Method for in-situ ecological simulation of double-layer agar culture

Abstract

The invention discloses a layered gel culture technology constructed by using environmental extracts, and belongs to the field of environmental microorganism culture. According to the technology, an environmental sample is separated into a particle phase and a liquid phase through water extraction, the particle phase and the liquid phase are respectively prepared into a lower-layer solid-phase matrix and an upper-layer gel phase with gel (preferably agar), a vertical nutrition / signal slow-release gradient is formed in a culture dish, and particle attachment sites are provided; and inoculating the environmental microorganisms on the upper layer or the surface for culture. Compared with nutrient agar or extremely simple water agar, the method has the advantages that more bacterial colonies and higher distinguishable form number can be obtained under the repeatable condition, and the community diversity and the OTU / ASV overlapping degree with an original sample are improved; rare / slow-growing groups can be stably separated, and subsequent 16S / metagenome analysis and agar block bacteriostasis primary screening are facilitated. Meanwhile, the invention provides a corresponding culture medium composition and a kit / prefabricated layered culture dish. According to the scheme, operation is easy and convenient, parameters can be standardized, and the method is suitable for isolated culture and ecological function research of environmental samples such as soil, sediments and low-solid water samples.

Description

Technical fields:

[0001] This invention belongs to the field of environmental microbiology and microbial culture technology, specifically relating to a method for constructing a double-layer agar (gel) culture using environmental extracts, its culture medium composition, and its uses, for improving the culturability of environmentally derived microorganisms. Background technology:

[0002] Microbiology has long suffered from the "Great Plate Count Anomaly": of the vast number of environmental microorganisms visible under a microscope, only a very small fraction can form colonies under standard laboratory culture conditions; the culturable proportion is generally considered to be no more than 1%. This phenomenon has been systematically reviewed and attributed to multiple limiting factors, including the lack of nutrients and signaling factors, dependence on symbiotic / mutually beneficial metabolites, difficulty in relieving stress or dormancy, and the lack of suitable physical attachment and spatial structure in the experimental system.

[0003] To overcome these bottlenecks, researchers have proposed various in-situ or near-in-situ culture strategies, such as diffusion chambers / membrane separation devices and iChip technology. These technologies connect the inoculum to the real environment via a semi-permeable medium, allowing small molecule nutrients and signals to diffuse across the membrane, thereby promoting the growth of recalcitrant microorganisms under conditions close to the natural microenvironment. These methods have shown potential in antibiotic discovery and the isolation of new species (e.g., the discovery of teixobactin). However, such devices typically involve specialized materials, processing, and field / site operations, resulting in high costs and complex procedures. Furthermore, there are practical obstacles to standardization and large-scale application across different laboratories.

[0004] On the other hand, environmental extract / soil extract culture media attempt to reproduce the chemical composition of the original environment in vitro to improve culturability and community representativeness. For example, the "novel soil extract culture medium" (NSE / ISEM) prepared by the new methanol method can significantly improve the isolation efficiency of previously uncultured bacteria and expand the range of phyla that can be isolated, demonstrating that "introducing soluble factors from the environment" plays an important role in the revival of rare and slow-growing taxa.

[0005] In summary, existing technologies either rely on dedicated in-situ devices, which limit their widespread adoption and standardization; or they only introduce environmentally soluble components into a monolayer gel system, making it difficult to simultaneously reconstruct the vertical nutrient / signal release gradient. Therefore, there is an urgent need for a culture method that can be implemented at the scale of conventional petri dishes, with simplified equipment and easy standardization, to balance environmental chemical fidelity and spatial structure simulation, thereby further improving the culturability of environmental microorganisms. These technical requirements are precisely the problems that this invention aims to solve. Summary of the Invention:

[0006] I. Technical problems to be solved

[0007] This invention aims to provide a culture method and culture medium composition that can reconstruct the environmental chemical composition and spatial structure characteristics at the scale of ordinary petri dishes, overcoming the technical defects of existing technologies that rely on dedicated in-situ devices, have complex processes that are difficult to standardize, and lack vertical nutrient / signal slow-release gradients in monolayer gel systems, resulting in insufficient recovery of rare / slow-growing groups.

