Method for identifying microbial metabolic molecular markers in a biological treatment process

Abstract

The application provides a method for identifying microbial metabolic molecular markers in a biological treatment process, comprising: determining a plurality of organic molecules of microbial metabolites in a preset molecular mass range from a target biological treatment process by using high-resolution mass spectrometry; pairing each two organic molecules to form a plurality of organic molecule pairs; screening a first target organic molecule pair from the plurality of organic molecule pairs according to first molecular mass difference information and second molecular mass difference information, wherein the first molecular mass difference information is determined according to the organic molecule pair, and the second molecular mass difference information is determined according to a whole genome and a metabolic pathway database; constructing a preset network according to the first target organic molecule pair, wherein the preset network comprises a plurality of nodes; screening a target node from the plurality of nodes according to a preset condition; and determining a microbial metabolic molecular marker in the target biological treatment process according to the target node.

Description

Technical Field

[0001] The present invention relates to the technical field of wastewater and waste pollution control, and in particular to a method for identifying microbial metabolic molecular markers in a biological treatment process. Background Art

[0002] Biological treatment is a key technology for the treatment and disposal of domestic sewage, organic wastewater, and organic waste. It forms a core component of treatment processes, including pollutant removal and organic matter stabilization in sewage / wastewater and waste. Examples include the activated sludge process commonly used in sewage treatment plants, anaerobic / aerobic biological treatment of organic wastewater, and aerobic composting and anaerobic digestion of organic waste. Biological treatment utilizes widely occurring microorganisms in nature, domesticating and regulating dominant microbial populations under controlled conditions to decompose and transform specific substances and pollutants. It is a near-natural treatment technology characterized by low treatment costs, controllable processes, and widespread application.

[0003] Monitoring the biological treatment process is crucial for regulating the stable operation of the process and improving treatment efficiency. Currently, indicators such as pH, temperature, conductivity, oxygen content, and redox potential monitored in biological treatment processes are primarily environmental factors that affect the metabolic activity of specific microbial groups. The relationship between these environmental factors and the stable operation and treatment efficiency of the biological treatment process, established through experiments and engineering experience, enables process regulation, process monitoring, and problem diagnosis. While the monitoring technology for these macro-level environmental factor indicators is mature, they often lag behind abnormal changes in microbial metabolism during the biological treatment process, making it difficult to detect abnormalities in microbial metabolism at an early stage. For example, during anaerobic digestion, methanogenic metabolism is inhibited, volatile fatty acids accumulate, and the pH decreases. However, due to the presence of ions such as carbonates and ammonia, the decrease in pH significantly lags behind abnormalities in microbial metabolism.

[0004] Early warning of abnormal microbial metabolism is a crucial foundation for improving the stable operation of biological treatment processes and even for future intelligent monitoring and control. This relies on indicators that can quickly and sensitively reflect abnormal microbial metabolism. Microbial metabolism produces a large number of small organic molecules, namely microbial metabolites. Microbial metabolites can accurately and rapidly reflect the state of microbial metabolism and serve as molecular markers that can indicate abnormal metabolic changes, such as the volatile fatty acids produced by microbial metabolism during anaerobic biological treatment. However, there is a lack of understanding of molecular markers of microbial metabolism in various biological treatment processes. Summary of the Invention

[0005] In view of this, the main purpose of the present invention is to provide a method for identifying molecular markers of microbial metabolism in a biological treatment process, in order to at least partially solve one of the above-mentioned technical problems.

[0006] The present invention provides a method for identifying molecular markers of microbial metabolism in a biological treatment process, comprising:

[0007] Using high-resolution mass spectrometry, multiple organic molecules of microbial metabolites within a preset molecular mass range are identified from the target biological process;

[0008] Pair every two organic molecules to form multiple organic molecule pairs;

[0009] screening a first target organic molecule pair from a plurality of organic molecule pairs according to the first molecular mass difference information and the second molecular mass difference information, wherein the first molecular mass difference information is determined based on the organic molecule pair, and the second molecular mass difference information is determined based on a whole genome and metabolic pathway database;

[0010] Constructing a preset network according to the first target organic molecule pair, wherein the preset network includes a plurality of nodes;

[0011] Filtering target nodes from multiple nodes according to preset conditions; and

[0012] According to the target node, the molecular markers of microbial metabolism in the target biological treatment process are determined.

[0013] According to an embodiment of the present invention, the present invention further includes:

[0014] Determine the molecular reaction information of metabolites in the microbial metabolic pathways during the target biological treatment process based on the whole genome and metabolic pathway database;

[0015] Determining a metabolic molecule reaction pair according to the metabolite molecular reaction information, wherein the metabolic molecule reaction pair includes a metabolic molecule reactant and a metabolic molecule product; and

[0016] According to the metabolic molecule reaction pair, second molecular mass difference information is determined.

[0017] According to an embodiment of the present invention, the preset network includes a preset molecular reaction network and / or a preset co-metabolism network between organic molecules and microorganisms;

[0018] Based on the first target organic molecule pair, a preset network is constructed, including:

[0019] When it is determined that the preset network includes a preset molecular reaction network, the preset molecular reaction network is constructed using the first target organic molecule in the first target organic molecule pair as a node and the molecular mass difference of the first target organic molecule pair as an edge;

[0020] In a case where it is determined that the preset network comprises a preset co-metabolic network between organic molecules and microorganisms, a second target organic molecule is determined according to the first target organic molecule in the pair of the metabolite molecule and the first target organic molecule, wherein the metabolite molecule is determined according to metabolite molecule reaction information in a metabolic pathway of microorganisms in the target biological treatment process and determined from a whole genome and metabolic pathway database;

[0021] A metabolic microorganism related to the second target organic molecule in the whole genome and metabolic pathway database is determined according to the second target organic molecule; and

[0022] A preset co-metabolic network between organic molecules and microorganisms is constructed with the second target organic molecule and the metabolic microorganism as nodes and the relationship between the second target organic molecule and the metabolic microorganism as edges.

