CN118497149A - A bacteriophage composition and its application in enhancing soil carbon fixation - Google Patents

Abstract

A bacteriophage composition and its application in strengthening soil carbon fixation, including three strains of bacteriophages, namely, a multivalent bacteriophage φYSZKP that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa; a Bacillus cereus phage φYSZBA1; and a Bacillus cereus phage φYSZBA2. The present invention injects the bacteriophage composition into the target soil, and uses the bacteriophage injection AMGs to cooperate with the host bacteria to strengthen the soil carbon fixation path. At the same time, the bacteriophage has the function of regulating the soil microbial community from the bottom up, further optimizing the indigenous bacterial symbiotic network, and improving the soil carbon fixation potential. This technology is an environmental improvement technology with good carbon fixation effect, low price and green friendliness.

Description

A bacteriophage composition and its application in enhancing soil carbon fixation

Technical Field

The present invention belongs to the technical field of bacteriophages, and in particular relates to a bacteriophage composition and its application in enhancing soil carbon fixation.

Background Art

Cross-kingdom interactions between microbial groups can promote the production and accumulation of microbial carbon. Bacteriophages (referred to as phages) and host bacteria are typical examples of cross-kingdom interactions. Bacteriophages are a class of organisms that survive by preying on living host bacteria. They are widely distributed in soil, air, and water, and their total number is estimated to reach 10 ^31. Bacteriophages can transfer auxiliary metabolic genes (AMGs) through horizontal gene transfer to compensate for the expression of multiple physiological functions of the host. Currently, there are two known ways for phages to participate in soil carbon cycling: lysing host communities to release metabolites, thereby promoting the accumulation of environmental organic carbon; or participating in the host-mediated carbon biogeochemical cycle through lysogeny, assisting the host in biosynthesis, and ultimately achieving macroscopic carbon fixation.

Phage transplantation refers to an emerging biological technology that efficiently enriches the entire phage community in a habitat, then transplants it into a designated target host environment and allows it to colonize and function. Soil phage transplantation is derived from the concept of fecal microbiota transplantation in the medical field to treat human diseases. The advantages of soil phage transplantation technology are: it can quantitatively identify the role and function of phages in the soil microbiome, and facilitates the transplantation of lysogenic phages known to carry carbon fixation function AMGs into the target soil to successfully assist host bacteria in strengthening their own carbon synthesis metabolic pathways.

Through relevant literature review and patent search, no public publication or acceptance of biotechnology related to optimizing soil microbial carbon fixation system and enhancing soil carbon fixation efficiency by means of bacteriophage transplantation was found. The existing method closest to the present invention is: preparing microbial agents to promote soil carbon fixation, such as invention publication number CN102775201A is to prepare carbon-fixing high-activity microbial organic fertilizer, and the microbial agents used include Bacillus thuringiensis and Nocardia; and patent publication numbers CN116813406A, CN117000756A, CN114916277A, CN101607838A, and CN117209183A are to sequentially apply microbial agents with microplastics, clay minerals, iron oxide, water-absorbing and water-retaining agents, and multi-source solid waste, so as to strengthen soil carbon fixation from a physical and chemical level by ensuring the living space of microorganisms and improving soil structure. The bacterial species mentioned in the above inventions include nitrate-reducing ferrous oxidizing microorganisms, lactic acid bacteria, soil photosynthetic microorganisms or ammonifying Thermovibrio, and do not involve cross-kingdom interactions of microorganisms. In addition to the improvement of bacterial agents, there are also a number of patents involving the design of soil microbial carbon fixation systems, such as the invention publication number: CN117296507A, which uses microorganisms to treat livestock manure, and then calculates and adds treated organic fertilizers to the actual soil conditions to enhance the soil carbon fixation potential. CN117256286A manufactures a drip irrigation system suitable for photosynthetic bacteria proliferation and soil carbon fixation by adding a jet suction liquid photosynthetic bacteria compound fertilizer module to the drip irrigation system. These methods all point out that microbial agents are an important way to fix soil carbon at present, but there is a general lack of application to the phage-host collaborative carbon fixation pathway that is widely present in the soil.

The main defects of the existing technology are: Most of the existing soil microbial carbon fixation technologies only mix some carbon-fixing bacteria and cultivable bacteria, ignoring the effects of the bacterial community or the overall microbial community. In addition, the functional bacterial community that has been artificially enhanced may have the risk of secondary biological contamination in the process of promoting soil carbon fixation, either the bacterial community itself or its metabolites. The artificial preparation process is complex and the preparation cycle is long. The research and development of carbon fixation equipment generally has the problems of high cost and lack of universality.

