Complex microbial inoculant for promoting microbial quorum sensing and application of complex microbial inoculant in compost

Abstract

The invention belongs to the field of microbial preparations, and particularly relates to a complex microbial inoculant for promoting microbial quorum sensing and application of the complex microbial inoculant in compost. The composite microbial agent comprises pseudomonas A3 and paenibacillus polymyxa 21-2, and the preservation number of the pseudomonas A3 is CCTCC (China Center for Type Culture Collection) NO: M20251394. The pseudomonas A3 can express key genes such as rhlI and lasI of a gram-negative bacteria AHL quorum sensing system and signal molecules of the key genes, and the paenibacillus polymyxa 21-2 can express luxS genes of a gram-positive bacteria AI-2 signal system and signal molecules DPD of the luxS genes. In the pig manure compost, the complex microbial inoculant is added, so that the expression of AI-2 and AHLs system related genes in the compost and the content of signal molecules DPD are remarkably improved, the microbial population sensing process of the compost is promoted, finally, the compost temperature is increased by about 6 DEG C, the compost carbon-nitrogen ratio is reduced, and the compost maturity is promoted.

Description

Technical Field

[0001] The present invention belongs to the field of microbial preparations, and in particular relates to a composite bacterial agent for promoting microbial quorum sensing and application thereof in composting. Background Art

[0002] Quorum sensing (QS) is a mechanism for information transmission between microorganisms that relies on the release and perception of signaling molecules to regulate a series of group behaviors, such as biofilm formation, free radical release, and horizontal gene transfer. Composting is a process of microbial fermentation. Quorum sensing, as a communication mechanism that enables mutual communication between microorganisms, mediates the regulation of microbial group behavior through the synthesis, perception, and response of autoinducers, and may have a certain impact on the microbial fermentation process of composting. Currently, research on aerobic composting focuses on the macro-control of environmental parameters, and understanding of intra- and interspecies communication between microorganisms is relatively scarce. Therefore, studying the role of quorum sensing in composting is of great significance.

[0003] Quorum Sensing Signal Molecules (QSSMs) mediate quorum sensing, are excreted into the extracellular environment, and accumulate with increasing population density. When their density reaches a response threshold, the signaling molecules bind to receptors, and through this interaction, they regulate gene expression and several physiological activities, including biofilm formation, expression of virulence factors, and synthesis of antimicrobial substances. QSSMs are ubiquitous in microorganisms such as bacteria and fungi, and their diversity is vast. Based on their chemical structure and functional mechanisms, QSSMs primarily include the following representative classes: acyl-homoserine lactones (AHLs), autoinducing peptides (AIPs), autoinducer-2 (AI-2), and other signaling molecules (such as PQS and DSF).

[0004] Although quorum sensing (QS) systems have been widely reported in a variety of microorganisms, it is important to emphasize that not all strains have the ability to express QS signaling molecules. Studies have shown that the expression of QS signaling molecules is strain-specific and not a universal property of microorganisms. Not all strains can activate the QS system. In a study of submarine hydrothermal vents, only 18 strains (about 11%) were detected to have the ability to produce AHL. [1] Not all strains can be used as compound preparations. For example, in lactic acid bacteria, the genes related to the ability to form biofilms and adhere to cells determine the ability of lactic acid bacteria to inhibit Campylobacter jejuni. [2] .

[0005] [1]Yin F, Gao D, Yue L, et al. Diversity of Bacteria with Quorum Sensing and Quenching Activities from Hydrothermal Vents in the Okinawa Trough. Microorganisms. 2023; 11(3):748

[0006] [2] Jin Xing. Study on the mechanism of lactic acid bacteria antagonizing Campylobacter jejuni. Doctoral dissertation of Jiangnan University, 2020 Summary of the Invention

[0007] The present invention aims to provide a composite bacterial agent for promoting microbial quorum sensing and its application in composting. The composite bacterial agent comprises Pseudomonas A3 and Paenibacillus polymyxa 21-2. The preservation number of Pseudomonas A3 is CCTCC NO: M20251394, and the preservation number of Paenibacillus polymyxa 21-2 is CCTCC NO: M 2022346.

[0008] Another object of the present invention is to provide the use of the above-mentioned composite bacterial agent in composting.