[0008] II. Technical Solution

[0009] This invention provides a bilayer gel culture method for environmental extracts, corresponding culture medium compositions, and applications / kits. The core principle is to construct a microenvironment for slow-release diffusion within a culture dish, using a homologous precipitate solid phase as the lower layer and a homologous supernatant gel as the upper layer, thereby improving culturability and community representativeness.

[0010] (I) Method

[0011] 1. Environmental extraction and separation: Mix environmental samples (such as soil and sediment) with sterile water at a ratio of 1:3–1:20 (preferably 1:5–1:10), shake at 25–30℃ for 30–120 min (preferably 60 min); separate the environmental supernatant and environmental precipitate by standing or centrifugation (1000–4000×g, 5–20 min).

[0012] 2. Supernatant sterilization and sedimentation sterilization: The supernatant is sterilized by a 0.22μm microporous membrane; the sediment is sterilized by autoclaving at 121℃ for 20–40 min, or by gamma irradiation (10–25kGy) or intermittent sterilization in a heat-sensitive environment.

[0013] 3. Construction of bilayer culture medium: Agar is selected as the gelling agent (gellan gum, κ-carrageenan, low-melting-point agarose, etc. can also be used as substitutes); Lower layer: Sterile precipitate is mixed with gelling agent (gel mass fraction 0.8–2.0%, preferably 1.2–1.6%) and poured into sterile petri dishes to form a solid matrix with a thickness of 3–8 mm; Upper layer: Filtered supernatant is mixed with gelling agent (same concentration as above) and covered on the lower layer to form a gel layer with a thickness of 2–5 mm; The thickness ratio of the upper and lower layers is preferably 1.2–3:1; The pH of the system is 6.0–8.0 (preferably 6.5–7.5) or the pH value of the in-situ environment.

[0014] 4. Inoculation and Culture: Target inoculation load 10 2 -10 4 CFU / plate; temperature 20–30℃ (preferably 25–30℃); select aerobic / anaerobic conditions according to the sample source; culture period 3–30 days.

[0015] 5. Evaluation and Downstream: Performance is evaluated using indicators such as total colony count, number of distinguishable morphologies, community diversity index (Shannon / Simpson), and overlap ratio with the original sample OTU / ASV. Examples can be validated by combining 16S rRNA amplicon / metagenomics and can be integrated with agar block antimicrobial methods, fermentation amplification, and physicochemical analysis processes.

[0016] (II) Culture medium composition

[0017] 1. Lower solid matrix: composed of environmental precipitates and gelling agents, providing a nutrient / signal sustained-release reservoir;

[0018] 2. Upper gel phase: Composed of environmental supernatant filtered through 0.22 μm and gelling agent, providing low-intensity soluble nutrients and chemical signals;

[0019] (III) Uses

[0020] It is used to improve the culturability of environmental microorganisms, isolate rare / slow-growing groups, improve the fidelity of OTU / ASV with the original community, and serve as a preprocessing platform for subsequent functional screening and omics analysis.

[0021] III. Beneficial Effects

[0022] Compared with nutrient agar or minimal water agar controls, the method of the present invention has the following advantages:

[0023] 1. Structural advantages: The introduction of a vertical nutrient / signal slow-release gradient within the culture dish more closely resembles the natural microenvironment-driven mechanism;

[0024] 2. Performance advantages: Increased total number of colonies and number of morphologies, higher diversity index, and greater overlap with the original sample's OTU / ASV (see examples for details);

[0025] 3. Universality: Applicable to soil, sediment, and low-solids water samples; it can be used for general upstream culture and is also convenient for downstream functional screening.

[0026] 4. Industrial applicability: The raw materials are readily available, the process is simple, and the parameter range is wide, making it easy to prepare kits and apply them in a standardized manner in teaching and scientific research.

