Method for screening sensitizing substrate for improving biotoxicity determination sensitivity of electrochemical active bacteria and method for determining biotoxicity by electrochemical active bacteria

Abstract

The invention discloses a method for screening a sensitizing substrate capable of improving biotoxicity determination sensitivity of electrochemical active bacteria and a method for determining biotoxicity by the electrochemical active bacteria, and belongs to the technical field of the electrochemical active bacteria. The method comprises the following steps: screening down-regulated metabolites under the condition that the sensitivity of electrochemical active bacteria is reduced after the electrochemical active bacteria are exposed by toxic pollutants, analyzing a metabolic pathway enriched by the down-regulated metabolites by using enrichment analysis, and using a substrate of the metabolic pathway enriched by the down-regulated metabolites as a sensitizing substrate; the sensitivity of the electrochemical active bacteria for determining the biotoxicity of the toxic pollutants is improved by using a sensitizing substrate. According to the method, an evolutionary biology method is adopted, the method is comprehensive and accurate, adapts to different strains and pollutants and is high in scientificity, the obtained sensitizing substrate is applied to biotoxicity determination, sensitivity can be remarkably improved, result accuracy and reliability can be enhanced, the application range is widened, and the method has wide application prospects in the field of application of electrochemical active bacteria to biotoxicity monitoring.

Description

Technical Field

[0001] This invention belongs to the field of electrochemically active bacteria technology, and particularly relates to a method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in determining biotoxicity, and a method for determining biotoxicity of electrochemically active bacteria. Background Technology

[0002] Preventing water environment risks is a crucial aspect of water quality monitoring. Simply detecting the concentration of specific chemicals in water (i.e., chemical monitoring) has limitations. Firstly, water bodies may contain tens to hundreds of thousands of chemicals, while conventional manual pollutant analysis typically only uses around a hundred indicators, failing to cover all potential pollutants. Many unknown or toxic pollutants may therefore be missed. Secondly, even if the concentration of each individual pollutant is below safety limits, the coexistence of multiple pollutants can produce a synergistic "toxic effect," with the overall biological toxicity potentially far exceeding expectations—something difficult to assess with chemical monitoring. Therefore, biotoxicity monitoring is needed to directly reflect the comprehensive biological effects of all pollutants by observing the overall response of organisms to water samples, thus overcoming the aforementioned shortcomings.

[0003] Electrochemically active bacteria (EAB) are promising test organisms for biotoxicity. During metabolism, EABs can transfer electrons to electrodes on their exterior, generating a measurable current signal. The intensity of this current signal directly reflects the bacterial's metabolic activity. When water samples containing toxic substances (such as heavy metals or pesticides) come into contact with EABs, their metabolic activity is inhibited or impaired, leading to a reduction in the number of electrons transferred to the electrodes and consequently a decrease in the current signal. By monitoring the degree of change in EAB current, the overall toxicity and biotoxicity of the water sample can be rapidly assessed. Compared to other test organisms, EABs are the only ones capable of directly converting biotoxicity into an electrical signal, possessing the unique advantage of being resistant to interference from water color and turbidity. However, the relatively low sensitivity of EABs in biotoxicity determination limits their practical application.

[0004] In the medical field, it has been discovered that using sensitivity metabolic markers as substrates can significantly increase the sensitivity of pathogenic bacteria to antibiotics; these sensitivity metabolic markers are also known as sensitizing substrates. In 2011, Kyle R. Allison et al. first reported in *Nature* a study on improving the antibiotic sensitivity of pathogenic bacteria by adding exogenous sensitizing substrates. In this study, the exogenous addition of mannitol as a sensitizing substrate increased the sensitivity of *Escherichia coli* to gentamicin by >1000 times; the exogenous addition of fructose as a sensitizing substrate increased the sensitivity of *Staphylococcus aureus* to gentamicin by >300 times. In fact, the sensitivity of EAB (Expert Analyzer) in detecting water biotoxicity is essentially the sensitivity of bacteria to toxic pollutants.