[0009] In order to achieve the above object, the present invention adopts the following technical measures:

[0010] After the applicant screened the 17 bacterial strains currently preserved in the laboratory for strains expressing QS genes, they conducted antagonism experiments between the strains and finally screened out two strains, including Pseudomonas A3 and Paenibacillus polymyxa 21-2. These two strains can be used as composite microbial preparations. Among them, the preservation number of Paenibacillus polymyxa 21-2 is CCTCC NO: M2022346, which has been disclosed in CN115125165A; Pseudomonas A3 has been deposited in the China Center for Type Collection on June 16, 2025, with the classification name: Pseudomonas aeruginosa A3, preservation number: CCTCC NO: M20251394, address: Wuhan University, Wuhan, China.

[0011] Pseudomonas aeruginosa A3 is a Gram-negative bacterium with neat colony edges, a smooth surface, and a light green color. Its optimal growth temperature is around 37°C. It typically enters the logarithmic growth phase when cultured in LB liquid medium with shaking at 180 rpm for 10–18 hours.

[0012] The protection scope of the present invention includes:

[0013] A composite bacterial agent for promoting microbial quorum sensing, comprising Pseudomonas A3 and Paenibacillus polymyxa 21-2, wherein the Pseudomonas A3 has a preservation number of CCTCC NO: M20251394, and the Paenibacillus polymyxa 21-2 has a preservation number of CCTCC NO: M 2022346.

[0014] In the composite bacterial agent described above, the effective bacterial concentration ratio of Pseudomonas A3 and Paenibacillus polymyxa 21-2 is 1 to 2:1, and the optimal ratio is 1:1.

[0015] Application of the above composite bacterial agent in promoting composting;

[0016] In the above-mentioned application, the composite bacterial agent promotes compost maturity by promoting microbial quorum sensing.

[0017] Application of the above composite bacterial agent in the preparation of compost microbial preparation.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The composite bacterial agent described in the present invention expresses key genes such as rhlI and lasI of the Gram-negative bacteria AHL quorum sensing system and its signal molecule C4-HSL, while Paenibacillus polymyxa 21-2 expresses the luxS gene of the Gram-positive bacteria AI-2 signaling system and its signal molecule DPD. This broad coverage can simultaneously promote the expression of quorum sensing systems and signal molecules for both Gram-positive and Gram-positive bacteria in microbial communities, enhancing information exchange between microorganisms. Adding the composite bacterial agent to pig manure compost significantly increased the expression of genes related to the AI-2 and AHLs systems and the content of the signal molecule DPD, promoting the quorum sensing process of compost microorganisms. Ultimately, it increased the compost temperature by approximately 6°C, reduced the carbon-nitrogen ratio of the compost, and promoted compost maturity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The growth curves of Pseudomonas A3 and Paenibacillus polymyxa 21-2 at different ratios are shown;

[0021] The total amount of microbial agent added was 1%.

[0022] Figure 2 The growth curve of the composite bacterial agent and the changes in the content of the secreted signal molecules;

[0023] Among them: A: Pseudomonas A3 growth curve and C4-HSL content changes; B: Pseudomonas A3 growth curve and 3OC12-HSL changes; C: Paenibacillus polymyxa 21-2 growth curve and DPD content changes.

[0024] Figure 3The effect of the composite bacterial agent of the present invention on the temperature of aerobic composting of pig manure;

[0025] Among them: CK is the control group; T3 is the composite bacterial agent, the same below.

[0026] Figure 4 The C / N changes at different composting periods;

[0027] Figure 5 To study the changes of DPD content in aerobic composting with the addition of functional bacteria;

[0028] The same letters indicate that the differences did not reach the significant level (p≥0.05), and different letters indicate that the differences reached the significant level (p<0.05), the same below.

[0029] Figure 6 The absolute abundance of AI-2 related genes in aerobic composting with different treatments (heat map).

[0030] Figure 7 The absolute abundance of AHLs-related genes in aerobic composting under different treatments (heat map). DETAILED DESCRIPTION

[0031] The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art; the reagents and materials described, unless otherwise specified, are commercially available. The deposit number of Paenibacillus polymyxa 21-2 involved in the present invention is CCTCCNO: M 2022346, which has been disclosed in CN115125165A.

[0032] Example 1:

[0033] 1) Screening of functional bacteria expressing quorum sensing genes

[0034] Seventeen bacterial strains currently preserved in the laboratory were screened for functional strains expressing QS genes. As shown in Table 1, six strains were screened out that could express QS-related genes, of which five strains could express the gene luxS that directs AI-2 synthesis, namely Bacillus subtilis BS12, Bacillus subtilis BS13, Paenibacillus polymyxa 21-2, Bacillus licheniformis 6-2, and Enterobacter cloacae N7. Only one Gram-negative bacterium, Pseudomonas aeruginosa A3, could express key genes such as rhlI and lasI of the AHL quorum sensing system.