[0027] IV. Terminology Definition

[0028] 1. Environmental supernatant components: The clarified liquid obtained after water extraction and standing / centrifugation of environmental samples contains soluble nutrients and signaling molecules, and is sterilized by 0.22μm.

[0029] 2. Environmental sediment components: The solid phase or particulate concentrates obtained from the above separation contain mineral / organic particles and adsorbed environmental molecules.

[0030] 3. Bilayer gel culture medium: A culture medium system consisting of a lower solid phase containing environmental precipitate and an upper gel layer containing environmental supernatant.

[0031] 4. Culturability / Fidelity: These refer to the number of colonies / morphological types obtained per unit time and per unit area, respectively, and the degree of overlap between the cultured community and the original community at the OTU / ASV level.

[0032] V. Optional Implementation Methods

[0033] 1. Three-layer variant: lower layer precipitate matrix / middle thin layer soft gel or inert membrane / upper layer supernatant gel, to enhance interfacial stability;

[0034] 2. Low-solids water sample preparation: No sedimentation is required. Simply add an appropriate concentration of gelling agent such as agar, sterilize, and then prepare the culture medium.

[0035] 3. Selective fine-tuning: Adding low doses of environmentally compatible trace elements / vitamins (×0.01–×0.1) to the upper gel to target the recovery of specific groups without introducing eutrophic effects;

[0036] 4. Alternative gelling agents: gellan gum other than agar, κ-carrageenan, low-melting-point agarose, etc. are alternatives;

[0037] 5. Downstream integration: Platform-based integration of agar block antibacterial screening, fermentation amplification, and omics analysis processes. Attached Figure Description

[0038] Figure 1 The preparation process of the environmental extract double-layer agar medium of the present invention

[0039] Figure 2 This study compares the culturing effects of the present invention with those of a traditional control culture medium. A: Experimental group, double-layer sludge culture medium (lower layer: sediment with slow-release nutrients, upper layer: environmental filtrate agar); B: Nutrient-rich control (NA + 1.5% agar); C: Low-nutrient minimally invasive culture medium control (ddH2O + 1.5% agar). Group A showed numerous and morphologically diverse small colonies, indicating a good recovery ability for rare / slow-growing groups; Group B was dominated by a few rapidly growing large colonies, which were prone to merging, indicating a selective effect under nutrient-rich conditions; Group C showed sparse, small, and transparent colonies, suggesting slow growth under low-nutrient conditions. Negative controls were included in each group, and no colonies were observed, verifying the absence of exogenous contamination. In summary, double-layer culture is more conducive to obtaining a high quantity and variety of culturable colonies within the same timeframe. Figure 3Comparison of diversity in the original lake bottom sediment community with three culture conditions. Lake bottom sediment (Mud) was collected and culturable communities were obtained under three culture conditions: environmental extract bilayer medium (DL_Agar), minimal water agar (H2O_Agar), and nutrient agar (NA_Agar). A–B (Chao, Sobs) showed that lake bottom sediment had the highest species richness; among the culturable communities, DL_Agar > H2O_Agar > NA_Agar. C (Simpson value, lower values ​​indicate higher diversity) and D (Shannon value, higher values ​​indicate higher diversity) results were consistent, with DL_Agar showing the highest diversity, followed by H2O_Agar, and NA_Agar showing the lowest. Kruskal–Wallis H test indicated that most pairwise comparisons were statistically significant (**P<0.01; ***P<0.001). The results indicate that the bilayer culture method is better than minimal water agar and nutrient agar in maintaining / restoring the richness and diversity of environmentally derived communities, and is a superior strategy for enhancing culturability. Figure 4 .16S rRNA sequencing analysis revealed community differences and fidelity under different culture strategies. A (Venn diagram): The original silt (Mud) contained the most and a large number of unique OTUs; among the three culturable communities, the double-layer medium (DL_Agar) had the largest overlap with the silt, indicating its higher recovery and expansion of the environmental lineage; H2O_Agar and NA_Agar had less overlap with the silt. Data showed that DL_Agar could culture 13.2% of the bacteria in the silt, while NA_Agar could only culture 3.5% of the bacteria in the silt, increasing the culture efficiency by approximately 3 times. B (Genus-level heatmap): Clustering by genus abundance, Mud samples clustered into one group, clearly separated from the culture group; NA_Agar showed high abundance dominance of a few fast-growing genera (such as Bacillus, Enterobacter, Stenotrophomonas, etc.), while H2O_Agar had a narrower overall abundance and phylogenetic width; DL_Agar showed a more balanced distribution across multiple phyla (such as Actinobacteria, Proteobacteria, etc.), retaining more environment-related phylogenetics. Overall, the two-layer culture method better balances diversity recovery and environmental fidelity preservation than both H2O_Agar and NA_Agar. Figure 5The antibacterial activity of representative functional strains against indicator pathogens was assessed. The in vitro activity of isolated strains was evaluated using the agar block diffusion method. Each circular agar block represented the product source of a candidate strain, placed on the surface of an evenly spread plate of indicator bacteria. After incubation, activity was determined by the clear inhibition zone, and its diameter was recorded. A: Representative strain showing a clear inhibition zone against *Escherichia coli* (ATCC 25922); B: Representative strain showing strong inhibition against *Staphylococcus aureus* (ATCC 6538); C: Representative strain producing a significant inhibition zone against *Candida albicans* (ATCC10231); D: Representative strain showing inhibitory activity against *Pseudomonas aeruginosa*. The results showed that multiple strains exhibited activity against Gram-negative, Gram-positive, and fungal pathogens, suggesting the existence of broad-spectrum or multi-target candidate products, providing priority targets for subsequent fermentation scale-up and chemical tracing. Detailed implementation method:

