental improvement technique that achieves a good carbon fixation effect, has a low price and is environment-friendly.

Claims

1. A method of enhancing soil carbon fixation, the method comprising applying a phage composition to the soil, wherein the phage composition comprises: Klebsiella pneumoniae and Pseudomonas aeruginosa phage YSZKP, having accession number CCTCC NO. M 2018514; Bacillus cereus phage YSZBA1, having accession number CCTCC NO. M 2018517; and Bacillus cereus phage YSZBA2, having accession number CCTCC NO. M 2018518.

2. The method of claim 1, wherein the soil is used for planting Chinese cabbage, lettuce, carrot or Capsicum frutescens.

3. The method of claim 1, wherein the phage composition comprises the Klebsiella pneumoniae and Pseudomonas aeruginosa phage YSZKP, Bacillus cereus phage YSZBA1 and Bacillus cereus phage YSZBA2 at a concentration ratio of 1:1:1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram of a technique used to promote promoting soil carbon fixation through phage transplantation;

(2) FIG. 2 is a TEM image of a phage YSZKP;

(3) FIG. 3 is a TEM image of a phage YSZBA1;

(4) FIG. 4 is a TEM image of a phage YSZBA2;

(5) FIG. 5 shows a one-step growth curve of phages YSZKP, YSZBA1 and YSZBA2);

(6) FIG. 6 shows the abundance of a related chitin synthetic gene GT2 in the soil of a vegetable base 70 days after microbial inoculum injection;

(7) FIG. 7 shows the average content of chitin in the soil of the vegetable base 70 days after microbial inoculum injection;

(8) FIG. 8 shows the abundance of the chitin synthetic gene GT2 in the soil of Fengqiu Station in Henan Province 70 days after microbial inoculum injection;

(9) FIG. 9 shows the average content of chitin in the soil of Fengqiu Station in Henan Province 70 days after microbial inoculum injection;

(10) FIG. 10 shows the abundance of the chitin synthetic gene GT2 in the soil of a pesticide contaminated relocation site in Inner Mongolia 60 days after microbial inoculum injection;

(11) FIG. 11 shows the average content of chitin in the soil of the pesticide contaminated relocation site in Inner Mongolia 60 days after microbial inoculum injection;

(12) FIG. 12 shows the abundance of the chitin synthetic gene GT2 in the soil of a vegetable base in Zhangye city of Gansu Province 45 days after microbial inoculum injection;

(13) FIG. 13 shows the average content of chitin in the soil of the vegetable base in Zhangye city of Gansu Province 45 days after microbial inoculum injection;

(14) FIG. 14 shows the abundance of the chitin synthetic gene GT2 in the soil of a contaminated site of a heavy metal enterprise in Xi'an city of Shaanxi Province 70 days after microbial inoculum injection; and

(15) FIG. 15 shows the average content of chitin in the soil of the contaminated site of the heavy metal enterprise in Xi'an city of Shaanxi Province 70 days after microbial inoculum injection.

(16) The multivalent phage YSZKP that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa was preserved on Aug. 1, 2018 in China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and assigned with a CCTCC NO. M 2018514.

(17) The Bacillus cereus phage YSZBA1 was preserved on Aug. 1, 2018 in China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and assigned with a CCTCC NO. M 2018517.

(18) The Bacillus cereus phage YSZBA2 was preserved on Aug. 1, 2018 in China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and assigned with a CCTCC NO. M 2018518.

EMBODIMENTS

(19) The following detailed embodiments is not intended to limit the technical solution of the present invention in any way, and all technical solutions obtained through equivalent replacement or equivalent transformation should be deemed as falling within the scope of protection of the present invention.

(20) The subject phages have been deposited under conditions that assure that access to the deposits will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

(21) Further, the subject deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least 30 (thirty) years after the date of deposits or for the enforceable life of any patent which may issue disclosing the deposits. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject deposits will be irrevocably removed upon the granting of a patent disclosing them.