[0005] Therefore, how to provide a method to improve the sensitivity of biotoxicity determination by electrochemically active bacteria is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a method for screening sensitizing substrates to enhance the sensitivity of biotoxicity determination using electrochemically active bacteria, and a method for determining biotoxicity using electrochemically active bacteria.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in biotoxicity assays includes the following steps: Metabolomics was used to screen for downregulated metabolites in electrochemically active bacteria after exposure to toxic pollutants, which reduced their sensitivity. The metabolic pathways enriched by the downregulated metabolites were analyzed, and the substrates of the downregulated metabolites enriched the metabolic pathways were used as sensitizing substrates to complete the screening of sensitizing substrates.

[0008] Beneficial Effects: According to the principles of evolutionary biology, after exposure to toxic pollutants, bacteria undergo metabolic reprogramming to reduce their sensitivity to these pollutants, thus increasing their resistance. Therefore, the downregulated metabolic pathways during metabolic reprogramming are detrimental to bacterial resistance to toxic pollutants. This invention, through substrate addition, avoids the downregulation of these metabolic pathways, making it more conducive to the recognition of toxic pollutants by electrochemically active bacteria. Therefore, this invention uses substrates representing downregulated metabolic pathways that have decreased sensitivity after exposure to toxic pollutants. By adding these substrates, the activation or upregulation of these pathways can improve bacterial sensitivity to toxic pollutants, leading to better identification of toxic pollutants and more sensitive determination of biotoxicity.

[0009] Preferably, the electrochemically active bacteria include Acinetobacter baylyi or Shewanella loihica PV-4.

[0010] Preferably, when the electrochemically active bacteria is Acinetobacter baylyi, the toxic pollutant is a heavy metal pollutant; or, When the electrochemically active bacteria is Shewanella loihica PV-4, the toxic pollutant is a benzene series pollutant.

[0011] Preferably, the sensitizing substrate includes one or more of tyrosine, tryptophan, and phenylalanine.

[0012] Preferably, the sensitizing substrate is tyrosine and / or tryptophan.

[0013] More preferably, when the electrochemically active bacteria is Acinetobacter baylyi, the toxic pollutant is a heavy metal pollutant, and the sensitizing substrate is tryptophan; or, When the electrochemically active bacteria is Shewanella loihica PV-4, the toxic pollutant is a benzene series pollutant, and the sensitizing substrate is tyrosine.

[0014] Beneficial Effects: The changes in intracellular metabolites of the same bacteria after exposure to different toxic pollutants vary. Given the wide variety of hazardous pollutants in the aquatic environment, it is clearly impossible to screen for sensitizing substrates for every single pollutant. Therefore, this invention analyzes the commonalities in the changes in intracellular metabolites of the same bacteria after exposure to different types of toxic pollutants, screens for common downregulated metabolic pathways after exposure to the same type of pollutant, and uses these pathways as sensitizing substrates to improve sensitivity to a class of toxic pollutants, rather than just a single pollutant.

[0015] Preferably, the metabolomics is non-targeted metabolomics.

[0016] Preferably, the method for analyzing metabolic pathways that downregulate metabolite enrichment is KEGG enrichment analysis (Kyoto Encyclopedia of Genes and Genomes enrichment analysis).

[0017] Beneficial Effects: Bacterial intracellular metabolites are diverse, and analyzing only a few metabolites is clearly insufficient to accurately evaluate the downregulated metabolic pathways in bacteria after exposure to toxic pollutants. Instead, high-throughput metabolomics analysis is necessary. Common metabolomics methods are divided into targeted metabolomics, targeted metabolomics, and non-targeted metabolomics. Among these, non-targeted metabolomics can analyze the widest variety of metabolites. Therefore, when the downregulated metabolic pathway is not clearly anticipated, non-targeted metabolomics is more likely to screen for the downregulated pathway.

[0018] Application of a sensitizing substrate obtained by the method described above in improving the sensitivity of biotoxicity determination in electrochemically active bacteria.