The main reasons for the defects are: Although it is recognized that soil can serve as an important global carbon sink, the impact of human activities on soil carbon fixation has been increasing in recent years, and early scientific researchers have paid relatively little attention to and researched the carbon fixation interaction system of soil microbial communities. Due to the competitive relationship between bacteria, the traditional technology of adding exogenous carbon-fixing functional bacteria to the soil is easily affected by the dominant ecological niche of the indigenous flora, resulting in a significant decrease in the effect and durability of the functional carbon-fixing bacteria. In the inoculation of carbon-fixing bacteria remediation technology, the fermentation, production, and amplification process of the bacterial agent are complex and costly. Exogenous addition of soil additives will also pose a survival risk to the indigenous flora, and it is difficult to assess the long-term negative ecological impact. Therefore, it is of great significance to add bacteriophages with carbon fixation functions, develop non-bacterial carbon-fixing bacteriophage soil additives, improve carbon fixation efficiency, and reduce ecological risks. Soil microbial carbon fixation technology.

Summary of the invention

Technical problem solved: In view of the above-mentioned defects of the prior art, the invention provides a bacteriophage composition and its application in enhancing soil carbon fixation. The present invention injects the bacteriophage composition into the target soil, and uses the bacteriophage injection AMGs to cooperate with the host bacteria to strengthen the soil carbon fixation pathway. At the same time, the bacteriophage has the function of regulating the soil microbial community from the bottom up, further optimizing the indigenous bacterial symbiotic network, and improving the soil carbon fixation potential. This technology is an environmental improvement technology with good carbon fixation effect, low price and green friendliness.

Technical solution: A phage composition contains three phages, which were all deposited in the China Center for Type Culture Collection on August 1, 2018, and are respectively a multivalent phage φYSZKP attacking Klebsiella pneumoniae and Pseudomonas aeruginosa, with a deposit number of CCTCC NO:M 2018514, and a classification name of Klebsiella and Pseudomonas aeruginosa phage φYSZKP; a Bacillus cereus phage φYSZBA1, with a deposit number of CCTCC NO:M2018517, and a classification name of Bacillus cereus phage φYSZBA1; and a Bacillus cereus phage φYSZBA2, with a deposit number of CCTCC NO:M 2018518, and a classification name of Bacillus cereus phage φYSZBA2.

Preferably, the concentration ratio of the three bacteriophages in the composition is 1:1:1.

The method for preparing the above-mentioned phage composition comprises mixing three phages so that their concentrations in the composition are 1:1:1.

The use of the above-mentioned bacteriophage composition in the preparation of products for enhancing soil carbon fixation.

The above soil is soil for growing cabbage, lettuce, carrot or chili pepper.

A soil conditioner for enhancing soil carbon fixation, comprising the above-mentioned bacteriophage composition and biologically active ingredients that help soil carbon fixation.

The above-mentioned biologically active ingredients include microbial agents, humic acid, amino acids or biochar.

The application of the soil conditioner described above involves injecting the soil conditioner into the target soil.

The working principle of the present invention is: 1. Bacteriophage is a type of bacterial virus composed of protein capsid (60%) and nucleic acid (40%), without a complete mature cell structure, and can be divided into two types: lytic and lysogenic; 2. Lysogenic phage can inject its own genetic material into the bacteria, affecting the metabolic process of the host bacterial community. Auxiliary metabolic genes (AMGs) are gene fragments present in the phage genome, which are generally randomly packaged and assembled into the phage genome during the synthesis of phage particles. AMGs can be expressed by the host during phage infection and play a key role, such as central carbon metabolism, energy metabolism and other processes; 3. Broad-spectrum phages refer to phages that can infect two or more "species" with similar homology or host bacteria between different species, which is conducive to the extensive use of the synergistic metabolic potential of phages; 4. The phages selected for phage transplantation technology are based on the natural (temperature, moisture, pH) and non-natural (heavy metals, pesticides, antibiotics) environmental conditions of the in situ soil. The phages selected for this technology have a broad-spectrum lysogenic phage with carbon fixation function. This phage can be well integrated into the microbial network of the region, while minimizing ecological risks, achieving a good phage-host synergistic carbon fixation effect, ensuring the diversity and stability of local microbial ecological functions; 5. The length of the phage is about 20~200μm, which is equivalent to hundreds and thousands of bacteria, so it can achieve extensive migration in the soil, which is conducive to saving application costs.