(22) The resistance gene GT2 refers to a carbon fixation function gene (anabolic gene) carried by the phages detected in soil and a carbon fixation gene carried by in-situ host bacteria.

(23) TABLE-US-00001 TABLE 1 Determination of Optimal Multiplicity of Infection (OMOI) Multiplicity of Phage Count Bacterial Titer A Titer B Titer C Test Infection (MOI) (PFU .Math. Count (PFU .Math. (PFU .Math. (PFU .Math. Tube (PFU .Math. CFU.sup.1) mL.sup.1) (CFU .Math. mL.sup.1) mL.sup.1) mL.sup.1) mL.sup.1) 1 100:1 1 10.sup.9 1 10.sup.7 3.2 10.sup.6 1.2 10.sup.8 5.2 10.sup.8 2 10:1 1 10.sup.8 1 10.sup.7 6.1 10.sup.6 1.7 10.sup.8 6.1 10.sup.8 3 1:1 1 10.sup.7 1 10.sup.7 0.8 10.sup.7 2.8 10.sup.7 6.9 10.sup.8 4 1:10 1 10.sup.6 1 10.sup.7 4.3 10.sup.7 5.3 10.sup.6 1.6 10.sup.9 5 1:100 1 10.sup.5 1 10.sup.7 3.9 10.sup.8 1.2 10.sup.6 2.8 10.sup.9 6 1:1000 1 10.sup.4 1 10.sup.7 4.7 10.sup.8 4.9 10.sup.5 5.2 10.sup.9 7 1:10000 1 10.sup.3 1 10.sup.7 7.6 10.sup.7 4.3 10.sup.5 3.1 10.sup.9 Note: A: titer of phage YSZKP; B: titer of phage YSZBA1; C: titer of phage YSZBA2.

Example 1

(24) Test soil for potted plants is collected from Hengliang vegetable base in Nanjing, Jiangsu Province. The planted vegetable was Hongpin No. 1 Capsicum frutescens var from Qianshu Baihua Seeds Co., Ltd. The basic physical and chemical properties of the soil are as follows: sand grain: 26.8%, soil grain: 37.4%, clay grain: 31.82%, pH: 7.24, total nitrogen: 1.44 g.Math.kg.sup.1, water-soluble nitrogen: 1.62 g.Math.kg.sup.1, total phosphorus: 1.50 g.Math.kg.sup.1, total potassium: 18.52 g.Math.kg.sup.1, and CEC: 16.43 cmol.Math.kg.sup.1.

(25) 0.5 g soil is weighed and loaded into a 15 mL sterile centrifuge tube. 4.5 mL liquid culture medium is added to the sample, the mixture is mixed thoroughly, incubated for 1 hour, and then centrifuged. The supernatant obtained by centrifugation is filtered through a tangential flow filtration system, the filtrate obtained after tangential flow filtration is a phage suspension, in which free phage is suspended in liquid component; frequent turn is needed during incubation. Separation of the filtered phage: 100 L carbon fixation host bacteria (Klebsiella) in a logarithmic phase, 200 L phage suspension, and 5 mL semi-solid culture medium are added, the mixture is poured into a solid LB culture medium, and is cultured at 30-37 C. in an inverted state after the mixture solidifies. Further phage purification: 100 L diluent and 100 L bacteria are sucked and spread on a double-layer plate and purified continuously for 3-5 times, until the plaques on the plate are in the same size. After the culture, the plaques on the Petri dish are transparent at the center, with no halo around it, and has a diameter of about 2-3 mm; the phage YSZKP is obtained. A single clear and transparent plaque is picked for enrichment, mixed with 50% glycerol at a ratio of 1:1, and stored at 80 C. for later use.