[0019] A method for determining the biotoxicity of electrochemically active bacteria, wherein the biotoxicity is determined by using a sensitizing substrate obtained by the above method and the electrochemically active bacteria under the condition of using the sensitizing substrate as the sole carbon source for metabolism.

[0020] Preferably, a sensitizing substrate is used as the sole carbon source.

[0021] Beneficial effects: Electrochemically active bacteria can metabolize common substrates to generate bioelectric signals. In this invention, the screening of sensitizing substrates is based on the metabolomics analysis of bacterial strains. Electrochemically active bacteria have metabolic pathways for metabolizing sensitizing substrates. This invention uses the metabolic pathways of sensitizing substrates instead of the metabolic pathways of common substrates as the sole carbon source, which can ensure the activation and more significant upregulation of the metabolic pathways of sensitizing substrates.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects: This invention employs evolutionary biology methods that are comprehensive, precise, and adaptable to different bacterial species and contaminants, demonstrating strong scientific rigor. The resulting sensitizing substrates, when applied to biotoxicity assays, significantly improve sensitivity, enhance the accuracy and reliability of results, and broaden their application scope. This invention optimizes the substrate screening process, improving the sensitization effect while ensuring stable and reproducible results. Furthermore, the appropriate dosage of the sensitizing substrate in this invention achieves optimal sensitization, ensuring accurate and stable results, while also saving costs and improving economic efficiency. It has broad application prospects in the field of biomonitoring of electrochemically active bacteria. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 In Example 1 Acinetobacter baylyi Principal component analysis of metabolites after toxic exposure and without exposure, including Cd 2+ It is CdSO4·8 / 3 hydrate; Cr 6+ K2Cr2O7; Hg 2+ It is HgNO3 hydrate; Pb is PbCl2; Figure 2 In Example 1 Acinetobacter baylyi Clustering heatmap analysis of metabolites after toxic exposure and without toxic exposure; Figure 3 In Example 1 Acinetobacter baylyi Cr 6+ KEGG-based pathway enrichment analysis of differentially regulated metabolites after exposure; Figure 4 In Example 1 Acinetobacter baylyi via Hg 2+ KEGG-based enrichment analysis of metabolic pathways that downregulate differential metabolites after exposure; Figure 5 In Example 1 Acinetobacter baylyi Cd 2+ KEGG-based enrichment analysis of metabolic pathways that downregulate differential metabolites after exposure; Figure 6 In Example 1 Acinetobacter baylyi via Pb 2+ KEGG-based enrichment analysis of metabolic pathways that downregulate differential metabolites after exposure; Figure 7 The main body diagram (a) and exploded diagram (b) of the sensor in Examples 2 and 4 are shown below; The following are the labels: upper chamber 1, lower chamber 2, working electrode 3, counter electrode 4, reference electrode 5, titanium wire 6, water inlet 7, water outlet 8, ring 9, and titanium wire hole 10. Figure 8 In Example 2 Acinetobacter baylyi Hg was determined using sodium acetate as the sole carbon source. 2+ Biotoxicity; Figure 9 In Example 2 Acinetobacter baylyi Pb was determined using sodium acetate as the sole carbon source. 2+ Biotoxicity; Figure 10 In Example 2 Acinetobacter baylyi Cd was determined using sodium acetate as the sole carbon source. 2+ Biotoxicity; Figure 11 In Example 2 Acinetobacter baylyi Figure showing the results of determining the biotoxicity inhibition rate using sodium acetate as the sole carbon source; Figure 12 In Example 2 Acinetobacter baylyi Hg was determined using tryptophan as the sole carbon source. 2+ Biotoxicity; Figure 13 In Example 2 Acinetobacter baylyi Pb was determined using tryptophan as the sole carbon source. 2+ Biotoxicity; Figure 14 In Example 2 Acinetobacter baylyi Cd determination using tryptophan as the sole carbon source 2+ Biotoxicity; Figure 15 In Example 2 Acinetobacter baylyi Figure showing the results of determining the biotoxicity inhibition rate using tryptophan as the sole carbon source; Figure 16 In Example 3 Shewanella loihica Principal component analysis of PV-4 metabolites after exposure to trichlorobenzene and benzene and before exposure to toxicity; Where A is Shewanella loihica The metabolite of PV-4 after exposure to trichlorobenzene, B is... Shewanella loihica The metabolites of PV-4 after benzene exposure, C is Shewanella loihica PV-4 does not produce metabolites following exposure to toxic pollutants; Figure 17 In Example 3 Shewanella loihica Clustering heatmap analysis of PV-4 metabolites after exposure to trichlorobenzene and benzene and before exposure to toxicity; Figure 18 In Example 3 Shewanella loihica KEGG-based enrichment analysis of differentially downregulated metabolites of PV-4 after trichlorobenzene exposure; Figure 19 In Example 3 Shewanella loihica KEGG-based enrichment analysis of differentially downregulated metabolites of PV-4 after benzene exposure; Figure 20 In Example 4 Shewanella loihica PV-4 used sodium lactate as a substrate to determine the biotoxicity of 1 mg / L trichlorobenzene; Figure 21 In Example 4 Shewanella loihica PV-4 used sodium lactate as a substrate to determine the biotoxicity of 5 mg / L trichlorobenzene; Figure 22 In Example 4 Shewanella loihica Statistical results of the determination of the biotoxicity inhibition rate of trichlorobenzene using sodium lactate as a substrate in PV-4; Figure 23 In Example 4 Shewanella loihica PV-4 used tryptophan as a substrate to determine the biotoxicity of 1 mg / L trichlorobenzene; Figure 24 In Example 4 Shewanella loihica PV-4 used tryptophan as a substrate to determine the biotoxicity of 5 mg / L trichlorobenzene; Figure 25 In Example 4 Shewanella loihica Statistical results of the biotoxicity inhibition rate of trichlorobenzene determined by PV-4 using tryptophan as a substrate. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] This invention discloses a method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in determining biotoxicity, comprising the following steps: Metabolomics was used to screen for downregulated metabolites in electrochemically active bacteria after exposure to toxic pollutants, which reduced their sensitivity. The metabolic pathways enriched by the downregulated metabolites were analyzed, and the substrates of the downregulated metabolites enriched the metabolic pathways were used as sensitizing substrates to complete the screening of sensitizing substrates.