[0023] According to an embodiment of the present application, target nodes are screened from a plurality of nodes according to preset conditions, which include:

[0024] In a case where it is determined that the preset network comprises a preset molecular reaction network, topological analysis is performed on the preset molecular reaction network to determine network structure attributes of the preset molecular reaction network;

[0025] The correlation values of each node in the preset molecular reaction network are calculated according to the network structure attributes;

[0026] Target nodes are screened from each node according to the threshold value and the correlation value;

[0027] In a case where it is determined that the preset network comprises a preset co-metabolic network between organic molecules and microorganisms, an initial microorganism obtained through third-party operation in the target biological treatment process is acquired;

[0028] Target nodes are screened from each node of the preset co-metabolic network between organic molecules and microorganisms according to the initial microorganism and the metabolic microorganism.

[0029] According to an embodiment of the present application, in a case where it is determined that the preset network comprises a preset co-metabolic network between organic molecules and microorganisms,

[0030] The method further comprises:

[0031] A script is written by using a language, and metabolite molecule reaction information in a metabolic pathway of microorganisms in the target biological treatment process is acquired from a whole genome and metabolic pathway database in combination with a preset program package; and

[0032] The metabolite molecule is identified from the metabolite molecule reaction information.

[0033] According to the embodiment of the present application, in the case that the preset network comprises a preset co-metabolic network between organic molecules and microorganisms, the second target organic molecule is determined according to the first target organic molecule in the pair of metabolite molecules and the first target organic molecule, comprising:

[0034] The molecular mass of the first target organic molecule is matched with the molecular mass of the metabolite molecule by using a language script; and

[0035] The first target organic molecule with the same molecular mass as the metabolite molecule is matched as the second target organic molecule.

[0036] According to the embodiment of the present application, the target node is screened from each node of the preset co-metabolic network between organic molecules and microorganisms according to the initial microorganism and the metabolizing microorganism, comprising:

[0037] The initial microorganism and the metabolizing microorganism are matched to obtain a matched microorganism; and

[0038] According to the matched microorganism, a node related to the matched microorganism is screened from each node of the preset co-metabolic network between organic molecules and microorganisms as the target node.

[0039] According to the embodiment of the present application, the network structure attribute comprises at least one of the following: the degree of the network node, the network path length, the network centrality, and the network module.

[0040] According to the embodiment of the present application, a plurality of organic molecules of the metabolite of the microorganism in the preset molecular mass range are determined from the target biological treatment process by using high-resolution mass spectrometry, comprising:

[0041] Dissolved organic matter obtained by third-party operation in the target biological treatment process is obtained;

[0042] The dissolved organic matter is tested by using high-resolution mass spectrometry to obtain mass spectrometry test results in the preset molecular mass range, wherein the mass spectrometry test results comprise a plurality of high-resolution mass spectrometry peaks; and

[0043] Each high-resolution mass spectrometry peak is matched with a molecular formula according to a preset analysis method to obtain a plurality of organic molecules.

[0044] According to the embodiment of the present application, the first target organic molecule pair is screened from a plurality of organic molecule pairs according to the first molecular mass difference information and the second molecular mass difference information, comprising:

[0045] For each organic molecule pair:

[0046] The first molecular mass difference information and the second molecular mass difference information are matched to obtain a matching result; and

[0047] When it is determined that the first molecular mass difference information and the second molecular mass difference information are consistent in the matching result, the organic molecule pair is taken as the first target organic molecule pair.

[0048] According to an embodiment of the present invention, by utilizing high-resolution mass spectrometry, a plurality of organic molecules are formed from a plurality of microbial metabolites within a preset molecular mass range determined in a target biological treatment process; a first target organic molecule pair is screened from the plurality of organic molecule pairs based on the first molecular mass difference information determined for the organic molecule pair and the second molecular mass difference information determined based on the whole genome and metabolic pathway database; a preset network comprising a plurality of nodes is constructed based on the first target organic molecule pair to screen the target node and determine the microbial metabolic molecular marker in the target biological treatment process. Based on the high resolution and accuracy of high-resolution mass spectrometry, accurate identification of complex microbial metabolic molecules in the biological treatment process is achieved; based on the constructed preset network comprising a plurality of nodes, the target node among the thousands or tens of thousands of metabolic molecules in the biological reaction process is rapidly identified, and the molecule corresponding to the target node is used as the microbial metabolic molecular marker in the biological treatment process, providing a new method for early warning indicator discrimination and intelligent monitoring of microbial metabolic changes in the biological treatment process. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 A flow chart of a method for identifying microbial metabolic molecular markers in a biological treatment process according to an embodiment of the present invention is schematically shown;

[0050] Figure 2 (a) schematically shows the results of high-resolution mass spectrometry testing according to Example 1 of the present invention;

[0051] Figure 2 (b) schematically shows a molecular reaction network constructed according to Example 1 of the present invention;

