Method for identifying strains participating in synthesis process of medium-chain organic acid in anaerobic fermentation and metabolic potential of strains
By using a combined platform of enrichment of functional microorganisms-metagenomics-box assembly-comparative genomics, anaerobic fermentation conditions were optimized, the metabolic potential of carbon chain elongation microorganisms was identified and evaluated, the problem of difficulty in enriching carbon chain elongation microorganisms was solved, and the efficiency of medium chain fatty acid production was improved.
Patent Information
- Application Number
- CN202511363615.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-23
- Publication Date
- 2025-12-23
AI Technical Summary
In anaerobic fermentation, the enrichment of carbon chain elongated microorganisms is difficult, which limits the efficient production of medium-chain fatty acids. Existing technologies are unable to effectively identify and utilize the metabolic potential of these microorganisms.
Using a combined platform of enriched functional microorganisms-metagenomics-box assembly-comparative genomics, we identified and evaluated the metabolic potential of carbon chain elongation functional microorganisms by optimizing the molar ratio of electron donors and acceptors and pH conditions, combined with DNA extraction and sequencing analysis.
It has enabled the enrichment of carbon chain elongation functional microorganisms from anaerobic bacteria, providing a reference for regulating the fermentation conditions of organic waste, improving the production efficiency of medium-chain fatty acids, and is suitable for processing samples of unknown species that are difficult to cultivate.
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Figure CN121182931A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for identifying microorganisms with carbon chain elongation functions. More specifically, it relates to a method for identifying microbial species and their metabolic potential involved in the synthesis of medium-chain organic acids during the anaerobic fermentation of organic waste. Background Technology
[0002] In recent years, with the rapid growth of the global economy and the increasing scarcity of fossil fuels, the world's demand for and dependence on energy has surged, leading to a significant focus on renewable energy and chemicals. Simultaneously, to achieve sustainable social development and maintain the health and stability of ecosystems, the efficient and stable degradation and further resource recycling of organic waste has become a research hotspot. Based on anaerobic microbial systems, this type of organic waste can be stably degraded and transformed into fuels and chemicals.
[0003] Under the action of anaerobic microorganisms, complex organic matter is first converted into intermediate products—short-chain fatty acids, which can then be further converted into methane. Compared with conventional anaerobic products, medium-chain fatty acids have longer carbon chains, making them more hydrophobic and easier to separate and extract; moreover, they have higher economic value and higher energy density, making them of great significance in both scientific research and practical applications.
[0004] Fermentation of organic waste requires the synergistic action of multiple microbial species. For example, fermenting bacteria transform macromolecules into monomers, which are then utilized by acid-producing bacteria to produce short-chain fatty acids. At this point, carbon chain elongating microorganisms can utilize various electron donors (such as ethanol, lactic acid, and syngas) to provide energy, thereby elongating the carbon chain of electron acceptors and ultimately obtaining medium-chain fatty acids.
[0005] Because carbon-chain elongating microorganisms have fewer substrates available than other bacteria, they are more susceptible to the effects of fermentation conditions. This makes the enrichment of carbon-chain elongating microorganisms difficult, greatly limiting the practical application of this technology. Therefore, it is necessary to identify carbon-chain elongating functional microorganisms in different anaerobic systems. Summary of the Invention
[0006] Based on the above-mentioned technical problems, the present invention provides a method for identifying microbial species and their metabolic potential involved in the synthesis of medium-chain organic acids in the anaerobic fermentation of organic waste by using a platform that combines enriched functional microorganisms-metagenomics-box assembly-comparative genomics.
[0007] The present invention adopts the following technical solution: A method for identifying microbial species involved in the synthesis of medium-chain organic acids in anaerobic fermentation and their metabolic potential, comprising the following steps: (1) Pretreatment of mixed microbial community: The anaerobic microbial inoculum is pretreated to inhibit the activity of methanogenic archaea, and then the anaerobic microbial inoculum is inoculated into an inorganic culture medium and cultured anaerobically until the background organic matter of the inoculum is consumed to obtain the inoculated microorganisms; (2) Shaking culture: The inoculated microorganisms in step (1) are inoculated into a reactor containing different molar ratios of electron donors and electron acceptors and pH and cultured until the concentration of all substances no longer changes; (3) Enrichment of carbon chain elongation microorganisms: Based on the yield and selectivity of medium-chain organic acids, the best reaction conditions are selected to obtain a group of bacteria with carbon chain elongation ability, that is, enriched carbon chain elongation functional microorganisms. (3) DNA extraction: Extract total microbial DNA from the enriched carbon chain elongation functional microorganisms; (4) Metagenomics: Sequencing the obtained total DNA to obtain raw metagenomic data of carbon chain elongation samples; (5) Identification of microorganisms with carbon chain elongation function: By analyzing metagenomic data and combining the optimal reaction conditions for carbon chain elongation, microorganisms with carbon chain elongation function can be identified; (6) Comparative genomics analysis: Comparative genomics analysis was performed on the identified carbon chain elongation functional microorganisms and known carbon chain elongation functional microorganisms to assess the metabolic potential of the identified carbon chain elongation functional microorganisms.
