Method for evaluating elasticity of microbial system

Abstract

The invention relates to a method for evaluating the elasticity of a microbial system, and belongs to the field of microbiology. The method defines the elasticity of the microbial community as a synergistic effect of resistance and functional redundancy based on a reconstructed elastic concept framework. Through metagenomics and metatranscriptomics technologies, resistance and function redundancy are quantified, and a corresponding evaluation methodology is constructed. The method comprises the following steps: firstly, obtaining microbiota related to a target function through multi-omics data acquisition and screening of microbiota with a specific function; then, respectively evaluating the diversity and cohesion of the microbiota in each process in the metabolic pathway so as to quantify the resistance; and calculating functional redundancy according to the discrimination of the redundant species. And finally, carrying out linear fitting on the resistance and the function redundancy, calculating the elasticity based on the resistance, the function redundancy and an area enclosed by a fitting line, and further calculating the elasticity maintenance. According to the method, the function maintaining capability of the microbial system in response to external disturbance can be evaluated, and an important technical support is provided for environmental microorganism research and ecological system management.

Description

Technical Field

[0001] The invention belongs to the field of microbiology and relates to a method for evaluating the elasticity of a microbial system. Background Art

[0002] The term resilience was first introduced into ecology in 1973, and it was defined as the ability of a system to absorb and adapt to disturbances while maintaining its basic structure and function. Microbial systems have structure and function and are adaptable to external disturbances. Resilience can be used to characterize the rapid response of microbial systems to disturbances. However, since the 1990s, large-scale field experiments in ecology have only transformed into microscopic experiments, resulting in a lag in the study of microbial systems in various ecosystems, especially the assessment of the resilience of microbial systems.

[0003] There are three types of methods available for assessing the resilience of microbial systems: one is to draw on the assessment methods of other ecosystems and use the difference in changes in a certain indicator before, during and after the disturbance as resistance, functional redundancy and resilience; the second is to use the average variability to evaluate the stable state of the system; the third is to use potential functions to calculate indicators such as potential energy depth and exit time. However, the currently available methods cannot truly assess resilience, and lack a reasonable methodology, making it difficult to capture dynamics at the microscopic level. This has, to a certain extent, limited the in-depth study of the response of microbial systems to external disturbances and the development of resilience assessment in microbial systems. Summary of the invention

[0004] In view of the above, an object of the present invention is to provide a method for evaluating the resilience of a microbial system.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for assessing the resilience of a microbial system, the method comprising the following steps:

[0007] S1: Multi-omics data acquisition: Acquire metagenomics and metatranscriptomics data of microbial communities;

[0008] S2: Screening of microbial communities with specific functions: Screening out functional microorganisms involved in specific metabolic pathways based on target functions;

[0009] S3: Evaluate resistance: Calculate the Shannon diversity and cohesion of the microbial community in each process in the metabolic pathway and calculate resistance accordingly;

[0010] S4: Evaluate functional redundancy: Based on the definition of functional redundancy and the determination of redundant species, calculate the functional redundancy of each process in the metabolic pathway of the microbial community;

[0011] S5: Evaluate elasticity: Perform a linear fit between resistance and functional redundancy, and calculate elasticity based on resistance, functional redundancy and the area enclosed by the fitting line, and further calculate elasticity maintenance.

[0012] The approach includes the reconstruction of a conceptual framework and the construction of a methodology. The conceptual framework is based on the consensus that has gradually been reached in the process of resilience research, that is, the resilience of microbial communities is determined by two parts, namely resistance and resilience. Resistance refers to the ability of microbial communities to maintain community structure and system functions in the face of external disturbances; resilience refers to the tendency of ecosystem functions to stabilize or quickly recover to pre-disturbance levels after being disturbed. On the other hand, functional redundancy describes the ability of microbial communities to replace each other without affecting performance, which ultimately affects the recovery potential of microbial communities. Ultimately, resilience is determined by both "resistance" and "functional redundancy", and this concept is characterized under the stability domain based on the ball and cup heuristic. Multi-omics technologies provide opportunities to explore the resilience and nonlinear characteristics of microbial communities. Metagenomics contains the complete genetic composition of its members and can be used to evaluate total genomic DNA and its metabolic potential. Metatranscriptomics data are related to the subset of genes expressed by microorganisms, providing sequence-based expression profiles and capturing a snapshot of genes that are essential for survival in a specific environment. Decoding the complex relationship between microbial community structure and function in metagenomics and metatranscriptomics can bring new insights into resilience. From this, the range of resilience can be obtained and whether the microbial community adapts to external perturbations to maintain functionality can be determined.