[0052] Figure 2 (c) schematically shows a schematic diagram of target molecule nodes screened based on network topology analysis according to Example 1 of the present invention;

[0053] Figure 2 (d) schematically shows the reaction relationship between target molecule nodes according to Example 1 of the present invention;

[0054] Figure 3 The diagram schematically shows a co-metabolism network between organic molecules and microorganisms established after interactive search with the KEGG database in Example 1 of the present invention. DETAILED DESCRIPTION

[0055] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0056] During the implementation of the present invention, it was discovered that the lack of knowledge about molecular markers of microbial metabolism in various bioprocesses is primarily due to the lack of effective methods for identifying these molecular markers in actual bioprocesses. This difficulty arises from the complex systems and diverse microbial populations involved in the biotreatment of wastewater and waste, making the resulting microbial metabolites difficult to identify at the molecular level. Furthermore, there is no method for identifying molecular markers of significant importance from among the thousands or tens of thousands of microbial metabolites.

[0057] Based on this, the present invention provides a method for identifying molecular markers of microbial metabolism in a biological treatment process, comprising: using high-resolution mass spectrometry to determine multiple organic molecules of microbial metabolites within a preset molecular mass range from a target biological treatment process; pairing every two organic molecules to form multiple organic molecule pairs; screening a first target organic molecule pair from the multiple organic molecule pairs based on first molecular mass difference information and second molecular mass difference information, wherein the first molecular mass difference information is determined based on the organic molecule pair, and the second molecular mass difference information is determined based on a whole genome and metabolic pathway database; constructing a preset network based on the first target organic molecule pair, wherein the preset network includes multiple nodes; screening a target node from the multiple nodes based on preset conditions; and determining the molecular markers of microbial metabolism in the target biological treatment process based on the target node.

[0058] The following schematically illustrates a method for identifying microbial metabolic molecular markers in a biological treatment process. It should be noted that this example is only a specific embodiment of the present invention and does not limit the scope of protection of the present invention.

[0059] Figure 1 The flowchart of the method for identifying microbial metabolic molecular markers in a biological treatment process according to an embodiment of the present invention is schematically shown.

[0060] According to an embodiment of the present invention, the method 100 for identifying microbial metabolic molecular markers in a biological treatment process includes operations S101 to S106.

[0061] In operation S101 , a plurality of organic molecules of microbial metabolites within a preset molecular mass range are determined from a target biological process using high-resolution mass spectrometry.

[0062] According to embodiments of the present invention, high-resolution mass spectrometry includes, but is not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICS MS). The preset molecular mass range can be determined based on the molecular mass of small organic molecules. Target biological treatment processes can include, but are not limited to, sewage biological treatment processes, wastewater biological treatment processes, and waste biological treatment processes.

[0063] According to embodiments of the present invention, samples can be collected from a target biological process. Dissolved organic matter in the sample can then be enriched, separated, and purified using pre-treatment methods such as electrodialysis and solid-phase extraction. High-resolution mass spectrometry can then be used to analyze the dissolved organic matter within a predetermined molecular weight range to identify multiple organic molecules that are microbial metabolites.

[0064] For example, the preset molecular mass range can be 100Da to 1000Da or 100Da to 2000Da. FT-ICS MS can be used to test the obtained soluble organic matter within the range of 100Da to 1000Da or 100Da to 2000Da to obtain the mass spectrometry results of small molecule organic matter. Molecular formula matching can be performed on a single high-resolution mass spectrum peak based on computing software or program packages such as Data Analysis (DA) or TRFu to determine multiple small molecule organic matter formulas. Based on the multiple small molecule organic matter formulas, multiple organic matter molecules of microbial metabolites can be obtained.

[0065] In operation S102 , every two organic molecules are paired to form a plurality of organic molecule pairs.

[0066] According to an embodiment of the present invention, the plurality of organic molecules obtained in operation S101 may be paired in pairs to obtain a plurality of organic molecule pairs.

[0067] In operation S103, a first target organic molecule pair is screened from a plurality of organic molecule pairs based on the first molecular mass difference information and the second molecular mass difference information, wherein the first molecular mass difference information is determined based on the organic molecule pair, and the second molecular mass difference information is determined based on the whole genome and metabolic pathway database.

[0068] According to an embodiment of the present invention, the first molecular mass difference information is determined based on the organic molecule pairs, which may include: calculating the difference between the molecular masses corresponding to each organic molecule pair to obtain the first molecular mass difference information.

[0069] According to an embodiment of the present invention, the second molecular mass difference information is determined based on the whole genome and metabolic pathway database, which can include writing a script in a language such as R / Python, combined with the use of the KEGGREST program package, to obtain and organize the metabolic molecular reactants and products of all metabolic reaction processes involved in the microbial metabolic pathway in the target biological treatment process from the whole genome and metabolic pathway database. The metabolic molecular reactants and products are used to form reaction pairs between reactant molecules and product molecules. According to the molecular masses corresponding to the respective molecular masses within the reaction pairs between the reactant molecules and the product molecules, the difference is calculated to obtain the second molecular mass difference information.

[0070] It should be noted that the script is written based on the R / Python language, and real-time online interactive search with the whole genome and metabolic pathway database (KEGG database) is synchronized with the latest information in the database. As the database of microbial metabolic pathways is continuously updated and improved, it supports the use of the microbial metabolic molecular marker identification method for the biological treatment process provided by the present invention to identify new molecular markers.