[0008] This invention achieves the enrichment of carbon chain elongation functional microorganisms from anaerobic bacterial communities by optimizing the molar ratio of electron donors and electron acceptors and pH conditions. It also combines a metagenomics-box assembly-comparative genomics platform to identify microbial species involved in the synthesis of medium-chain organic acids in the anaerobic fermentation of organic waste and their metabolic potential.
[0009] Furthermore, in step (1), the anaerobic microbial inoculum is a mixed microbial community, and the pretreatment is high-pressure steam sterilization at 100-120℃ for 30-60 min and the addition of 50-100 mM sodium 2-bromoethanesulfonate.
[0010] Further, in step (1), the composition of each 1L of the inorganic culture medium is as follows: NH4Cl, 1g / L; NaCl, 0.1g / L; MgCl2, 0.1g / L; anhydrous CaCl2, 0.05g / L; K2HPO4, 0.4g / L; FeCl2, 0.002g / L; H3BO3, 0.1mg / L; CoCl2, 0.1mg / L; ZnCl2, 0.1mg / L; NiCl2, 0.1mg / L; CuCl2, 0.06mg / L; ethylenediaminetetraacetic acid, 0.001g / L; MnCl2, 0.1mg / L; H2SeO4, 0.1mg / L; AlCl3, 0.1mg / L; the balance is water.
[0011] Furthermore, in step (1), the anaerobic culture is carried out by purging with nitrogen and sealing during the culture process.
[0012] Furthermore, in step (2), the pH of each reactor group is set to 5-10, the molar ratio of electron donor to electron acceptor is 2-10, and the COD load is 50 g / L.
[0013] Furthermore, in step (2), the electron donor is one of ethanol, lactic acid, methanol, propanol, butanol, pyruvic acid, amino acid, polypeptide, sweet alcohol, sugar, carbon monoxide, and hydrogen; the electron acceptor is one or more of formic acid, acetic acid, propionic acid, butyric acid, and valeric acid.
[0014] Furthermore, in step (4), the raw metagenomic data of the obtained carbon chain elongation sample is subjected to data quality control to remove low-quality reads.
[0015] Furthermore, in step (5), the analysis of metagenomic data includes: splicing the quality-controlled sequence data to obtain contigs, detecting the size and quality of the contigs, then binning the contigs to assemble them, removing redundancy and extracting MAGs, detecting the integrity and contamination of MAGs, performing biological classification and gene prediction on high-quality MAGs, and calculating the relative abundance of MAGs.
[0016] Furthermore, in step (5), identifying microorganisms with carbon chain elongation function specifically includes: 1) Focus on MAGs with an integrity level >50% and a contamination level <10%; 2) Focus on MAGs with a relative abundance > 0.8; 3) Select MAGs containing genes related to acetyl-CoA synthesis and the reverse β-oxidation pathway; 4) Among the selected MAGs, those with complete energy metabolism are microorganisms that have the ability to produce medium-chain organic acids by extending carbon chains.
[0017] Furthermore, in step (6), assessing the metabolic potential of the identified carbon chain elongation functional microorganisms specifically includes: 1) Download the complete gene sequences of related microorganisms and known microorganisms with carbon chain elongation functions from NCBI; 2) Perform a genome-wide comparison between the identified MAGs and the downloaded gene sequences; 3) Based on the presence or absence of key functional genes, determine the metabolic potential of identified MAGs to achieve carbon chain elongation for different electron donors.