Beneficial effects: Aiming at the need to strengthen soil carbon fixation, the present invention provides a phage composition and its application in strengthening soil carbon fixation. Its advantages are: 1. Phage can promote the host carbon fixation process through AMGs injection, effectively exerting the soil carbon fixation potential; 2. Lysogenic phage weakens the interspecies competition problem existing in traditional carbon fixation bacteria, and optimizes the microbial community structure from the perspective of cross-border cooperation; 3. Buffer preparation materials are easy to obtain, easy to store, convenient to transport, simple to use and operate, and can effectively control costs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the application technology of bacteriophage transplantation to promote soil carbon fixation.

FIG2 is a transmission electron micrograph of bacteriophage φYSZKP.

FIG3 is a transmission electron micrograph of bacteriophage φYSZBA1.

FIG4 is a transmission electron micrograph of bacteriophage φYSZBA2.

FIG5 is a one-step growth curve of bacteriophages (φYSZKP, φYSZBA1, φYSZBA2).

Figure 6 shows the abundance of the chitin synthesis gene GT2 in the soil of the vegetable base 70 days after the injection of the bacterial agent.

Figure 7 shows the average chitin content in the soil of the vegetable base 70 days after the injection of the bacterial agent.

Figure 8 shows the abundance of chitin synthesis gene GT2 in soil 70 days after the injection of bacterial agent.

Figure 9 shows the average chitin content in the soil 70 days after the bacterial agent was injected into the soil.

Figure 10 shows the abundance of chitin synthesis gene GT2 in the soil of the fungicide and pesticide contaminated relocation site 60 days after the injection of fungicides.

Figure 11 shows the average chitin content in the soil of the fungicide and pesticide contaminated relocation site 60 days after the injection of fungicides.

Figure 12 shows the abundance of chitin synthesis gene GT2 in the soil 45 days after the injection of bacterial agent.

Figure 13 shows the average chitin content in the soil 45 days after the bacterial agent was injected into the soil.

Figure 14 shows the abundance of chitin synthesis gene GT2 in the soil of the contaminated site 70 days after the injection of the bacterial agent.

Figure 15 shows the average chitin content in the soil of the contaminated site 70 days after the injection of the bacterial agent.

The polyvalent bacteriophage φYSZKP that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa has a deposit number of CCTCCNO:M 2018514 and was deposited in China Center for Type Culture Collection on August 1, 2018, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Wuhan University, China Center for Type Culture Collection.

Bacillus cereus phage φYSZBA1, with the deposit number: CCTCC NO:M 2018517, was deposited on August 1, 2018 in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Wuhan University, China Center for Type Culture Collection.

Bacillus cereus phage φYSZBA2, with the deposit number: CCTCC NO:M 2018518, was deposited on August 1, 2018 in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Wuhan University, China Center for Type Culture Collection.

DETAILED DESCRIPTION

The following specific implementation methods do not limit the technical solutions of the present invention in any form, and all technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

The resistance gene GT2 refers to the carbon fixation functional genes (anabolism genes) carried by the bacteriophage detected in the soil and the carbon fixation genes carried by the in situ host bacteria.

Table 1 Determination of optimal multiplicity of infection (OMOI)

Note: A: titer of phage φYSZKP; B: titer of phage φYSZBA1; C: titer of phage φYSZBA2.

Embodiment 1:

The vegetables planted were Capsicum frutescens var . Hongpin No. ^{1, from Qianshubaihua Seeds. The basic physical and chemical properties of the test soil were as follows: sand 26.8%, soil 37.4%, clay 31.82%, pH 7.24, total nitrogen 1.44 g·kg -1} , water-soluble nitrogen 1.62 g·kg ^-1 , total phosphorus 1.50 g·kg ^-1 , total potassium 18.52 g·kg ^-1 , and CEC 16.43 cmol·kg ^-1 .