[0013] The methodology was constructed by separately assessing functional redundancy, resistance and resilience.

[0014] Functional redundancy refers to the ability of multiple different organisms to perform common metabolic functions. The assessment of functional redundancy in this article is based on this definition, so the focus of the assessment is to quantify redundant species. There are many processes in a metabolic pathway, and each process is performed by different functional genes. Based on multi-omics technology, there are two types of determination of redundant species: Ⅰ) In the metagenomics or metatranscriptomics layer, if a species is involved in the execution of at least two processes, the species is considered redundant; Ⅱ) Under the same metabolic pathway, between the metagenomics layer and the metatranscriptomics layer, if a species appears in the same process, the species is considered redundant. The more redundant species there are, the higher the degree of functional redundancy. The assessment value of functional redundancy is determined according to the number of redundant species. Functional redundancy R func The evaluation is as follows:

[0015]

[0016] Where a is the ath process in the metabolic pathway; N a,MG and N a,MTare the number of species in the ath process at the metagenomics (MG) layer and metatranscriptomics (MT) layer respectively; N a,r(MG) and N a,r(MT) is the number of redundant species in the metagenomics layer or metatranscriptomics layer in process a; N a,r(MG∩MT) It refers to the number of redundant species between the metagenomics layer and the metatranscriptomics layer in process a.

[0017] Resistance stru It is quantified based on the theoretical contribution of Robert May. Factors such as species diversity and their connection strength play a role in determining resistance. The evaluation of species diversity and connection strength is also refined to each process of each metabolic pathway at the metagenomics layer and metatranscriptomics layer. Here, the connection strength is measured by cohesion Coh.

[0018] R stru =H shan -Coh

[0019]

[0020] In the formula, H shan is Shannon diversity; S obs is the number of species observed; a i is the abundance of the i-th species in sample j; A is the sum of the abundances of all species; indicates that two species occupy similar ecological niches and respond to disturbances in similar patterns. Indicates intense competition among species for nutrients or ecological niches. and are the positive connection strength and negative connection strength respectively.

[0021] Elasticity is determined by resistance and functional redundancy. In the local stability domain, the elasticity of a microbial system with nonlinear characteristics can be regarded as linear, so resistance and functional redundancy are linearly fitted. Since there are two types of functional redundancy, elasticity also includes type I and type II.

[0022] Type I resilience is measured by resistance and functional redundancy at the metagenomic level and resistance and functional redundancy at the metatranscriptomic level, respectively. In Type I, we assume that the more resistant a microbial system is, the less functional redundancy is required. Based on this assumption, three scenarios emerge:

[0023] 1) The microbial system is resistant and completely undisturbed, and no functional redundancy is required;

[0024] 2) The resistance of the microbial system is disturbed and weakened, and functional redundancy appears to support functional recovery;

[0025] 3) If the microbial system is completely affected by the disturbance, functional redundancy is enhanced to maintain the normal functioning of the system.

[0026] The resilience of type II is measured by the resistance of the metagenomic layer and the functional redundancy between the metagenomic layer and the metatranscriptomic layer, and the resistance of the metatranscriptomic layer and the functional redundancy between the metagenomic layer and the metatranscriptomic layer. In type II, we assume that the stronger the resistance of the microbial system, the more species involved in the same process, and the stronger the functional redundancy between the metagenomic layer and the metatranscriptomic layer. According to this assumption, there are also three situations:

[0027] 1) If the microbiome is resistant to external interference at both the metagenomic and metatranscriptomic layers, then the functional redundancy between the metagenomic and metatranscriptomic layers is strong;

[0028] 2) The resistance of the microbiome is weakened by disturbances at both the metagenomic and metatranscriptomic layers, the number of species involved in a certain process is reduced, and the functional redundancy between the metagenomic and metatranscriptomic layers is also reduced;

[0029] 3) When resistance is completely affected by disturbance, functional redundancy is weak and will be exerted by new species in the same process between metagenomic and metatranscriptomic layers.

[0030] The range of resistance and functional redundancy is the same as the resistance and functional redundancy fitting line as the boundary, and the area enclosed is used to measure the elasticity R es At the same time, elasticity maintains R es,main A positive value indicates that the microbiome functions normally.