[0040] The present invention will be further described below with reference to the embodiments. These embodiments are only for illustrating the present invention and not for limiting its scope; any modifications or equivalent substitutions made under the guidance of the present invention should fall within the protection scope of the present invention.

[0041] Example 1: Comparison of the effects of the double-layer culture method and the traditional culture method

[0042] 1. Environmental Sample Preparation: Select representative soil samples as the source of environmental materials and the source of microorganisms to be cultured. For example, collect 1 kg of fresh soil from the top 5–10 cm of forest land, removing stones and large organic debris. After mixing the sample, divide it into two equal parts: one part is used to prepare the culture medium (to provide precipitate and supernatant), and the other part is reserved for subsequent inoculation and control analysis.

[0043] 2. Extraction of environmental precipitate and supernatant: Take 500g of soil sample for culture medium preparation, add sterile distilled water at a ratio of soil sample to sterile distilled water of 1:9, and shake on a shaker at 25℃ and 220rpm for 1 hour to dissolve soluble substances. After the suspension has stood for 2 hours, take the clear upper layer of "environmental supernatant" into a new conical flask; leave the precipitate in the conical flask for later use.

[0044] 3. Preparation of Bilayer Agar Culture Medium: Prepare an appropriate amount of agar solution as a solidifying agent. The lower layer of the medium is prepared by mixing environmental precipitate with agar (agar concentration approximately 1.5%), and the upper layer is prepared by mixing environmental supernatant with agar (agar concentration approximately 1.5%). During preparation, first dissolve the agar and heat until completely melted. Then, add the environmental precipitate and supernatant separately, mix well, and autoclave at 121℃ for 30 minutes. After sterilization, while still hot, pour the lower layer of medium into sterile petri dishes to form a layer approximately 5 mm thick, and allow it to solidify naturally. After the lower layer has solidified, slowly pour in the upper layer of medium (approximately 3–4 mm thick), avoiding washing the surface of the lower layer, and allow it to solidify. The resulting bilayer culture medium has a distinct interface: the lower layer is opaque and earthy in color, and the upper layer is a transparent light brown gel.