(26) Based on the aforesaid phage YSZKP obtained from the host bacteria Klebsiella, a process of accelerating expression of the phage in a broad host spectrum is performed: 600 L preserved stock solution of the phage is taken, and is added together with a mixed bacterial suspension of 200 L Klebsiella and 200 mL Bacillus cereus into 99 mL LB liquid culture medium, then solid calcium chloride is added to adjust the final concentration to 1 mmol.Math.L.sup.1; the mixture is cultured at 37 C. while shaking at 150 rpm for 96 hours, and is sampled once every 8 hours; the phage obtained by centrifugal filtration and Bacillus cereus are poured on a double-layer plate for verification by observing the plaques; appearance of plaques proves that the directed evolution is successful. Thus, phages YSZBA1 and YSZBA2 are obtained. A single clear and transparent plaque is selected for enrichment, mixed with 50% glycerol at a ratio of 1:1, and stored at 80 C. for later use.

(27) Based on the above operations, two specific phage strains are obtained, namely: Klebsiella phage YSZKP and Bacillus cereus phages YSZBA1 and YSZBA2.

(28) Five groups are arranged in the experiment: (1) control group (CK): three plants of capsicum frutescens var are planted in each pot (the seeds are covered with soil having a thickness of 0.5-1 cm, at room temperature 252 C.); (2) single phage treatment (V1): on the basis of the control group, 100 mL suspension of phage YSZKP at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (3) single phage treatment (V2): on the basis of the control group, 100 mL suspension of phage YSZBA1 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (4) single phage treatment (V3): on the basis of the control group, 100 mL suspension of phage YSZBA2 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (5) hybrid phage treatment (V): on the basis of the control group, 100 mL mixed suspension of the above three phages at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated. Soil samples are collected in situ 70 days after the plants of Capsicum frutescens var are planted, and the abundances of a chitin synthetic gene GT2 in the soil under the treatments of the five groups CK, V1, V2, V3 and V are 4.0810.sup.5 copies.Math.g.sup.1, 7.2410.sup.7 copies.Math.g.sup.1, 3.3810.sup.8 copies.Math.g.sup.1, 2.0110.sup.8 copies.Math.g.sup.1, and 8.1910.sup.8 copies.Math.g.sup.1 respectively (FIG. 6). Compared with the control group, the abundance of carbon fixation resistant gene GT2 in the group V is increased by three orders of magnitude (p<0.05). For the control group and treatment groups, the average contents of chitin in the soil are 228.32 mg.Math.kg.sup.1, 429.66 mg.Math.kg.sup.1, 310.75 mg.Math.kg.sup.1, 337.00 mg.Math.kg.sup.1, and 443.67 mg.Math.kg.sup.1 respectively (FIG. 7). Compared with the treatment without phage transplantation, for the treatment with hybrid phage transplantation, the content of soil organic matters is increased by 5 g/kg to 310 g/kg after 70 days culture, indicating that the addition of phage suspension has a significant effect on enriching microbial carbon anabolic genes and promoting soil carbon fixation.

Example 2

(29) Target soil: soil from Fengqiu Station in Henan Province. The basic physical and chemical properties of the soil are as follows: pH: 6.8, organic matters: 21.1 g.Math.kg.sup.1, total nitrogen: 4.8 g.Math.kg.sup.1, total phosphorus: 2.1 g.Math.kg.sup.1; mechanical composition of the soil: 45.1% sand grain (sandy soil), 22.8% soil grain and 32.1% clay grain.

(30) Five groups are arranged in the experiment: (1) control group (CK): three plants of carrot are planted in each pot (the seeds are covered with soil having a thickness of 0.5-1 cm, at room temperature 252 C.); (2) single phage treatment (V1): on the basis of the control group, 100 mL suspension of phage YSZKP at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (3) single phage treatment (V2): on the basis of the control group, 100 mL suspension of phage YSZBA1 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (4) single phage treatment (V3): on the basis of the control group, 100 mL suspension of phage YSZBA2 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (5) hybrid phage treatment (V): on the basis of the control group, 100 mL mixed suspension of the above three phages at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated. Soil samples are collected in situ 70 days after the plants of carrot are planted, and the abundances of the chitin synthetic gene GT2 in the soil under the treatments of the five groups CK, V1, V2, V3 and V are 5.2710.sup.5 copies.Math.g.sup.1, 5.2310.sup.7 copies.Math.g.sup.1, 2.2810.sup.8 copies.Math.g.sup.1, 1.2510.sup.8 copies.Math.g.sup.1, and 7.6810.sup.8 copies.Math.g.sup.1 respectively (FIG. 8). Compared with the control group, the abundance of carbon fixation resistant gene GT2 in the treatment group V is increased by three orders of magnitude (p<0.05). For the control group and treatment groups, the average contents of chitin in the soil are 564.75 mg.Math.kg.sup.1, 647.99 mg.Math.kg.sup.1, 628.37 mg.Math.kg.sup.1, 564.73 mg.Math.kg.sup.1, and 695.12 mg.Math.kg.sup.1 respectively (FIG. 9). Compared with the treatment without phage transplantation, for the treatment with hybrid phage transplantation, the content of soil organic matters is increased by 10 g/kg to 22 g/kg after 70 days culture.