[0031] The calculation formula for Type I elasticity is:

[0032] R es,MG =∫∫f(x,y) MG dxdy

[0033] R es,MT =∫∫f(x,y) MT dxdy

[0034] R es,main =R es,MT -R es,MG

[0035] In the formula, R es,MG and R es,MT are the elasticities quantified at the metagenomics layer and the metatranscriptomics layer, respectively; f(x,y) MGand f(x,y) MT are the fitting lines of resistance and functional redundancy at the metagenomics layer and metatranscriptomics layer, respectively; x is the resistance range; y is the functional redundancy range; R es,main YesR es,MT and R es,MG The difference.

[0036] The calculation formula for type II elasticity is:

[0037] R es,G =∫∫f(x MG ,y MG∩MT )dxdy

[0038] R es,T =∫∫f(x MT ,y MG∩MT )dxdy

[0039] R es,main =R es,T -R es,G

[0040] In the formula, R es,G is the elasticity quantified at the metagenomics layer and between the metagenomics and metatranscriptomics layers; R es,T is the elasticity quantified at the metatranscriptomics layer and between the metagenomics and metatranscriptomics layers; f(x MG ,y MG∩MT ) is the fitting line of metagenomic layer resistance and functional redundancy between metagenomic and metatranscriptomic layers; f(x MT ,y MG∩MT ) is the fitting line of the resistance of the metatranscriptomics layer and the functional redundancy between the metagenomics and metatranscriptomics layers; x is the resistance range; y is the functional redundancy range; R es,main YesR es,T and R es,G The difference.

[0041] This method only considers the elasticity of the microbial system under any stable domain, and the stable domain may be a new stable domain. Since the evaluation of resistance, functional redundancy and elasticity is refined to each process of the metabolic pathway, the premise of the evaluation is to know the specific metabolic pathway, and there are at least 2 metabolic processes in the metabolic pathway. If amplicon sequencing is used to obtain the composition of the microbial community and the abundance of specific genes, the accuracy of the elasticity evaluation will be affected due to the lack of sequencing depth. This constraint applies to the entire content of the present invention and will not be repeated below.

[0042] The beneficial effects of the present invention are:

[0043] (1) Through metagenomics and metatranscriptomics technologies, we can comprehensively analyze the genetic composition and expression of microbial communities, deeply understand the structure and function of microbial communities, and provide a basis for revealing the resilience of microbial systems.

[0044] (2) Metatranscriptomics technology can capture the dynamic changes in gene expression of microbial communities, thereby more accurately evaluating the response of microbial systems to external perturbations and improving the accuracy of resilience assessment.

[0045] (3) Metagenomics and metatranscriptomics technologies can identify redundant species involved in the same function and quantify their participation in metabolic pathways, thereby more accurately assessing the degree of functional redundancy and providing an important basis for resilience assessment.

[0046] (4) Compared with traditional experimental methods, metagenomics and metatranscriptomics have higher-resolution microbial data and can significantly improve research depth.

[0047] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0049] Figure 1 It is a conceptual framework diagram of the elasticity of the present invention;

[0050] Figure 2 This is a schematic diagram of redundant species determination of the present invention;

[0051] Figure 3 It is a schematic diagram of elasticity evaluation of the present invention. DETAILED DESCRIPTION

[0052] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0053] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0054] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0055] like Figure 1 Based on the conceptual framework of resilience shown in the figure, the present invention provides a method for assessing the resilience of a microbial system, comprising the following steps:

[0056] S1. Multi-omics data acquisition

[0057] The activated sludge mixed liquor collected from a sewage treatment plant was used as the initial sample. The initial sample was filtered through a 0.22μm mixed cellulose ester membrane and stored in a cryopreservation tube for DNA and RNA extraction, sequencing, assembly, and species and functional annotation, thereby obtaining basic metagenomics and metatranscriptomics data.

[0058] S2. Screening of microbiota with specific functions

[0059] (1) Find and identify metabolic pathways with specific functions;

[0060] (2) Select the abundance and expression of functional genes involved in each metabolic process in the metabolic pathway and the species that play a role in each metabolic process in metagenomics and metatranscriptomics respectively;

[0061] (3) If the processes of the metabolic pathway are complete in metagenomics and metatranscriptomics, and at least one functional gene is involved in each process and the abundance and expression of the functional gene are not 0, then it can be shown that the screening of the microbiome with specific functions is successful, and other microbiome with specific functions can be screened according to the same process;

[0062] (4) Arrange the species and their abundance, genes and their abundance and expression levels of the screened microbial community in the order of their participation in the metabolic pathway for subsequent use.