[0018] The present invention has the following beneficial effects: This invention provides a method for identifying microbial species involved in the synthesis of medium-chain organic acids (MCA) during anaerobic fermentation and their metabolic potential. The process involves extracting total DNA from a reactor enriched with functional microorganisms, sequencing to obtain raw metagenomic data from the carbon chain elongation reactor, analyzing the metagenomic results to identify microorganisms with carbon chain elongation functions, and finally using comparative genomics to elucidate the metabolic potential of the identified functional microorganisms. This provides a reference for regulating fermentation conditions in the production of MCA from organic waste and allows for biofortification using the identified functional microorganisms to achieve efficient production of MCA. The entire process does not rely on traditional cell culture techniques and is suitable for handling samples of unknown species that are difficult to culture. Attached Figure Description
[0019] Figure 1 The changes in organic acids at different pH values are given when the molar ratio of lactic acid to butyric acid is 2, where AD is at pH=5.5, pH=7, pH=6, and pH=9.5 respectively.
[0020] Figure 2 The changes in organic acids at different pH values are given when the molar ratio of lactic acid to butyric acid is 3, where A and D are respectively pH=5.5, pH=7, pH=6, and pH=9.5.
[0021] Figure 3 For carbon chain elongation microorganisms identified based on metagenomics-binding assembly.
[0022] Figure 4 To predict the metabolic potential of functional microorganisms based on comparative omics. Detailed Implementation
[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.
[0024] Example 1 A method for identifying microbial species involved in the synthesis of medium-chain organic acids in anaerobic fermentation and their metabolic potential, comprising the following steps: (1) Add 20 mL of anaerobic inoculum and 180 mL of inorganic culture medium after high-pressure steam sterilization at 120℃ for 1 h to the anaerobic reactor. Maintain the temperature at 35℃. After the organic matter is consumed, the inoculated microorganism is obtained.
[0025] The anaerobic inoculum was taken from sludge from the cassava alcohol wastewater treatment plant in Taicang, Jiangsu.
[0026] The composition of each 1L of the inorganic culture medium is as follows: NH4Cl, 1g / L; NaCl, 0.1g / L; MgCl2, 0.1g / L; anhydrous CaCl2, 0.05g / L; K2HPO4, 0.4g / L; FeCl2, 0.002g / L; H3BO3, 0.1mg / L; CoCl2, 0.1mg / L; ZnCl2, 0.1mg / L; NiCl2, 0.1mg / L; CuCl2, 0.06mg / L; ethylenediaminetetraacetic acid, 0.001g / L; MnCl2, 0.1mg / L; H2SeO4, 0.1mg / L; AlCl3, 0.1mg / L; the balance is water.
[0027] (2) The microorganisms obtained in step (1) were divided into 6 groups, using lactic acid as an electron donor and butyric acid as an electron acceptor, with a total organic load of 50 g COD / L. The molar ratio of lactic acid to butyric acid in the first, third and fifth groups was 2, and the pH was set to 6, 7 and 9.5, respectively; the molar ratio of lactic acid to butyric acid in the second, fourth and sixth groups was 3, and the pH was set to 6, 7 and 9.5, respectively; 100 mM sodium 2-bromoethanesulfonate was added to inhibit methanogenic bacteria, the serum bottle reactor was purged with nitrogen for 5 minutes, and an anaerobic environment was created by sealing with butyl rubber stoppers and aluminum rolled edges. All reactors were made in triplicate and placed in a constant temperature shaker at 35℃ and 150 r / min until the concentration of all substances no longer changed.
[0028] (3) Repeat step (2) 5 times to obtain the enrichment of microorganisms that can utilize lactic acid and butyric acid under different conditions, such as Figure 1 and Figure 2 As shown, at pH 9.5, the carbon chain elongation process is almost impossible, producing only low concentrations of valeric acid and hexanoic acid (57.2±2.9 and 16.3±0.5 mM C, respectively). The optimal conditions for the production of medium-chain fatty acids from lactic and butyric acids are pH 6 and a lactic acid / butyric acid ratio of 2, resulting in the highest concentration and selectivity of hexanoic acid (796±23 mM C and 64%, respectively).
[0029] (4) Extract the total microbial DNA from the microorganisms enriched in step (3).
[0030] (5) Sequencing the total DNA obtained in step (4) to obtain the metagenomic raw base sequence (reads) data of the carbon chain elongation sample.
[0031] (6) Identify microorganisms with carbon chain elongation function: By analyzing metagenomic data and combining the optimal reaction conditions for carbon chain elongation, identify microorganisms with carbon chain elongation function.
[0032] (7) Comparative genomics analysis: Comparative genomics analysis was performed on the identified carbon chain elongation functional microorganisms and known carbon chain elongation functional microorganisms to assess the metabolic potential of the identified carbon chain elongation functional microorganisms.