[0045] 4. Sample inoculation: Take the reserved supernatant as a bacterial suspension and serially dilute it to 10⁻⁶. -2 -10 -4 The inoculated agar was evenly spread on the upper surface of the double-layer culture medium. Nutrient-rich control (nutrient agar NA), minimal control (water agar), and negative control (no inoculation) plates were prepared in parallel. After inoculation, the plates were inverted and incubated at 30°C.

[0046] 5. Cultivation and Observation: Cultivate for approximately 2 weeks, recording the time of colony appearance, the final total colony count, and the number of colony morphological types. Visible colonies typically appear on the second day of the double-layer medium, and the count stabilizes by the 14th day. In contrast, NA medium produces a large number of colonies within 24–48 hours, but fast-growing bacteria often dominate the entire plate, while minimally invasive medium results in later colony appearance and a smaller number of colonies.

[0047] 6. Microbial Community Analysis: After cultivation, representative colonies were randomly selected from each plate for purification and identification. All colonies were scraped, mixed, and genomic DNA was extracted for metagenomic or 16S rRNA amplicon sequencing. DNA was extracted directly from the original soil sample before cultivation as a control. Bioinformatics analysis was used to compare community diversity and composition under different cultivation conditions.

[0048] 16S rRNA sequencing results and analysis:

[0049] To verify the enrichment effect of the "double-layer culture method" on rare / slow-growing microorganisms, we performed 16S rRNA amplicon sequencing on four groups of samples: original sludge samples (Mud), double-layer culture plates (DL_Agar), nutrient agar controls (NA_Agar), and water agar controls (H2O_Agar). The sequencing results showed:

[0050] (1) Species richness (Chao and Sobs indices) was highest in the original silt sample; in the culturable community, the richness of the double-layer culture group was significantly higher than that of H2O_Agar and NA_Agar, while the richness of the control group was the lowest. Simpson and Shannon diversity indices showed that the double-layer culture group had both high diversity and good evenness, close to that of the original sample.

[0051] (2) Venn diagram analysis showed that the overlap of OTUs between the double-layer culture group and the original sludge was the largest, and about 13.2% of the bacterial groups in the original sample could be cultured, while some of the double-layer culture-specific bacterial groups were also retained; the overlap of H2O_Agar and NA_Agar with the original sample was relatively low.

[0052] (3) Community composition analysis of each culture group revealed that the double-layer culture group restored the major phyla such as Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Acidobacteria, and detected some groups with low abundance in the original sample, such as Nitrospira and Cellulolytic Bacteria, while NA_Agar was mainly occupied by a few fast-growing Pseudomonas and Bacillus.

[0053] The following image is a visualization of the 16S rRNA sequencing results:

[0054] Figure 3 This study compared the diversity of the original lake bottom sediment community with that under three culture conditions. Lake bottom sediment (Mud) was collected and culturable communities were obtained under three culture conditions: environmental extract bilayer medium (DL_Agar), minimal water agar (H2O_Agar), and nutrient agar (NA_Agar). A–B (Chao, Sobs) showed that lake bottom sediment had the highest species richness; among the culturable communities, DL_Agar > H2O_Agar > NA_Agar. C (Simpson value, lower values ​​indicate higher diversity) and D (Shannon value, higher values ​​indicate higher diversity) results were consistent, with DL_Agar showing the highest diversity, followed by H2O_Agar, and NA_Agar showing the lowest. The Kruskal–Wallis H test indicated that most pairwise comparisons were statistically significant (**P<0.01; ***P<0.001). The results indicate that the bilayer culture method is better than minimal water agar and nutrient agar in maintaining / restoring the richness and diversity of environmentally derived communities, and is a superior strategy for enhancing culturability.