[0063] S3. Evaluate resistance

[0064] The assessment of resistance involves the calculation of indices of diversity and cohesion of processes in metabolic pathways.

[0065] (1) Based on the tables in S2, the Shannon diversity of each process in the metabolic pathway was calculated in metagenomics and metatranscriptomics using the species and abundance data of the microbiome;

[0066] (2) The data table in S2 was reorganized into a table of species and their abundances involved in the entire metabolic pathway, and a Spearman correlation molecular ecological network of logarithmically transformed species abundances was constructed. The reorganized species and their abundance table was input, and the correlation critical value of the microbial community with a specific function was automatically determined using a method based on random matrix theory;

[0067] (3) Using the “taxa shuffle” null model, the correlation cutoff value and the species and their abundance table in S2 were input to calculate the cohesion of the microbiome in each process in the metabolic pathway;

[0068] (4) Substitute Shannon diversity and cohesion into the resistance calculation formula to calculate the resistance of each process in the metabolic pathway.

[0069] S4. Evaluate functional redundancy

[0070] The assessment of functional redundancy focuses on identifying redundant species and evaluating functional redundancy by quantifying the participation of redundant species in metabolic pathways, such as Figure 2 shown.

[0071] (1) The redundant species of type I were determined in the metagenomics layer and the metatranscriptomics layer respectively. According to the table in S2, we checked which species were involved in at least two processes in each process of the metabolic pathway, and they were the redundant species of the entire metabolic pathway.

[0072] (2) Determination of redundant species of type II: In the same process in the same metabolic pathway, if the same species is involved in the process in the metagenomics layer and the metatranscriptomics layer, it is a redundant species in the process in the metabolic pathway.

[0073] (3) According to the functional redundancy calculation formula, the number of redundant species and the total number of species involved in the process are substituted to calculate the functional redundancy of each process in the metabolic pathway.

[0074] S5. Evaluate Resilience

[0075] Resilience is determined by resistance and functional redundancy. Based on the two types of functional redundancy, there are also two types of resilience assessment, such as Figure 3 shown.

[0076] (1) The two types of resilience assessments differ only in the data used, and the implementation process is the same. Type I resilience uses the data calculated at the metagenomics layer and the metatranscriptomics layer and the type I functional redundancy; Type II resilience uses the data calculated at the metagenomics layer and the metatranscriptomics layer and the type II functional redundancy;

[0077] (2) Linearly fit the resistance and functional redundancy of each process in the metabolic pathway obtained in S3 and S4, integrate the fitting line within the range of actual resistance and functional redundancy, and the result is elasticity;

[0078] (3) The elasticity obtained from the metatranscriptomics layer and the metagenomics layer is further subtracted to obtain elasticity maintenance.

[0079] The method of the present invention realizes the assessment of the resilience of microbial systems based on the conceptual framework of resilience. By establishing a methodology of resistance, functional redundancy and resilience, the ability of microorganisms to maintain normal functions under external disturbances is explored, providing important support for conducting environmental microbial research.

[0080] This method is suitable for the assessment of the resilience of environmental microbial systems such as sewage treatment plants, soil, and water bodies.

[0081] Example

[0082] Target function: Nitrogen cycle

[0083] Multi-omics data acquisition: Activated sludge samples were collected from sewage treatment plants, and metagenomics and metatranscriptomics sequencing were performed to obtain nitrogen cycle-related genes and microbial community data.

[0084] Screening of specific functional microbial communities: Microbial communities involved in nitrification were screened out and organized in metagenomic and metatranscriptomic data respectively.

[0085] Assessing resistance: The operations are the same for metagenomic and metatranscriptomics data, and metagenomics is used as an example below.

[0086] Shannon diversity of nitrification metabolic pathways: The Shannon diversity values ​​of the five nitrification processes were calculated separately.

[0087] Nitrification metabolic pathway cohesion: Calculate the cohesion values ​​of the five nitrification processes separately.

[0088] Resistance of nitrification metabolic pathway: The resistance values ​​of the five nitrification processes were added together to obtain the total resistance of the microbial community.