[0033] The identification of microorganisms with carbon chain elongation function includes the following steps: Step 1: Use FastQC software to assess the quality of the DNA sequence, and use Trimmomatic and bbduk software for data quality control to remove low-quality reads.
[0034] Step 2: Use Megahit software to splice the quality-controlled sequence data to obtain contigs, and use Quast software to detect the size and quality of the contigs.
[0035] Step 3: Binning assembly of contigs was performed using bowtie2, CONCOCT, Metabat2, MetaBAT1, VAMB, MaxBin2, and DAS_Tool. Redundancy was removed using dRep to extract metagenomics and assemble genomes (MAGs). The integrity and contamination level of MAGs were detected using checkM. In this example, a total of 227 MAGs were obtained.
[0036] Step 4: Perform biological classification of high-quality MAGs using GTDB-Tk and CAT / BAT, and calculate the relative abundance of MAGs using bowtie2, checkM coverage, and checkM profile.
[0037] Step 5: Combine COG, KEGG, Pfam, dbCAN, and use Prodigal, GhostKOALA, KofamKOALA, and eggNOG mapper to annotate the MAGs assembled data.
[0038] Step 6: Based on the metagenomic data analysis results, identify microorganisms with carbon chain elongation function through the following steps: Step 1: Focus on MAGs with an integrity level >50% and a contamination level <10%.
[0039] Step 2: Focus on MAGs with relatively high abundance (>0.8). Based on the above criteria, this embodiment yields 44 MAGs ( Figure 3 ).
[0040] Step 3: Select MAGs containing genes related to acetyl-CoA synthesis (LDH, iLDH, por) and reverse β-oxidation pathway (Thl, BHBD, Crt, Cat) in a reaction system with pH 6 and a molar ratio of lactic acid to butyric acid of 2.
[0041] Step 4: Among the selected MAGs, those with complete energy metabolism (EtfA-B; RnfA-E) are microorganisms capable of producing medium-chain organic acids through carbon chain elongation. Based on the above criteria, Clostridium sp. BUCT163 can be identified as a lactate-driven carbon chain elongation functional microorganism.
[0042] Predicting the metabolic potential of carbon chain elongation functional microorganisms includes the following steps: Step 1: In this embodiment, the lactic acid-driven carbon chain elongation functional microorganisms are classified into the genus […]. Clostridium Currently belonging to Clostridium Only in China C. kluyveri It is an ethanol-driven, carbon chain elongation-functional microorganism, therefore downloaded from NCBI. C.kluyveri The complete gene sequence of the bacterial strain, i.e. C.kluyveri NBRC 12016 , C. kluyveri DSM 555 , C. kluyveri BJN0002, and C. kluyveri JZZ .
[0043] Step 2: Use Roray software to perform whole-genome comparisons between the identified MAGs and the four downloaded gene sequences.
[0044] Step 3: Based on the presence or absence of key functional genes (ADH, LDH, iLDH, por, Thl, BHBD, Crt, Cat), determine the metabolic potential of the identified MAGs for carbon chain elongation using different electron donors. C. kluyveri The four strains did not possess the ability to drive carbon chain elongation with lactate, while Clostridium sp. BUCT163 possesses the ability to drive carbon chain elongation simultaneously with lactic acid and ethanol, and this strain can be used for biofortification of systems where both lactic acid and ethanol drive carbon chain elongation. Figure 4 ).
[0045] Example 2 A method for identifying microbial species involved in the synthesis of medium-chain organic acids in anaerobic fermentation and their metabolic potential, comprising the following steps: (1) Add the anaerobic sludge (same as in Example 1) sterilized by high pressure steam at 120℃ for 1h and the inorganic culture medium into the anaerobic reactor. Keep the temperature at 35℃. After the organic matter is consumed, the inoculated microorganisms are obtained.
[0046] (2) The microorganisms obtained in step (1) were divided into 6 groups, using ethanol as an electron donor and acetic acid as an electron acceptor, with a total organic load of 50 g COD / L. The molar ratio of ethanol to acetic acid in the first, third and fifth groups was 10, and the pH was set to 6, 7 and 9.5, respectively; the molar ratio of ethanol to acetic acid in the second, fourth and sixth groups was 2, and the pH was set to 6, 7 and 9.5, respectively; 100 mM sodium 2-bromoethanesulfonate was added to inhibit methanogens, the serum bottle reactor was purged with nitrogen for 5 minutes, and an anaerobic environment was created by sealing with butyl rubber stoppers and aluminum rolled edges. All reactors were made in triplicate and placed in a constant temperature shaker at 35℃ and 150 r / min until the concentration of all substances no longer changed.