[0071] According to an embodiment of the present invention, the first molecular mass difference information and the second molecular mass difference information can be matched, and the organic molecule pairs corresponding to the first molecular mass difference information that match the second molecular mass difference information can be selected as the first target organic molecule pairs. The allowable error for the matching can be ±0.00001, ±0.0001, or ±0.001.

[0072] In operation S104 , a preset network is constructed according to the first target organic molecule pair, wherein the preset network includes a plurality of nodes.

[0073] According to an embodiment of the present invention, the preset network may include a preset molecular reaction network. The preset molecular reaction network may be constructed using the first target organic molecule in the first target organic molecule pair as a node and the molecular mass difference of the first target organic molecule pair as an edge.

[0074] According to an embodiment of the present invention, the preset network may include a preset co-metabolism network between organic molecules and microorganisms. The metabolite molecule can be determined based on the metabolite molecule reaction information in the microbial metabolic pathway during the target biological treatment process determined by the whole genome and metabolic pathway database. The molecular mass of the metabolite molecule is matched with the molecular mass of the first target organic molecule in the first target organic molecule pair to obtain a second target organic molecule. Based on the second target organic molecule, the metabolic microorganism related to the second target organic molecule in the whole genome and metabolic pathway database is determined; and the preset co-metabolism network between organic molecules and microorganisms is constructed with the second target organic molecule and the metabolic microorganism as nodes and the relationship between the second target organic molecule and the metabolic microorganism as an edge.

[0075] According to an embodiment of the present invention, the preset network may include a preset molecular reaction network and a preset co-metabolism network between organic molecules and microorganisms.

[0076] In operation S105 , a target node is screened from a plurality of nodes according to a preset condition.

[0077] According to an embodiment of the present invention, if the preset network is a preset molecular reaction network, a topological analysis can be performed on the preset molecular reaction network to determine the network structure properties of the preset molecular reaction network; based on the network structure properties, the correlation value of each node in the preset molecular reaction network is calculated; and based on the threshold and the correlation value, the target node is screened from each node.

[0078] The network structure attributes may include at least one of the following: network node degree, network path length, network centrality, and network module. The relevant values ​​of each node may include intra-module connectivity and inter-module connectivity. The threshold value may be determined based on actual analysis.

[0079] For example, network structural properties can be calculated using Gephi, Cytoscape, or other publicly available software packages such as igraph. The intra-module connectivity (z) and inter-module connectivity (p) values ​​of each node in the preset molecular reaction network are calculated according to the following equations (1) and (2), respectively.

[0080]

[0081]

[0082] Among them, k ib It can be expressed as the number of connections between node i and other nodes in module b, k b and It can be expressed as the mean and standard deviation of module b’s connectivity, k i It can be expressed as the number of connections between node i and other nodes in the entire network, k ic It can be expressed as the number of connections between node i and other nodes in module c, N M is the number of modules in the network.

[0083] You can z i ≤2.5 and p i ≤0.62 as a general node; z i ≤2.5 and p i >0.62 is considered an important connection node; z i >2.5 and p i ≤0.62 as an important node in the module, z i >2.5 and p i>0.62 as the important nodes of the entire network, and screen out the important nodes in the preset molecular reaction network (except general nodes, other nodes can be regarded as important nodes).

[0084] According to an embodiment of the present invention, if the preset network is a preset co-metabolism network between organic molecules and microorganisms, the initial microorganisms obtained through third-party operation during the target biological treatment process can be obtained; based on the initial microorganisms and metabolic microorganisms, the target nodes are screened from the various nodes of the preset co-metabolism network between organic molecules and microorganisms.

[0085] The third-party operation can be a human experimental operation in the target biological treatment process. Metabolic microorganisms can be determined based on the metabolic microorganisms involved in the microbial metabolic pathways in the target biological treatment process through the whole genome and metabolic pathway database.

[0086] For example, based on the initial microorganisms (e.g., 16S rRNA, metagenome), the microorganisms present in the preset co-metabolism network between organic molecules and microorganisms can be retained. Nodes related to the microorganism can be selected from the various nodes in the preset co-metabolism network between organic molecules and microorganisms as target nodes.

[0087] According to an embodiment of the present invention, if the preset network is a preset molecular reaction network and a preset co-metabolism network between organic molecules and microorganisms, a first target node can be selected from multiple nodes based on preset conditions for the preset molecular reaction network. Then, a second target node can be selected from multiple nodes based on preset conditions for the preset co-metabolism network between organic molecules and microorganisms. The first target node and the second target node are then combined to obtain a target node.

[0088] In operation S106 , a microbial metabolic molecular marker in the target biological treatment process is determined according to the target node.

[0089] According to embodiments of the present invention, the target node can be determined as a molecular marker of microbial metabolism in the target biological treatment process. Furthermore, the response relationships between potential molecular markers and the operational performance of the biological reaction process under different time series can be further combined to ultimately verify the target node and determine the molecular marker of microbial metabolism in the biological reaction process.