(31) The inoculation of the phage can significantly promote the carbon fixation process of the soil microbe to a certain extent, and also is helpful for maintaining and improving the diversity and stability of soil microbial ecological functions after remediation.

Example 3

(32) Test soil for potted plants is collected from a relocation site contaminated by bactericides and pesticides in Inner Mongolia. The basic physical and chemical properties of the soil are as follows: pH: 5.5, organic matters: 22.4 g.Math.kg.sup.1, total nitrogen: 2.4 g.Math.kg.sup.1, total phosphorus: 0.6 g.Math.kg.sup.1, total potassium: 11.1 g.Math.kg.sup.1, and rapidly available potassium: 72.7 g.Math.kg.sup.1; mechanical composition of the soil: 52.3% sand grain (sandy soil), 20.6% soil grain and 27.1% clay grain. The average concentration of carbon-related metabolites in soil layer zero to one meter below ground is 428.2 mg.Math.kg.sup.1.

(33) Five groups are arranged in the experiment: (1) control group (CK): three plants of lettuce are planted in each pot (the seeds are covered with soil having a thickness of 0.5-1 cm, at room temperature 252 C.); (2) single phage treatment (V1): on the basis of the control group, 100 mL suspension of phage YSZKP at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (3) single phage treatment (V2): on the basis of the control group, 100 mL suspension of phage YSZBA1 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (4) single phage treatment (V3): on the basis of the control group, 100 mL suspension of phage YSZBA2 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (5) hybrid phage treatment (V): on the basis of the control group, 100 mL mixed suspension of the above three phages at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated. Soil samples are collected in situ 60 days after the plants of lettuce are planted, and the abundances of the chitin synthetic gene GT2 in the soil under the treatments of the five groups CK, V1, V2, V3 and V are 2.1510.sup.4 copies.Math.g.sup.1, 9.3610.sup.5 copies.Math.g.sup.1, 8.5010.sup.5 copies.Math.g.sup.1, 1.1510.sup.6 copies.Math.g.sup.1, and 2.2210.sup.6 copies.Math.g.sup.1 respectively (FIG. 10). For the treatment group and the control group, the average contents of chitin in the soil are 424.92 mg.Math.kg.sup.1, 480.91 mg.Math.kg.sup.1, 442.81 mg.Math.kg.sup.1, 473.30 mg.Math.kg.sup.1, and 521.33 mg.Math.kg.sup.1 respectively (FIG. 11). Compared with the treatment without phage transplantation, for the treatment with hybrid phage transplantation, the content of soil organic matters is increased by 8 g/kg to 17 g/kg after 60 days culture.

Example 4

(34) Target soil: soil from a vegetable base in Zhangye, Gansu. The basic physical and chemical properties of the soil are as follows: pH: 5.8, organic matters: 25.1 g.Math.kg.sup.1, total nitrogen: 6.8 g.Math.kg.sup.1, total phosphorus: 2.7 g.Math.kg.sup.1; mechanical composition of the soil: 39.1% sand grain (sandy soil), 22.8% soil grain and 38.1% clay grain.