[0047] (3) Repeat step (2) 5 times to obtain the enrichment of microorganisms that can utilize ethanol and acetic acid under different conditions. The directional synthesis of medium-chain fatty acids can be achieved when the pH is 6 and the molar ratio of ethanol to acetic acid is 10.
[0048] (4) Extract the total microbial DNA from the microorganisms enriched in step (3).
[0049] (5) Sequencing the total DNA obtained in step (4) to obtain raw metagenomic data of carbon chain elongation samples.
[0050] (6) Identification of microorganisms with carbon chain elongation function: By analyzing metagenomic data and combining the optimal reaction conditions for carbon chain elongation, MAGs with genes related to acetyl-CoA synthesis (adh) and reverse β-oxidation pathways (Thl, BHBD, Crt, Cat) and complete energy metabolism (EtfA-B; RnfA-E) were selected. Microorganisms with carbon chain elongation function were identified as... Treponema BUCT126.
[0051] (7) Comparative genomics analysis: This included analyzing the identified functional microorganisms with elongated carbon chains and their genus classification on NCBI. Treponema Comparative genomic analysis of strains with complete genes showed that the key gene for carbon chain elongation is not genus-specific. Treponema The core genes.
[0052] Example 3 A method for identifying microbial species involved in the synthesis of medium-chain organic acids in anaerobic fermentation and their metabolic potential, comprising the following steps: (1) Add the anaerobic sludge (same as in Example 1) sterilized by high pressure steam at 120℃ for 1h and the inorganic culture medium into the anaerobic reactor. Keep the temperature at 35℃. After the organic matter is consumed, the inoculated microorganisms are obtained.
[0053] (2) Divide the microorganisms obtained in step (1) into 6 groups, using CO as an electron donor and acetic acid as an electron acceptor, with a CO partial pressure of 1.5 bar. The acetic acid concentration of the first, third and fifth groups was set to 100 mM, and the pH was set to 6, 7 and 9.5, respectively; the acetic acid concentration of the second, fourth and sixth groups was set to 50 mM, and the pH was set to 6, 7 and 9.5, respectively; 100 mM of sodium 2-bromoethanesulfonate was added to inhibit methanogens, the serum bottle reactor was purged with nitrogen for 5 minutes, and an anaerobic environment was created by sealing with butyl rubber stoppers and aluminum rolled edges. All reactors were made in triplicate and placed in a constant temperature shaker at 35℃ and 150 r / min until the concentration of all substances no longer changed.
[0054] (3) Repeat step (2) 3 to 5 times to obtain the enrichment of microorganisms that can utilize CO and acetic acid under different conditions. The directional synthesis of medium-chain fatty acids can be achieved at pH 7 and acetic acid concentration of 100 mM.
[0055] (4) Extract the total microbial DNA from the microorganisms enriched in step (3).
[0056] (5) Sequencing the total DNA obtained in step (4) to obtain raw metagenomic data of carbon chain elongation samples.
[0057] (6) Identification of microorganisms with carbon chain elongation function: By analyzing metagenomic data and combining the optimal reaction conditions for carbon chain elongation, MAGs with genes related to acetyl-CoA synthesis (fdh, fhs, cdh) and reverse β-oxidation pathways (Thl, BHBD, Crt, Cat) and complete energy metabolism (EtfA-B; RnfA-E) were selected; microorganisms with carbon chain elongation function were identified as... Eubacterium BUCT11.
[0058] (7) Comparative genomics analysis: This included analyzing the identified functional microorganisms with elongated carbon chains and their genus classification on NCBI. Eubacterium Comparative genomic analysis of strains with complete genes showed that... Eubacterium BUCT11 can utilize both CO and methanol as electron donors to achieve carbon chain extension.
[0059] This result reveals the potential of different electron donor-driven carbon chain elongation functional microorganisms and their metabolic potential in anaerobic fermentation systems, expanding the current understanding of the ecological functions and carbon chain elongation functional microorganism library in anaerobic fermentation systems.
[0060] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.