[0089] It is assumed that the Shannon diversity of the five processes of nitrification are: 1.1, 1.2, 1.3, 1.4, 1.5;

[0090] It is assumed that the cohesion indexes of the five processes of nitrification are: -0.5, -0.6, -0.7, -0.8, -0.9;

[0091] Nitrification resistance: R stru =1.1 0.5 +1.2 0.6 +1.3 0.7 +1.4 0.8 +1.5 0.9 =6.12;

[0092] Evaluate functional redundancy: Determine the number of redundant species and the participating species tree in each nitrification process, calculate the functional redundancy of each process according to the formula, and then obtain the functional redundancy of the microbial community.

[0093] Assume that the number of redundant species in the five processes of nitrification are: 1, 2, 3, 4, and 5;

[0094] It is assumed that the number of redundant species between the metagenomics layer and the metatranscriptomics layer for the five processes of nitrification are: 0, 1, 2, 3, and 4;

[0095] Assume that the number of species involved in the five processes of nitrification are: 5, 6, 7, 8, and 9;

[0096] Functional redundancy of nitrification type I: R func =1 / 5+2 / 6+3 / 7+4 / 8+5 / 9=2.02;

[0097] Functional redundancy of nitrification type II: R func =0 / 5+1 / 6+2 / 7+3 / 8+4 / 9=1.27;

[0098] Evaluating resilience: The resilience of the microbial community was obtained by integrating the resistance and functional redundancy of the five processes of nitrification.

[0099] Assuming type I elasticity, the resistance and functional redundancy fitting lines are expressed as: y1 = 2x1 + b;

[0100] Type I elasticity:

[0101] Type II elasticity:

[0102] For elastic calculation of macrotranscriptomics, just repeat the above steps.

[0103] Assume that the elasticity calculation result of metatranscriptomics is:

[0104] Type I elasticity: R es,MT =0.8;

[0105] Type II elasticity: R es,T =0.7;

[0106] Type I elastic maintenance: R es,main =R es,MT -R es,MG =0.8-0.7362=0.0638;

[0107] Type II elastic maintenance: R es,main =R es,T -R es,G =0.7-0.7362=-0.0362.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A method for assessing the resilience of microbial systems, Features: The method comprises the following steps: S1: Multi-omics data acquisition: Acquire metagenomics and metatranscriptomics data of microbial communities; S2: Screening of microbial communities with specific functions: Screening out functional microorganisms involved in specific metabolic pathways based on target functions; S3: Evaluate resistance: Calculate the Shannon diversity and cohesion of the microbial community in each process in the metabolic pathway and calculate resistance accordingly; S4: Evaluate functional redundancy: Based on the determination of redundant species, calculate the functional redundancy of each process in the metabolic pathway of the microbial community; S5: Evaluate elasticity: Perform a linear fit between resistance and functional redundancy, and calculate elasticity based on resistance, functional redundancy and the area enclosed by the fitting line, and further calculate elasticity maintenance.

2. A method for evaluating the resilience of a microbial system according to claim 1, Features: In S1, the specific method for acquiring multi-omics data includes: Extraction of DNA and RNA from microbial communities; Sequencing and assembling DNA and RNA; Species and functional annotations were performed on sequencing data.

3. A method for evaluating the resilience of a microbial system according to claim 1, Features: In S2, the specific method for screening microbial communities with specific functions includes: Find and identify metabolic pathways of target functions; The species and functional genes involved in the metabolic pathway were screened through metagenomics and metatranscriptomics; Determine whether the screened species and functional genes meet the screening conditions; The species and their abundance, genes and their abundance and expression levels of the screened microbiome were sorted out.

4. A method for evaluating the resilience of a microbial system according to claim 1, Features: In S3, the specific method for evaluating resistance includes: Calculate the Shannon diversity of each process in the metabolic pathway; Constructing a molecular ecological network of microbial communities; Calculate the correlation cutoff value of microbial communities; The cohesion of the microbial community was calculated using the null model; Substitute Shannon diversity and cohesion to calculate resistance.

5. A method for evaluating the resilience of a microbial system according to claim 1, Features: In S4, the specific method for evaluating functional redundancy includes: Determine the definition of functional redundancy; Based on the definition of functional redundancy, determine which species in the microbial community are redundant; Calculate the participation of redundant species in metabolic pathways; Functional redundancy was calculated by substituting the number of redundant species and the total number of species involved in the process.

6. A method for evaluating the resilience of a microbial system according to claim 1, Features: In S5, the specific method of evaluating elasticity includes: Resistance and functional redundancy were linearly fitted; Elasticity was calculated based on resistance, functional redundancy, and the area enclosed by the fitted line; Resilience maintenance was further calculated using the difference in resilience between metatranscriptomics and metagenomics.

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