[0090] According to an embodiment of the present application, a plurality of organic molecule pairs of microorganism metabolites in a preset molecular mass interval determined from a target biological treatment process are formed by using high-resolution mass spectrometry; a first target organic molecule pair is screened from the plurality of organic molecule pairs according to first molecular mass difference information determined from the organic molecule pairs and second molecular mass difference information determined from a whole genome and metabolic pathway database; and a target node is screened from a preset network including a plurality of nodes constructed according to the first target organic molecule pair, so as to determine a microorganism metabolic molecule marker in the target biological treatment process. Based on the high resolution and accuracy of high-resolution mass spectrometry, the complex microorganism metabolic molecules in the biological treatment process are accurately identified; based on the preset network including a plurality of nodes, the target node is quickly identified from thousands or tens of thousands of metabolic molecules in the biological reaction process, and the molecule corresponding to the target node is taken as the microorganism metabolic molecule marker in the biological treatment process, thereby providing a new method for early warning indicator discrimination and intelligent monitoring of the microorganism metabolic changes in the biological treatment process.

[0091] It should be noted that the microorganism metabolic molecule marker identification method in the biological treatment process provided by the present application can realize software automation of the entire process by using a programming language, that is, the plurality of organic molecules of microorganism metabolites in the preset molecular mass interval determined from the high-resolution mass spectrometry are taken as input, and the microorganism metabolic molecule marker in the target biological treatment process is automatically output, so as to quickly lock the target and save time and experimental cost.

[0092] According to an embodiment of the present application, the microorganism metabolic molecule marker identification method in the biological treatment process can further include:

[0093] According to the whole genome and metabolic pathway database, metabolic molecule reaction information in a metabolic pathway of microorganisms in the target biological treatment process is determined; according to the metabolic molecule reaction information, a metabolic molecule reaction pair is determined, wherein the metabolic molecule reaction pair includes a metabolic molecule reactant and a metabolic molecule product; and second molecular mass difference information is determined according to the metabolic molecule reaction pair.

[0094] According to an embodiment of the present application, a script can be written by using a language such as R / Python, and metabolic molecule reactants and products of all metabolic reaction processes involved in the metabolic pathway of microorganisms in the target biological treatment process can be obtained and sorted by using a KEGGREST program package. The reactant molecules and product molecules are used to form a reaction pair between the reactant molecules and the product molecules. According to the respective molecular masses in the reaction pair between the reactant molecules and the product molecules, a difference is calculated to obtain the second molecular mass difference information.

[0095] According to an embodiment of the present application, based on metabolic reactions of known relevant metabolic pathways in the target biological treatment process, a metabolite molecular reaction pair mass difference relationship is constructed, which is applicable to different biological treatment processes and different metabolic molecular reaction networks are constructed differently.

[0096] According to an embodiment of the present application, the preset network can include a preset molecular reaction network and / or a preset co-metabolic network between organic molecules and microorganisms.

[0097] In the case where the preset network includes the preset molecular reaction network, the preset molecular reaction network is constructed with the first target organic molecule in the first target organic molecule pair as a node and the molecular mass difference of the first target organic molecule pair as an edge.

[0098] In the case where the preset network includes the preset molecular reaction network, the preset molecular reaction network is constructed with the first target organic molecule in the first target organic molecule pair as a node and the molecular mass difference of the first target organic molecule pair as an edge.

[0099] In the case where the preset network includes the preset co-metabolic network between organic molecules and microorganisms, a second target organic molecule is determined according to the metabolite molecule and the first target organic molecule in the first target organic molecule pair, wherein the metabolite molecule is determined according to the metabolic information of the metabolite molecule reaction in the metabolic pathway of the microorganism in the target biological treatment process in the whole genome and metabolic pathway database; a metabolic microorganism related to the second target organic molecule in the whole genome and metabolic pathway database is determined according to the second target organic molecule; and the preset co-metabolic network between organic molecules and microorganisms is constructed with the second target organic molecule and the metabolic microorganism as nodes and the relationship between the second target organic molecule and the metabolic microorganism as an edge.

[0100] It should be noted that in the case where the preset network includes the preset molecular reaction network and the preset co-metabolic network between organic molecules and microorganisms, the preset molecular reaction network and the preset co-metabolic network between organic molecules and microorganisms can be constructed respectively.

[0101] In the case where the preset network includes the preset molecular reaction network, the preset molecular reaction network is constructed with the first target organic molecule in the first target organic molecule pair as a node and the molecular mass difference of the first target organic molecule pair as an edge.

[0102] Constructing a co-metabolism network between a preset organic molecule and a microorganism can include: determining a second target organic molecule based on a first target organic molecule in a pair of a metabolite molecule and a first target organic molecule; determining a metabolic microorganism related to the second target organic molecule in a whole genome and metabolic pathway database based on the second target organic molecule; and constructing a co-metabolism network between the preset organic molecule and the microorganism with the second target organic molecule and the metabolic microorganism as nodes and the relationship between the second target organic molecule and the metabolic microorganism as an edge, wherein the metabolite molecule is determined based on the metabolite molecule reaction information in the microbial metabolic pathway during the target biological treatment process determined based on the whole genome and metabolic pathway database.

[0103] According to an embodiment of the present invention, by constructing a preset network, it is beneficial to accurately identify molecular markers of microbial metabolism in the biological treatment process based on the preset network, providing a new method for early warning indicator discrimination and intelligent monitoring of changes in microbial metabolism in the biological treatment process.

[0104] According to an embodiment of the present invention, screening a target node from a plurality of nodes according to a preset condition may include:

[0105] When it is determined that the preset network includes a preset molecular reaction network, a topological analysis is performed on the preset molecular reaction network to determine the network structure properties of the preset molecular reaction network; based on the network structure properties, the correlation value of each node in the preset molecular reaction network is calculated; and based on the threshold and the correlation value, the target node is screened from each node.