[0061] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A method for identifying microbial strains involved in the synthesis of medium-chain organic acids during anaerobic fermentation and their metabolic potential, characterized in that, Includes the following steps: (1) The anaerobic microbial inoculum is pretreated to inhibit the activity of methanogenic archaea, and then the anaerobic microbial inoculum is inoculated into an inorganic culture medium and cultured anaerobically until the background organic matter of the inoculum is consumed to obtain the inoculated microorganisms; (2) Inoculate the microorganisms from step (1) into reactors containing different molar ratios of electron donors and electron acceptors and different pH values and culture them until the concentrations of all substances no longer change; (3) Based on the yield and selectivity of medium-chain organic acids, the optimal reaction conditions are selected to obtain a group of bacteria with carbon chain elongation ability, that is, enriched carbon chain elongation functional microorganisms. (3) Extract total microbial DNA from the enriched carbon chain elongation functional microorganisms; (4) Sequencing the obtained total DNA to obtain raw metagenomic data of carbon chain elongation samples; (5) By analyzing metagenomic data and combining the optimal reaction conditions for carbon chain elongation, microorganisms with carbon chain elongation function can be identified; (6) Comparative genomic analysis was performed on the identified carbon chain elongation functional microorganisms and known carbon chain elongation functional microorganisms to assess the metabolic potential of the identified carbon chain elongation functional microorganisms.
2. The method according to claim 1, characterized in that, In step (1), the anaerobic microbial inoculum is a mixed bacterial community, and the pretreatment is high-pressure steam sterilization at 100-120℃ for 30-60 min and the addition of 50-100 mM sodium 2-bromoethanesulfonate.
3. The method according to claim 1, characterized in that, In step (1), the composition of each 1L of the inorganic culture medium is: NH4Cl, 1g / L; NaCl, 0.1g / L; MgCl2, 0.1g / L; anhydrous CaCl2, 0.05g / L; K2HPO4, 0.4g / L; FeCl2, 0.002g / L; H3BO3, 0.1mg / L; CoCl2, 0.1mg / L; ZnCl2, 0.1mg / L; NiCl2, 0.1mg / L; CuCl2, 0.06mg / L; ethylenediaminetetraacetic acid, 0.001g / L; MnCl2, 0.1mg / L; H2SeO4, 0.1mg / L; AlCl3, 0.1mg / L; the balance is water.
4. The method according to claim 1, characterized in that, In step (1), the anaerobic culture is performed by purging with nitrogen and then sealing the container during the culture process.
5. The method according to claim 1, characterized in that, In step (2), the pH of each reactor group is set to 5-10, the molar ratio of electron donor to electron acceptor is 2-10, and the COD load is 50 g / L.
6. The method according to claim 1, characterized in that, In step (2), the electron donor is one of ethanol, lactic acid, methanol, propanol, butanol, pyruvic acid, amino acid, polypeptide, sweet alcohol, sugar, carbon monoxide, and hydrogen; the electron acceptor is one or more of formic acid, acetic acid, propionic acid, butyric acid, and valeric acid.
7. The method according to claim 1, characterized in that, In step (4), the raw metagenomic data of the obtained carbon chain elongation sample is subjected to data quality control to remove low-quality reads.
8. The method according to claim 7, characterized in that, In step (5), the analysis of metagenomic data includes: splicing the quality-controlled sequence data to obtain contigs, detecting the size and quality of the contigs, then binning the contigs to assemble them, removing redundancy and extracting MAGs, detecting the integrity and contamination of MAGs, performing biological classification and gene prediction on high-quality MAGs, and calculating the relative abundance of MAGs.
9. The method according to claim 8, characterized in that, In step (5), identifying microorganisms with carbon chain elongation function specifically includes: 1) Focus on MAGs with an integrity level >50% and a contamination level <10%; 2) Focus on MAGs with a relative abundance > 0.8; 3) Select MAGs containing genes related to acetyl-CoA synthesis and the reverse β-oxidation pathway; 4) Among the selected MAGs, those with complete energy metabolism are microorganisms that have the ability to produce medium-chain organic acids by extending carbon chains.
10. The method according to claim 9, characterized in that, In step (6), assessing the metabolic potential of the identified carbon chain elongation functional microorganisms specifically includes: 1) Download the complete gene sequences of related microorganisms and known microorganisms with carbon chain elongation functions from NCBI; 2) Perform a genome-wide comparison between the identified MAGs and the downloaded gene sequences; 3) Based on the presence or absence of key functional genes, determine the metabolic potential of identified MAGs to achieve carbon chain elongation for different electron donors.