[0027] In a preferred embodiment, the electrochemically active bacteria include Acinetobacter baylyi or Shewanella loihica PV-4.

[0028] In a preferred embodiment, the electrochemically active bacteria are Acinetobacter baylyi At that time, the toxic pollutant was a heavy metal pollutant; or, The electrochemically active bacteria are Shewanella loihica In PV-4, the toxic pollutant is a benzene series pollutant.

[0029] In a preferred embodiment, the sensitizing substrate includes one or more of tyrosine, tryptophan, and phenylalanine.

[0030] In a preferred embodiment, the sensitizing substrate is tryptophan.

[0031] In a preferred embodiment, the metabolomics is targeted metabolomics, non-targeted metabolomics, or targeted metabolomics. More preferably, it is non-targeted metabolomics.

[0032] In a preferred embodiment, the method for analyzing the metabolic pathways that downregulate metabolite enrichment is KEGG enrichment analysis.

[0033] This invention also discloses the application of a sensitizing substrate obtained by the method described above in improving the sensitivity of biotoxicity determination in electrochemically active bacteria.

[0034] This invention also discloses a method for determining the biotoxicity of electrochemically active bacteria, using the sensitizing substrates obtained by screening using the above method.

[0035] In a preferred embodiment, the amount of the sensitizing substrate added is 1-10 mM.

[0036] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels; Acinetobacter baylyi The strain number is ATCC 33305.

[0037] Unless otherwise specified, room temperature or normal temperature in the embodiments of the present invention refers to 25±3℃.