[0035] Table 1 Screening of functional bacteria expressing QS genes

[0036]

[0037] Note: “+” indicates that the QS gene was detected; “-” indicates that the QS gene was not detected.

[0038] 2) Establishment of compound microbial agents

[0039] In order to explore whether there is antagonism between different functional strains, based on the 6 functional bacteria that can express AHL quorum sensing genes and AI-2 quorum sensing genes, Pseudomonas A3, Bacillus subtilis BS12 and BS13 and Paenibacillus polymyxa 21-2 were selected for antagonism test using the punching method. As shown in Table 2, Pseudomonas A3 has an antagonistic effect on both Bacillus subtilis BS12 and BS13 (+), but has no antagonistic effect on Paenibacillus polymyxa 21-2 (-). Bacillus subtilis BS12 and BS13 have mutual antagonism (+). Paenibacillus polymyxa 21-2 has no obvious antagonistic effect with other strains (-). Therefore, Pseudomonas A3 and Paenibacillus polymyxa 21-2 were selected as functional bacteria and added together into the subsequent composting experiment.

[0040] Among them, Pseudomonas A3 was deposited in the China Center for Type Culture Collection on June 16, 2025, with the classification name: Pseudomonas aeruginosa A3, the deposit number: CCTCC NO: M20251394, and the address: Wuhan University, Wuhan, China.

[0041] Table 2 Antagonism between functional strains

[0042]

[0043] Note: “+” indicates the presence of antagonism; “-” indicates the absence of antagonism.

[0044] Example 2:

[0045] Determination of the optimal ratio of compound microbial agents:

[0046] Preparation of seed solution of Pseudomonas A3 and Paenibacillus polymyxa 21-2: Incubate in LB liquid medium at 37°C, 180 rpm for about 12 h until OD 600 When the value is 1 (the final concentration is about 10 8 CFU / mL).

[0047] The two selected strains were co-cultured in LB liquid medium (37°C, 180 rpm) with a total addition of 1% (volume ratio of seed solution to culture medium). The growth density (OD 600 ) Determine the optimal ratio of bacterial agent addition.

[0048] The results are as follows Figure 1As shown in the figure, when the addition ratio of Pseudomonas A3 to Paenibacillus polymyxa 21-2 is 1:1 or 2:1, the growth density is the highest. When the addition ratio of Paenibacillus polymyxa 21-2 is too high, the growth of Pseudomonas A3 is inhibited. Therefore, the addition ratio of 1:1 is selected as the optimal ratio of functional strains and used in the following examples. The effective bacterial concentration of the composite bacterial agent is 10 8 CFU / mL.

[0049] Changes in the content of QSSMs secreted by Pseudomonas aeruginosa A3 and Paenibacillus polymyxa 21-2 during their proliferation

[0050] The two OD 600 The bacterial suspension with a value of about 1.0 was added to LB liquid culture medium at a ratio of 1%, and cultured at 37°C and 180 rpm. The growth density of functional strains and the changes in the content of QSSMs in the supernatant at different time periods were detected by UV spectrophotometry and LC-MS / MS, respectively. The concentration change of AI-2 was reflected by the change in the concentration of DPD, a precursor of AI-2. The results are shown in Figure 2. Figure 2 .in, Figure 2 A in the middle represents the growth curve of Pseudomonas A3 and the change in C4-HSL content. From 3h to 24h, the growth density of Pseudomonas first increases and then tends to be stable, which is a typical microbial growth law. The content of C4-HSL first increases and then decreases with the growth of Pseudomonas A3, reaches a peak in the logarithmic growth period, and then decreases. Figure 2 Figure B shows the growth curve of Pseudomonas and the changes in 3OC12-HSL. The change trend is similar to that of C4-HSL. In the middle and late stages of the logarithmic growth period (9h to 12h), the concentration of 3OC12-HSL increases significantly with the rapid growth of the bacteria, which is synchronized with the rapid increase in the OD value. Figure 2 Figure C shows the growth curve of Paenibacillus polymyxa and the changes in DPD content. Similar to Pseudomonas, Paenibacillus polymyxa is in its logarithmic growth phase between 3 and 9 hours, with low bacterial concentrations and DPD concentrations. DPD increases slightly as the bacteria grow. At the mid-stage of bacterial growth, bacterial proliferation reaches a high level, and DPD content rises significantly, then decreases during the stable growth phase.