[0106] When it is determined that the preset network includes a preset co-metabolism network between organic molecules and microorganisms, initial microorganisms obtained through third-party operations in the target biological treatment process are obtained; based on the initial microorganisms and metabolic microorganisms, target nodes are screened from various nodes of the preset co-metabolism network between organic molecules and microorganisms.

[0107] According to an embodiment of the present invention, network structure attributes may include at least one of the following: network node degree, network path length, network centrality, and network module. The relevant values ​​of each node may include intra-module connectivity and inter-module connectivity. Thresholds may be determined based on actual analysis.

[0108] According to an embodiment of the present invention, the third-party operation can be a human experimental operation in the target biological treatment process. The metabolic microorganisms can be determined based on the metabolic microorganisms involved in the microbial metabolic pathways in the target biological treatment process using a whole genome and metabolic pathway database.

[0109] According to an embodiment of the present invention, based on constructing a preset molecular reaction network combined with network structure topology analysis or based on constructing a preset co-metabolism network between organic molecules and microorganisms, it is possible to quickly identify target node molecules among thousands or tens of thousands of metabolic molecules in the biological reaction process.

[0110] According to an embodiment of the present invention, when it is determined that the preset network includes a preset co-metabolism network between organic molecules and microorganisms, the method of constructing the preset network based on the first target organic molecule pair may further include:

[0111] Use language to write scripts, combined with preset program packages, to obtain metabolite molecular reaction information in the microbial metabolic pathway during the target biological treatment process from the whole genome and metabolic pathway database; and identify metabolite molecules from the metabolite molecular reaction information.

[0112] According to an embodiment of the present invention, a script can be written in R / Python language and combined with the KEGGREST package to obtain metabolite molecular reaction information in the microbial metabolic pathway of the target biological treatment process from the whole genome and metabolic pathway database.

[0113] According to an embodiment of the present invention, language-based script writing and real-time online interactive search with the KEGG database are beneficial for synchronization with the latest information in the KEGG database, and as the microbial metabolic pathways in the KEGG database are continuously updated and improved, it helps to realize the identification of new molecular markers.

[0114] According to an embodiment of the present invention, when it is determined that the preset network includes a preset co-metabolism network between an organic molecule and a microorganism, determining the second target organic molecule based on the first target organic molecule in the pair of the metabolite molecule and the first target organic molecule may include:

[0115] A script is written using a language to match the molecular mass of the first target organic molecule with the molecular mass of the metabolite molecule; and the first target organic molecule whose molecular mass matches the molecular mass of the metabolite molecule is used as the second target organic molecule.

[0116] According to an embodiment of the present invention, a script can be written in a language such as R or Python to match the molecular mass of the first target organic molecule (to three decimal places) with the molecular mass of the metabolite molecule. If the matching results are consistent, the corresponding first target organic molecule can be used as the second target organic molecule.

[0117] According to an embodiment of the present invention, using a language to write a script to determine the second target organic molecule is conducive to constructing a co-metabolism network between the preset organic molecule and the microorganism, and further helps to accurately identify the target metabolite molecules known in the database during the microbial biological reaction process based on the preset co-metabolism network between the organic molecule and the microorganism.

[0118] According to an embodiment of the present invention, based on the initial microorganisms and the metabolizing microorganisms, screening target nodes from various nodes of a preset co-metabolism network between organic molecules and microorganisms may include:

[0119] The initial microorganisms and the metabolic microorganisms are matched to obtain matched microorganisms; and based on the matched microorganisms, nodes related to the matched microorganisms are screened from various nodes of the preset co-metabolism network between organic molecules and microorganisms as target nodes.

[0120] According to an embodiment of the present invention, based on a preset co-metabolism network between organic molecules and microorganisms, nodes in the database related to known initial microorganisms are accurately identified, thereby realizing rapid non-target screening of microbial metabolic molecular markers from complex biological treatment processes, and providing a new method for the early warning indicator discrimination and intelligent monitoring of microbial metabolic changes in biological treatment processes.

[0121] According to an embodiment of the present invention, using high-resolution mass spectrometry to determine multiple organic molecules of microbial metabolites within a preset molecular mass range from a target biological process may include:

[0122] Obtain dissolved organic matter obtained through third-party operations during the target biological treatment process; use high-resolution mass spectrometry to test the dissolved organic matter to obtain mass spectrometry test results within a preset molecular mass range, wherein the mass spectrometry test results include multiple high-resolution mass spectrometry peaks; and according to a preset analysis method, perform molecular formula matching on each high-resolution mass spectrometry peak to obtain multiple organic molecules.

[0123] According to an embodiment of the present invention, a sample from a target biological process can be collected and then enriched, separated, and purified using pre-treatment methods such as electrodialysis and solid phase extraction to extract dissolved organic matter from the sample. The pre-set analysis method can include computing software or program packages such as Data Analysis (DA) or TRFu.

[0124] For example, the method for extracting soluble organic matter can be to use commercially purchased or laboratory-filled solid-phase extraction columns to enrich the soluble organic matter in the sample and separate it from other inorganic ions and other components in the sample. The enrichment, separation and purification process includes treatment methods such as pH adjustment, organic solvent elution, water and nitrogen stripping or low-temperature drying. The purification effect of organic matter can also be improved by electrodialysis and other methods to increase the recovery rate of organic matter in the extraction process.