Weigh 0.5g of soil and place it in a 15mL sterile centrifuge tube. Add 4.5mL of liquid culture medium to the sample, mix thoroughly and incubate for 1h before centrifugation. The supernatant obtained by centrifugation is filtered using a tangential flow filtration system. The filtrate after tangential flow filtration is a phage suspension, so that free phages are suspended in the liquid component and frequently turned over during incubation. Isolate the filtered phage: add 100µL of carbon-fixing host bacteria (Klebsiella) in the logarithmic phase + 200µL phage suspension + 5mL semi-solid culture medium, pour into solid LB medium, invert and culture at 30-37℃ after solidification. Continue phage purification: aspirate 100μL of dilution and 100μL of bacteria on a double-layer plate for continuous purification 3-5 times until the plaque size on the plate is consistent. After culture, the plaques on the culture dish are transparent in the middle, without halos around them, and about 2 to 3 mm in diameter, and the phage φYSZKP is obtained. Pick a single clear and transparent plaque for enrichment, mix it with 50% glycerol in a 1:1 ratio, and store it at -80°C for later use.

Based on the phage φYSZKP obtained with Klebsiella as the host bacteria, the process of accelerating the expression of phage with a wide host spectrum was carried out: 600 μL of the preserved phage stock solution was taken, mixed with 200 μL of Klebsiella and 200 mL of Bacillus cereus bacterial suspension, and added into 99 mL of LB liquid culture medium, and then calcium chloride solid was added to adjust the final concentration to 1 mmol·L ^-1 , and cultured at 37°C, 150 rpm and shaking for 96 h, sampling was taken every 8 h, and the phage obtained by centrifugation filtration was verified by pouring double-layer plates with Bacillus cereus, and the plaques were observed. If plaques appeared, it proved that the directed evolution was successful, and phages φYSZBA1 and φYSZBA2 were obtained. Single clear and transparent plaques were picked for enrichment, mixed with 50% glycerol in a 1:1 ratio, and stored at -80°C for later use.

Based on the above operation, two exclusive phages were obtained, namely: the multivalent phage φYSZKP that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa, and the Bacillus cereus phages φYSZBA1 and φYSZBA2.

Five treatment groups were set up in the experiment: ① Control group (CK): 3 chili peppers were planted in each pot (0.5-1 cm of soil was covered on the seeds, and the room temperature was 25±2℃); ② Single phage treatment (V1): 100 mL of φYSZKP phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ③ Single phage treatment (V2): 100 mL of φYSZBA1 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ④ Single phage treatment (V3): 100 mL of φYSZBA2 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ⑤ Mixed phage treatment (V): 100 mL of the mixed suspension of the above three phages with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group. On the 70th day of the growth of chili pepper, the soil was sampled on site, and the abundance of chitin synthesis gene GT2 in the soil under the five treatments of CK, V1, V2, V3, and V was determined to be 4.08×10 ⁵ copies·g ^-1 , 7.24×10 ⁷ copies·g ^-1 , 3.38×10 ⁸ copies·g ^-1 , 2.01×10 ⁸ copies·g ^-1 , and 8.19×10 ⁸ copies·g ^-1 (Figure 6). The abundance of carbon fixation resistance gene GT2 in the soil of the V treatment group increased by 3 orders of magnitude compared with the control group ( p <0.05). The average chitin content in the soil of the treatment group and the control group was determined to be 228.32mg·kg ^-1 , 429.66mg·kg ^-1 , 310.75mg·kg ^-1 , 337.00mg·kg ^-1 , and 443.67mg·kg ^-1 (Figure 7). After the mixed phage transplantation, the soil organic matter content increased by 5‰ g/kg to 31‰ g/kg after 70 days of cultivation compared with the treatment without phage transplantation, indicating that the addition of phage suspension has a significant effect on enriching microbial carbon synthesis metabolism genes and promoting soil carbon fixation.

Embodiment 2:

The basic physical and chemical properties of the tested soil were as follows: pH 6.8, organic matter 21.1 g·kg ^-1 , total nitrogen 4.8 g·kg ^-1 , total phosphorus 2.1 g·kg ^-1 , and the mechanical composition of the soil was 45.1% sand (sandy soil), 22.8% soil particles, and 32.1% clay particles.