[0051] Example 3:

[0052] Application of composite bacterial agents that promote microbial quorum sensing in composting:

[0053] The test materials, pig manure, were obtained from the experimental pig farm of Huazhong Agricultural University, and sawdust was obtained from the veterinary hospital of Huazhong Agricultural University. The mixing ratio of pig manure and sawdust was formulated based on their carbon and nitrogen content. After mixing, the compost had a C / N ratio of 25:1 and a moisture content of 65%. The composite inoculant prepared in Example 2 was dissolved in sterile water and sprayed at the beginning of composting (a total of 2% (w / w) composite inoculant was added). The compost reactor was equipped with a ventilation system for aeration. The entire composting test was conducted at the test base under an external environment of approximately 10°C.

[0054] Here are the results:

[0055] 1) Changes in routine composting indicators

[0056] Temperature changes:

[0057] Depend on Figure 3 As can be seen, the temperature of all treatment groups rose rapidly within 2 to 4 days and entered a high temperature stage, with the highest temperature occurring around day 4 to 8. The maximum temperature in the group with functional microbial agent was even higher, about 6°C higher than that in the control group.

[0058] C / N changes:

[0059] Changes in C / N ratio in aerobic composting under different treatments Figure 4 As shown in the figure, as composting progresses, the microbial decomposition rate of carbon sources such as cellulose and hemicellulose is generally higher than the nitrogen loss rate, resulting in a gradual decrease in the C / N ratio, ultimately stabilizing at around 15% to 20%. The carbon-nitrogen ratio decreased more significantly in the treatment with functional bacterial agents, remaining consistently lower than that in the control group, indicating greater compost maturity.

[0060] 2) Changes in the content of AI-2 signaling molecules during aerobic composting

[0061] In order to explore the changes in AI-2 content in compost, the concentration of DPD, a precursor of AI-2, was detected by LC-MS / MS. Figure 5 As shown in the figure, DPD concentration was highest in the initial stage and decreased continuously as the composting process progressed, reaching its lowest level at the mature stage. The DPD concentration of the exogenously added inoculant was slightly higher than that of the other groups throughout the composting process. During the warming period, the DPD concentration of the inoculant group was significantly higher than that of the control group (p < 0.05), indicating a stronger promotion effect on AI-2 in the compost.

[0062] 3) Changes in absolute abundance of QS genes in compost

[0063] Absolute quantitative detection of AI-2 related genes in aerobic compost with different treatments, such as Figure 6As shown in the figure, during the composting process, the gene expression levels of luxS, lsrG, lsrF, and lsrR were relatively high in both the control group and the additive treatment groups. All genes showed a downward trend from the initial stage of composting to the mature stage. From the results of the heat map and bar chart, it can be seen that the total amount of AI-2 related genes in the control group did not change much at all in each period. The expression level was the lowest at the mature stage, about 10 4 copies / g~10 7 The gene expression levels of the treated groups with added microbial agents during the high temperature and mature stages were higher than those in the other treatment groups, indicating that exogenous microbial agents may have promoted the quorum sensing process of microorganisms, thereby maintaining AI-2-related genes at a higher level.

[0064] 4) Changes in the expression of AHLs-related genes in aerobic composting under different treatments

[0065] like Figure 7 As shown, throughout the composting process, AHL-related genes showed an increasing and then decreasing trend, with lowest gene expression levels during the mature stage. This may be due to the stabilization of organic matter decomposition, the decrease in temperature, and the weakening of some microbial activity. Compared with the control group, the expression levels of the three AHL-related genes in the inoculum group were significantly increased, suggesting that exogenous inoculum may promote microbial activity by stimulating endogenous QS pathways, thereby promoting AHL quorum sensing.

Claims

1. A composite bacterial agent for promoting microbial quorum sensing, comprising Pseudomonas ( Pseudomonas aeruginosa )A3 and Paenibacillus polymyxa ( Paenibacillus polymyxa ) 21-2, the preservation number of the Pseudomonas A3 is CCTCC NO: M20251394, and the preservation number of Bacillus polymyxa 21-2 is CCTCC NO: M 2022346.

2. The composite bacterial agent according to claim 1, wherein the effective bacterial concentration ratio of Pseudomonas A3 and Paenibacillus polymyxa 21-2 is 1-2:

1.

3. The composite bacterial agent according to claim 2, wherein the effective bacterial concentration ratio of Pseudomonas A3 and Paenibacillus polymyxa 21-2 is 1:

1.

4. Use of the composite bacterial agent according to claim 1 in promoting compost maturity.

5. The use according to claim 4, wherein the composite bacterial agent promotes compost maturity by promoting microbial quorum sensing.

6. Use of the composite bacterial agent according to claim 1 in the preparation of a compost microbial preparation.

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