[0125] According to embodiments of the present invention, high-resolution mass spectrometry can employ, but is not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICS MS) and UPLC-Q-Orbitrap, among other high-resolution mass spectrometry methods. During retesting, high-resolution mass spectrometry analysis parameters, such as instrument parameters such as positive and negative ion modes, can be optimized and adjusted based on sample source and composition characteristics.

[0126] According to the embodiments of the present invention, compared with commonly used visible light, fluorescence, near-infrared and other spectral methods, the high resolution and accuracy of high-resolution mass spectrometry are used to achieve accurate and non-target identification of complex microbial metabolic molecules in biological reaction processes.

[0127] According to an embodiment of the present invention, screening a first target organic molecule pair from a plurality of organic molecule pairs based on the first molecular mass difference information and the second molecular mass difference information may include:

[0128] For each organic molecule pair:

[0129] Matching the first molecular mass difference information and the second molecular mass difference information to obtain a matching result; and when it is determined that the first molecular mass difference information and the second molecular mass difference information are consistent in the matching result, taking the organic molecule pair as a first target organic molecule pair.

[0130] According to an embodiment of the present invention, the matching error may be ±0.00001. After performing the operation on all organic molecule pairs, a set of first target organic molecule pairs may be obtained.

[0131] According to an embodiment of the present invention, based on the metabolic reactions of the known related metabolic pathways in the target biological treatment process, the mass difference relationship of the metabolite molecular reaction is specifically constructed, which is applicable to differentially constructing different metabolic molecular reaction networks in different biological treatment processes.

[0132] The following is a more specific example to illustrate the method for identifying microbial metabolic molecular markers in a biological treatment process provided by the present invention. It should be noted that the example is only a specific embodiment of the present invention and does not limit the scope of protection of the present invention.

[0133] Example 1

[0134] This Example 1 provides a method for identifying microbial metabolic molecular markers in wastewater / waste biological treatment processes. Specific operations may include operations S1 to S7:

[0135] In operation S1, samples were collected from the anaerobic digestion reactor and the dissolved organic matter was enriched, separated and purified in the laboratory using solid phase extraction.

[0136] In operation S2, the organic molecules in the extracted dissolved organic matter were tested using FT-ICR MS in negative ion mode, and the results were as follows: Figure 2 (a) shows the mass spectrometry test results.

[0137] In operation S3 , the molecular formula represented by each mass spectrum peak is matched using computing software such as Data Analysis (DA) or TRFu or related software such as a program package.

[0138] In operation S4, a code is written in R language to obtain the molecular reaction relationship under the metabolic pathway involved in the anaerobic digestion biological reaction process in the KEGG database, and the mass difference of the molecular reaction pair is calculated.

[0139] In operation S5, the R language was used to write a code to calculate the mass difference between each two molecules in the molecular formula obtained by mass spectrometry test, and match it with the mass difference of the molecular reaction pair in operation S4. The tolerance was set to 0.00001, and the reaction relationship between the molecules in the molecular formula obtained by mass spectrometry test was screened to construct the following Figure 2 (b) The molecular reaction network shown.

[0140] In operation S6, R language is used to write code, and the igraph package is used to calculate the network topology attributes and calculate the screening target molecular nodes, such as Figure 2 (c) and combined with database search to explore the reaction relationship between target molecular nodes, such as Figure 2 (d) shown.

[0141] In operation S7, R language was used to write code to interactively search the KEGG database for microorganisms related to each molecular node, and compared with the microbial community in the anaerobic digestion reactor measured by 16s rRNA to establish a Figure 3 The co-metabolism network between organic molecules and microorganisms is shown, that is, the connection relationship between microorganisms and organic molecules in the reactor. Based on this connection relationship, it is further determined that there are some special organic molecules in the reactor, namely Figure 3 Some organic molecules (such as C 11 H 22 NO7PS、C 14 H 18 N4O3、C 13H 18 O7、C 17 H 20 N4O6、C9H 16 N5O8P, etc.). These organic molecules are important molecules for co-metabolism between microorganisms because they are connected with multiple microorganisms. They are considered to be a type of microbial metabolic molecular marker.

[0142] Through the above operations, the potential microbial metabolic molecular markers shown in Table 1 can be obtained. According to the temporal relationship between the methane production efficiency and the molecular markers during the anaerobic digestion process, as well as the existing theoretical knowledge, it can be judged that C 11 H 22 NO7PS molecule (Coenzyme B) is an important molecular marker indicating the metabolic process of anaerobic digestion microorganisms. It is a coenzyme for anaerobic digestion and methanogenesis and is directly related to the amount of methane produced.

[0143] Table 1

[0144]

[0145]

[0146] Example 2

[0147] In Example 2, samples were collected from the aerobic composting process, and solid phase extraction combined with electrodialysis pretreatment was used to enrich, separate, and purify the dissolved organic matter; other operations were the same as in Example 1.

[0148] Example 3

[0149] In Example 3, samples were collected from a biochemical reactor in a sewage treatment plant and tested using FT-ICR MS in positive and negative ion modes to obtain mass spectrometry results. The molecular formulas in the two modes were then matched and merged for subsequent analysis. Other operations were the same as in Example 1.

[0150] Example 4

[0151] Example 4: Samples were collected from an organic waste dry anaerobic digestion reactor. After pretreatment, the samples were analyzed using a UPLC-Q-Orbitrap mass spectrometer to obtain mass spectrometry results. Other operations were the same as in Example 1.