Five treatment groups were set up in the experiment: ① Control group (CK): 3 carrots were planted in each pot (0.5-1 cm of soil was covered on the seeds, and the room temperature was 25±2℃); ② Single phage treatment (V1): 100 mL of φYSZKP phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ③ Single phage treatment (V2): 100 mL of φYSZBA1 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ④ Single phage treatment (V3): 100 mL of φYSZBA2 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ⑤ Mixed phage treatment (V): 100 mL of the mixed suspension of the above three phages with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group. On the 70th day of carrot growth, soil samples were collected on site, and the abundance of chitin synthesis gene GT2 in the soil under the five treatments of CK, V1, V2, V3, and V was 5.27×10 ⁵ copies·g ^-1 , 5.23×10 ⁷ copies·g ^-1 , 2.28×10 ⁸ copies·g ^-1 , 1.25×10 ⁸ copies·g ^-1 , and 7.68×10 ⁸ copies·g ^-1 (Figure 8). The abundance of carbon fixation resistance gene GT2 in the soil of the V treatment group increased by 3 orders of magnitude compared with that of the control group ( p <0.05). The average chitin content in the soil of the treatment group and the control group was 564.75mg·kg ^-1 , 647.99mg·kg ^-1 , 628.37mg·kg ^-1 , 564.73mg·kg ^-1 , and 695.12mg·kg ^-1 (Figure 9). After the mixed phage transplantation, the soil organic matter content increased by 10‰ g/kg to 22‰ g/kg after 70 days of cultivation compared with the treatment without phage transplantation.

After inoculation with bacteriophages, the soil microbial carbon fixation process is significantly promoted to a certain extent, and it also helps to maintain and improve the diversity and stability of soil microbial ecological functions after restoration.

Embodiment 3:

The basic physical and chemical properties of the tested soil are as follows: pH 5.5, organic matter 22.4 g·kg ^-1 , total nitrogen 2.4 g·kg ^-1 , total phosphorus 0.6 g·kg ^-1 , total potassium 11.1 g·kg ^-1 , available potassium 72.7 g·kg ^-1 ; the mechanical composition of the soil is 52.3% sand (sandy soil), 20.6% soil particles, and 27.1% clay particles. The average concentration of carbon-related metabolites in the 0-1 m soil layer is 428.2 mg·kg ^-1 .

Five treatment groups were set up in the experiment: ① Control group (CK): 3 lettuces were planted in each pot (0.5-1 cm of soil was covered on the seeds, and the room temperature was 25±2℃); ② Single phage treatment (V1): 100 mL of φYSZKP phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ③ Single phage treatment (V2): 100 mL of φYSZBA1 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ④ Single phage treatment (V3): 100 mL of φYSZBA2 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ⑤ Mixed phage treatment (V): 100 mL of the mixed suspension of the above three phages with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group. On the 60th day of lettuce growth, soil samples were collected on site, and the abundance of chitin synthesis gene GT2 in the soil under the five treatments of CK, V1, V2, V3, and V was 2.15×10 ⁴ copies·g ^-1 , 9.36×10 ⁵ copies·g ^-1 , 8.50×10 ⁵ copies·g ^-1 , 1.15×10 ⁶ copies·g ^-1 , and 2.22×10 ⁶ copies·g ^-1 (Figure 10). The average chitin content in the soil of the treatment group and the control group was 424.92mg·kg ^-1 , 480.91mg·kg ^-1 , 442.81mg·kg ^-1 , 473.30mg·kg ^-1 , and 521.33mg·kg ^-1 (Figure 11). After 60 days of cultivation after mixed phage transplantation, the soil organic matter content increased by 8‰ g/kg compared with the treatment without phage transplantation, reaching 17‰ g/kg.

Embodiment 4:

The basic physical and chemical properties of the tested soil were as follows: pH 5.8, organic matter 25.1 g·kg ^-1 , total nitrogen 6.8 g·kg ^-1 , total phosphorus 2.7 g·kg ^-1 , and the mechanical composition of the soil was 39.1% sand (sandy soil), 22.8% soil particles, and 38.1% clay particles.

Five treatment groups were set up in the experiment: ① Control group (CK): 3 cabbages were planted in each pot (0.5-1 cm of soil was covered on the seeds, and the room temperature was 25±2℃); ② Single phage treatment (V1): 100 mL of φYSZKP phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ③ Single phage treatment (V2): 100 mL of φYSZBA1 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ④ Single phage treatment (V3): 100 mL of φYSZBA2 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ⑤ Mixed phage treatment (V): 100 mL of the mixed suspension of the above three phages with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group. On the 45th day after the cabbage was grown, the soil was sampled on site, and the abundance of chitin synthesis gene GT2 in the soil under the five treatments of CK, V1, V2, V3, and V was 2.82×10 ⁵ copies·g ^-1 , 8.38×10 ⁷ copies·g ^-1 , 1.96×10 ⁸ copies·g ^-1 , 8.04×10 ⁷ copies·g ^-1 , and 7.61×10 ⁸ copies·g ^-1 (Figure 12). The average chitin content in the soil of the treatment group and the control group was 369.65mg·kg ^-1 , 473.57mg·kg ^-1 , 421.81mg·kg ^-1 , 425.64mg·kg ^-1 , and 511.88mg·kg ^-1 (Figure 13). After 45 days of cultivation after mixed phage transplantation, the soil organic matter content increased by 10‰ g/kg compared with the treatment without phage transplantation, reaching 22‰ g/kg.