(35) Five groups are arranged in the experiment: (1) control group (CK): three plants of Chinese cabbage are planted in each pot (the seeds are covered with soil having a thickness of 0.5-1 cm, at room temperature 252 C.); (2) single phage treatment (V1): on the basis of the control group, 100 mL suspension of phage YSZKP at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (3) single phage treatment (V2): on the basis of the control group, 100 mL suspension of phage YSZBA1 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (4) single phage treatment (V3): on the basis of the control group, 100 mL suspension of phage YSZBA2 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (5) hybrid phage treatment (V): on the basis of the control group, 100 mL mixed suspension of the above three phages at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated. Soil samples are collected in situ 45 days after the plants of Chinese cabbage are planted, and the abundances of the chitin synthetic gene GT2 in the soil under the treatments of the five groups CK, V1, V2, V3 and V are 2.8210.sup.5 copies.Math.g.sup.1, 8.3810.sup.7 copies.Math.g.sup.1, 1.9610.sup.8 copies.Math.g.sup.1, 8.0410.sup.7 copies.Math.g.sup.1, and 7.6110.sup.8 copies.Math.g.sup.1 respectively (FIG. 12). For the treatment group and the control group, the average contents of chitin in the soil are 369.65 mg.Math.kg.sup.1, 473.57 mg.Math.kg.sup.1, 421.81 mg.Math.kg.sup.1, 425.64 mg.Math.kg.sup.1, and 511.88 mg.Math.kg.sup.1 respectively (FIG. 13). Compared with the treatment without phage transplantation, for the treatment with hybrid phage transplantation, the content of soil organic matters is increased by 10 g/kg to 22 g/kg after 45 days culture.

Example 5

(36) Target soil: soil from a polluted site of a heavy metal enterprise in Xi'an, Shaanxi. The basic physical and chemical properties of the soil are as follows: pH: 6.8, organic matters: 20.1 g.Math.kg.sup.1, total nitrogen: 7.8 g.Math.kg.sup.1, total phosphorus: 7.6 g.Math.kg.sup.1; mechanical composition of the soil: 42.1% sand grain (sandy soil), 22.8% soil grain and 35.1% clay grain.

(37) Five groups are arranged in the experiment: (1) control group (CK): three plants of lettuce are planted in each pot (the seeds are covered with soil having a thickness of 0.5-1 cm, at room temperature 252 C.); (2) single phage treatment (V1): on the basis of the control group, 100 mL suspension of phage YSZKP at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (3) single phage treatment (V2): on the basis of the control group, 100 mL suspension of phage YSZBA1 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (4) single phage treatment (V3): on the basis of the control group, 100 mL suspension of phage YSZBA2 at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated; (5) hybrid phage treatment (V): on the basis of the control group, 100 mL mixed suspension of the above three phages at 10.sup.6 pfu.Math.mL.sup.1 concentration is inoculated. Soil samples are collected in situ 70 days after the plants of lettuce are planted, and the abundances of the chitin synthetic gene GT2 in the soil under the treatments of the five groups CK, V1, V2, V3 and V are 2.3010.sup.5 copies.Math.g.sup.1, 1.9610.sup.6 copies.Math.g.sup.1, 2.8710.sup.6 copies.Math.g.sup.1, 2.0310.sup.6 copies.Math.g.sup.1, and 6.2110.sup.6 copies.Math.g.sup.1 respectively (FIG. 14). For the treatment groups and the control group, the average contents of chitin in the soil are 212.26 mg.Math.kg.sup.1, 308.67 mg.Math.kg.sup.1, 310.75 mg.Math.kg.sup.1, 312.27 mg.Math.kg.sup.1, and 432.78 mg.Math.kg.sup.1 respectively (FIG. 15). Compared with the treatment without phage transplantation, for the treatment with hybrid phage transplantation, the content of soil organic matters is increased by 9 g/kg to 16 g/kg after 50 days culture.

(38) The result proves that the applied technique of promoting soil carbon fixation by phage transplantation has the advantages of high broad-spectrum, low ecological risk and environmental friendliness, and it is a soil carbon storage optimization technique that has good application prospects.