[0152] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for identifying molecular markers of microbial metabolism in a biological treatment process, comprising: Using high-resolution mass spectrometry, multiple organic molecules of microbial metabolites within a preset molecular mass range are identified from the target biological process; pairing every two of the organic molecules to form a plurality of organic molecule pairs; screening a first target organic molecule pair from a plurality of organic molecule pairs according to first molecular mass difference information and second molecular mass difference information, wherein the first molecular mass difference information is determined based on the organic molecule pair, and the second molecular mass difference information is determined based on a whole genome and metabolic pathway database; Constructing a preset network according to the first target organic molecule pair, wherein the preset network includes a plurality of nodes; Filtering a target node from the plurality of nodes according to a preset condition; Determining, according to the target node, a molecular marker of microbial metabolism in the target biological treatment process; Determining metabolite molecular reaction information in the microbial metabolic pathway during the target biological treatment process based on the whole genome and metabolic pathway database; Determining a metabolic molecule reaction pair according to the metabolite molecule reaction information, wherein the metabolic molecule reaction pair includes a metabolic molecule reactant and a metabolic molecule product; determining the second molecular mass difference information according to the metabolic molecule reaction pair; Wherein, the preset network includes a preset molecular reaction network and / or a preset co-metabolism network between organic molecules and microorganisms; The step of constructing a preset network based on the first target organic molecule pair includes: When it is determined that the preset network includes the preset molecular reaction network, the preset molecular reaction network is constructed using the first target organic molecule in the first target organic molecule pair as a node and the molecular mass difference of the first target organic molecule pair as an edge; When it is determined that the preset network includes a co-metabolism network between the preset organic molecule and the microorganism, determining a second target organic molecule based on a metabolite molecule and a first target organic molecule in the pair of the first target organic molecule, wherein the metabolite molecule is determined based on metabolite molecule reaction information in the microbial metabolic pathway in the target biological treatment process determined based on the whole genome and metabolic pathway database; Determining, based on the second target organic molecule, metabolic microorganisms associated with the second target organic molecule in the whole genome and metabolic pathway database; and The second target organic molecule and the metabolizing microorganism are used as nodes, and the relationship between the second target organic molecule and the metabolizing microorganism is used as an edge to construct a co-metabolism network between the preset organic molecule and the microorganism.

2. The method according to claim 1, wherein The step of screening a target node from the plurality of nodes according to a preset condition includes: When it is determined that the preset network includes the preset molecular reaction network, performing a topological analysis on the preset molecular reaction network to determine a network structure property of the preset molecular reaction network; Calculating the correlation value of each node in the preset molecular reaction network according to the network structure attributes; Filtering the target node from each node according to the threshold and the correlation value; When it is determined that the preset network includes the preset co-metabolism network between the organic molecules and the microorganisms, obtaining initial microorganisms obtained through a third-party operation during the target biological treatment process; The target node is screened from various nodes of the preset co-metabolism network between the organic molecules and the microorganisms according to the initial microorganisms and the metabolizing microorganisms.

3. The method according to claim 1, wherein In the case where it is determined that the preset network includes the preset co-metabolism network between organic molecules and microorganisms, The method further comprises: Using a language to write a script, combined with a preset program package, to obtain metabolite molecular reaction information in the microbial metabolic pathway of the target biological treatment process from the whole genome and metabolic pathway database; and The metabolite molecule is identified from the metabolite molecule reaction information.

4. The method according to claim 1, wherein, when it is determined that the preset network includes a co-metabolism network between the preset organic molecule and the microorganism, determining the second target organic molecule based on the first target organic molecule in the pair of metabolite molecule and the first target organic molecule comprises: Using a language to write a script to match the molecular mass of the first target organic molecule with the molecular mass of the metabolite molecule; as well as The first target organic molecule whose molecular mass matches the molecular mass of the metabolite molecule is used as the second target organic molecule.

5. The method according to claim 2, wherein: The step of screening the target node from various nodes of the preset co-metabolism network between organic molecules and microorganisms based on the initial microorganisms and the metabolizing microorganisms includes: Matching the initial microorganism and the metabolizing microorganism to obtain a matched microorganism; and According to the matched microorganism, a node related to the matched microorganism is screened from each node of the preset co-metabolism network between organic molecules and microorganisms as the target node.

6. The method according to claim 2, wherein: The network structure attributes include at least one of the following: degree of network nodes, network path length, network centrality, and network module.

7. The method according to claim 1, wherein The method of using high-resolution mass spectrometry to determine multiple organic molecules of microbial metabolites within a preset molecular mass range from a target biological treatment process includes: Obtaining dissolved organic matter obtained during the target biological treatment process through third-party operations; Using the high-resolution mass spectrometer, testing the dissolved organic matter to obtain a mass spectrometry test result within the preset molecular mass range, wherein the mass spectrometry test result includes a plurality of high-resolution mass spectrometry peaks; and According to a preset analysis method, molecular formula matching is performed on each of the high-resolution mass spectrum peaks to obtain a plurality of the organic molecules.

8. The method according to claim 1, wherein The step of screening a first target organic molecule pair from a plurality of organic molecule pairs according to the first molecular mass difference information and the second molecular mass difference information includes: For each organic molecule pair: Matching the first molecular mass difference information with the second molecular mass difference information to obtain a matching result; and When it is determined that the first molecular mass difference information and the second molecular mass difference information are consistent in the matching result, the organic molecule pair is used as the first target organic molecule pair.

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