[0055] Figure 416S rRNA analysis revealed community differences and fidelity under different culture strategies. A (Venn diagram): The original mud (Mud) contained the most and a large number of unique OTUs. Among the three culturable communities, the double-layer medium (DL_Agar) had the largest overlap with the mud, while still retaining some DL_Agar-specific taxa, showing its higher recovery and expansion of the environmental lineage; H2O_Agar and NA_Agar had less overlap with the mud. Data showed that DL_Agar could culture 13.2% of the bacteria in the mud, while NA_Agar could only culture 3.5% of the bacteria in the mud, increasing the culture efficiency by approximately 3 times. B (Genus-level heatmap): Clustering by genus abundance, Mud samples clustered into one group, clearly separated from the culture group; NA_Agar showed high abundance dominance of a few fast-growing genera (such as Bacillus, Enterobacter, Stenotrophomonas, etc.), while H2O_Agar had a narrower overall abundance and phylogenetic width; DL_Agar showed a more balanced distribution across multiple phyla (such as Actinobacteria, Proteobacteria, etc.), retaining more environment-related phylogenetics. Overall, the two-layer culture method better balances diversity recovery and environmental fidelity preservation than both H2O_Agar and NA_Agar.

[0056] Example 2: Cultivating microorganisms with antibacterial activity using a double-layer culture method

[0057] This embodiment, based on the experimental data from Example 1, compares the effectiveness of the double-layer culture method with that of traditional culture media in culturing soil microorganisms. The double-layer culture medium not only produces a greater number of colonies but also exhibits a richer diversity of bacterial types. 16S rRNA amplicon analysis shows that the community diversity restored by the double-layer culture group is closer to that of the original sample, and it is enriched with some rare functional bacteria (such as nitrifying bacteria and nitrogen-fixing bacteria).

[0058] Single colony isolation and agar block antibacterial method experiment:

[0059] In the aforementioned two-layer culture system, we randomly selected single colonies from the plates and isolated and purified them one by one, obtaining more than 2000 representative strains. Each strain was amplified and cultured to prepare mature bacterial colonies. Agar blocks with a diameter of 8 mm were cut as the product source, and the agar block diffusion method was used for initial screening of four indicator bacteria: *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 6538, *Candida albicans* ATCC 10231, and *Pseudomonas aeruginosa* ATCC 9027. The experimental steps included:

[0060] 1. Producer culture: Each strain was cultured on NA solid medium until a bacterial colony formed; holes were punched in the bacterial colony using a sterile punch to obtain agar blocks with a diameter of 8 mm, which were used as test samples.

[0061] 2. Confrontation and evaluation: After the indicator bacteria are evenly spread on the plate or covered with soft agar, place the agar block containing the product on its surface; set up a positive control (known antibiotic or product strain) and a negative control (blank agar block), and perform three replicates for each combination; observe the clear inhibition zone and measure its diameter after 24–48 h of incubation.

[0062] The experimental results showed that approximately 12.3% of the strains produced a clear inhibition zone against at least one indicator bacterium, with the inhibition zone diameter ranging from 9 to 20 mm. Among these, 6 strains showed inhibition zones larger than 15 mm against *E. coli*, 8 strains showed strong inhibition against *S. aureus*, 5 strains showed significant inhibition against *C. albicans*, and 1 strain produced a visible inhibition zone against *P. aeruginosa*. Most positive strains were active against two or more indicator bacteria, suggesting that their products may have broad-spectrum antimicrobial activity. No inhibition zone was observed in the negative control, indicating that the experimental procedure was uncontaminated. The following figure illustrates the antimicrobial results of the agar diffusion method:

[0063] Figure 5 The antibacterial activity of representative functional strains against indicator pathogens was assessed using the agar block diffusion method. Each circular agar block represented the product source of a candidate bacterium, placed on the surface of an evenly spread plate of indicator bacteria, and the activity was determined by the clear inhibition zone after incubation, with the diameter recorded. A: Representative strain showing a clear inhibition zone against *Escherichia coli* ATCC 25922; B: Representative strain showing strong inhibition against *Staphylococcus aureus* ATCC 6538; C: Representative strain producing a significant inhibition zone against *Candida albicans* ATCC 10231; D: Representative strain showing inhibitory activity against *Pseudomonas aeruginosa*. The results showed that multiple strains exhibited activity against Gram-negative, Gram-positive, and fungal pathogens, suggesting the existence of broad-spectrum or multi-target candidate products, providing priority targets for subsequent fermentation scale-up and chemical tracing.