Embodiment 5:

The basic physical and chemical properties of the tested soil were as follows: pH 6.8, organic matter 20.1 g·kg ^-1 , total nitrogen 7.8 g·kg ^-1 , total phosphorus 7.6 g·kg ^-1 , and the mechanical composition of the soil was 42.1% sand (sandy soil), 22.8% soil particles, and 35.1% clay particles.

Five treatment groups were set up in the experiment: ① Control group (CK): 3 lettuces were planted in each pot (0.5-1 cm of soil was covered on the seeds, and the room temperature was 25±2℃); ② Single phage treatment (V1): 100 mL of φYSZKP phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ③ Single phage treatment (V2): 100 mL of φYSZBA1 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ④ Single phage treatment (V3): 100 mL of φYSZBA2 phage suspension with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group; ⑤ Mixed phage treatment (V): 100 mL of the mixed suspension of the above three phages with a concentration of 10 ⁶ pfu·mL ^-1 was inoculated on the basis of the control group. On the 70th day of lettuce growth, soil sampling was carried out on site, and the abundance of chitin synthesis gene GT2 in the soil under the five treatments of CK, V1, V2, V3, and V was determined as 2.30×10 ⁵ copies·g ^-1 , 1.96×10 ⁶ copies·g ^-1 , 2.87×10 ⁶ copies·g ^-1 , 2.03×10 ⁶ copies·g ^-1 , and 6.21×10 ⁶ copies·g ^-1 , respectively (Figure 14). The average chitin content in the soil of the treatment group and the control group were 212.26mg·kg ^-1 , 308.67mg·kg ^-1 , 310.75mg·kg ^-1 , 312.27mg·kg ^-1 , and 432.78mg·kg ^-1 ( Figure 15 ). After phage transplantation, the soil organic matter content increased by 9‰ g/kg to 16‰ g/kg after 50 days of cultivation, compared with the treatment without phage transplantation.

This shows that the application technology of bacteriophage transplantation to promote soil carbon sequestration has the advantages of high broad spectrum, low ecological risk and environmental friendliness. It is a soil carbon storage optimization technology with good application prospects.

Claims (8)

1. A phage composition, characterized in that it contains three phages, all of which were deposited in the China Center for Type Culture Collection on August 1, 2018, and are respectively a multivalent phage φYSZKP attacking Klebsiella pneumoniae and Pseudomonas aeruginosa, with a deposit number of CCTCC NO: M 2018514, and a classification name of Klebsiella and Pseudomonas aeruginosa phage φYSZKP; a Bacillus cereus phage φYSZBA1, with a deposit number of CCTCC NO: M2018517, and a classification name of Bacillus cereus phage φYSZBA1; a Bacillus cereus phage φYSZBA2, with a deposit number of CCTCC NO: M 2018518, and a classification name of Bacillus cereus phage φYSZBA2. 2. The phage composition according to claim 1, characterized in that the concentration ratio of the three phages in the composition is 1:1:1. 3. The method for preparing the phage composition according to claim 1 or 2, characterized in that it comprises mixing three phages so that their concentrations in the composition are 1:1:1. 4. Use of the bacteriophage composition according to claim 1 or 2 in the preparation of a product for enhancing soil carbon fixation. 5. The use according to claim 4, characterized in that the soil is soil for planting cabbage, lettuce, carrot or chili pepper. 6. A soil conditioner for enhancing soil carbon fixation, characterized in that it contains the bacteriophage composition of claim 1 or 2, and a biologically active ingredient that helps soil carbon fixation. 7. The soil conditioner according to claim 6, characterized in that the biologically active ingredients include microbial agents, humic acid, amino acids or biochar. 8. Use of a soil conditioner for enhancing soil carbon fixation, characterized in that the soil conditioner according to claim 6 is injected into target soil.