[0064] By using single-colony isolation and agar block inhibition, this invention has identified a group of functional bacterial strains with antibacterial potential from double-layer culture plates. The products of these strains will be further investigated for their active ingredients using OSMAC liquid fermentation, crude extraction, and physicochemical analysis.

Claims

1. A method for culturing environmentally derived microorganisms, characterized in that, The process includes the following steps: (1) contacting an environmental sample with a sterile extract to obtain an environmental extract comprising a particulate phase and a liquid phase, wherein the environmental sample is selected from soil, sediment, silt, river water, lake water, seawater, groundwater, biofilm, plant rhizosphere samples, or a combination thereof, and the mass or volume ratio of the environmental sample to the sterile extract is 1:3 to 1:20; (2) mixing the particulate phase with a gelling agent solution and sterilizing or removing bacteria to form a lower solid phase matrix; and mixing the liquid phase with the gelling agent solution after sterilization or removal of bacteria. (3) A lower solid phase matrix and an upper gel phase are sequentially laid in the culture container, so that the two are stacked in the vertical direction to establish a slow-release diffusion gradient from bottom to top; (4) The environmental microorganisms to be cultured are inoculated on the surface of the upper gel phase and cultured under suitable culture conditions until colonies are formed; wherein, the gelling agent is selected from at least one of agar, agarose, gellan gum, carrageenan, alginate or equivalents; the sterilization treatment includes filtration sterilization, heat sterilization, irradiation sterilization or a combination thereof.

2. A stratified culture medium composition for culturing environmentally derived microorganisms, characterized in that, The composition comprises: a) a lower solid phase matrix: a solid or semi-solid matrix formed by a particulate phase containing environmental extracts and a gelling agent; b) an upper gel phase: a gel layer formed by a liquid phase containing environmental extracts and a gelling agent; wherein the lower solid phase matrix and the upper gel phase are stacked vertically in use, thereby allowing nutrients and signaling molecules in the lower layer to diffuse slowly upwards.

3. The use of the method or composition as claimed in claim 1 or 2, characterized in that, Used to improve the culturability and community representativeness of environmentally sourced microorganisms, and / or to isolate rare or slow-growing groups, and / or for subsequent functional screening, including agar block or agar plug diffusion inhibition screening.

4. The method of claim 1, wherein the environmental sample is selected from soil, sediment, silt, river water, lake water, seawater, groundwater, biofilm, plant rhizosphere sample or a combination thereof, and the mass or volume ratio of the environmental sample to the sterile extract is 1:3 to 1:20 (preferably 1:5 to 1:10).

5. The method or composition of claim 1 or 2, wherein the mass fraction of the gelling agent is 0.8% to... 2.0% (w / v), preferably 1.2% to 1.6% (w / v).

6. The method or composition of claim 1 or 2, wherein the thicknesses of the lower solid matrix and the upper gel phase are 3-8 mm and 2-5 mm, respectively, and the ratio of the lower layer thickness to the upper layer thickness is 1.2-3:

1.

7. The method or composition of claim 1 or 2, wherein the particle size in the lower solid matrix is ​​10 to 300 micrometers and the loading on a dry weight basis is 2 to 200 g / L.

8. The method of claim 1, wherein the temperature of the culture system is 20-30°C, the culture period is 3-30 days (which can be extended to several months for slow-growing samples), and aerobic or anaerobic culture conditions are selected according to the sample source, and the pH of the system is 6.0-8.0 or close to the pH of the in-situ environment.

9. The method of claim 1, wherein after culturing in step (4), the obtained colonies are subjected to agar block diffusion method for initial functional screening to identify production strains with antimicrobial activity and to serve as the starting point for subsequent fermentation amplification and physicochemical analysis.

10. The method of claim 1, wherein a low concentration of nutrients, trace elements or vitamins at an order of 0.01 to 0.1 times may be optionally added to the upper gel phase to selectively revive specific groups without introducing an overnutrition effect.

Images