[0038] Example 1 A method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in biotoxicity assays includes the following steps: 1. Scale up culture using DM medium. Acinetobacter baylyi Each liter of DM medium contains 2.5 g NaHCO3, 0.08 g CaCl2·H2O, 1.0 g NH4Cl, 0.94 g MgCl2·6H2O, 10 g NaCl, and 7.2 g 4-hydroxyethylpiperazine ethanesulfonic acid. The culture temperature is set at 25℃, the shaking speed is 150 rpm, and the culture method is aerobic. When the growth reaches OD... 600 =1 is used for subsequent experiments. Based on whether heavy metal contaminants were added and the type of heavy metal, they were divided into 5 groups. The Con group had no additional heavy metal contaminants added to the culture medium; the Cr group had an additional 5 mg / L Cr added to the culture medium. 6+ The Hg group received an additional 5 mg / L Hg in the culture medium. 2+ The Cd group received an additional 5 mg / L of Cd in the culture medium. 2+ The Pb group received an additional 5 mg / L of Pb in the culture medium. 2+ Each group had four replicates, and the culture time was uniformly set to 2 days. After culture, non-targeted metabolomics was used to test bacterial metabolites, including principal component analysis and cluster heatmap analysis.

[0039] Non-targeted metabolomics, based on high-resolution mass spectrometry detection technology and combined with a high-quality secondary spectral information database, identifies molecular characteristic peaks by matching them, achieving high-throughput and high-quality identification of metabolites in vivo and reflecting the information of total metabolites to the greatest extent.

[0040] Raw data were preprocessed using XCMS software. First, peak extraction, peak quantification, and peak alignment were performed. Then, based on the set ppm and adduct ion information, the data was compared with a high-quality secondary spectral database for metabolite identification. Finally, metabolites with a coefficient of variation less than 30% from the quality control samples were retained as the final identification results for subsequent analysis.

[0041] The results are as follows Figure 1-2 As shown, from Figure 1 It can be seen that exposure to toxic pollutants significantly affected... Acinetobacter baylyi Metabolite production. Samples from different groups clustered in one area, while samples from different groups were distributed in different areas, indicating that exposure to heavy metal pollutants has a greater impact on metabolites than individual differences. Figure 2 As shown, the clustering heatmap further confirms the above conclusions. It can be seen that sample clustering is exactly the same as sample grouping; samples in the same group are clustered together. These results demonstrate that exposure to all four heavy metal pollutants significantly affects... Acinetobacter baylyi The metabolic process.

[0042] 2. Using the KEGG website, enrichment analysis was performed on the differential metabolites of each heavy metal and the Con group.

[0043] The results are as follows Figure 3-6 As shown, comparison Figure 3-6 Enrichment analysis revealed that tryptophan metabolism was present in the KEGG-based pathway enrichment analysis of the downregulated differential metabolites after exposure to the four heavy metal pollutants. This downregulation indicates that this metabolic process is detrimental to bacterial resistance to heavy metal stress, and the bacteria actively downregulated this pathway to maintain a higher level of resistance. In other words, when... Acinetobacter baylyi When using tryptophan metabolism Acinetobacter baylyi It may have even lower resistance to heavy metals, meaning tryptophan holds promise as a potential treatment. Acinetobacter baylyi Sensitizing substrates for the determination of heavy metal pollutants.

[0044] Example 2 The biotoxicity of three heavy metal pollutants was determined using previously established biotoxicity assay methods.

[0045] The method for determining biotoxicity specifically includes the following steps: use Figure 7The sensor shown has a structure similar to a three-electrode electrochemical system. Externally, it is cylindrical, and internally it includes an upper chamber 1 and a lower chamber 2, with a total internal volume of 176 mL. Both the upper and lower chambers are cavities formed by a combination of cylindrical and conical shapes, with the conical cavity located at the bottom. This conical cavity is designed to avoid significant dead zones during water flow. The sensor also includes three electrodes (…). Figure 7 Part b) consists of the working electrode 3, the counter electrode 4, and the reference electrode 5. The working electrode 3 is sandwiched between the upper chamber 1 and the lower chamber 2, and its electrical signal is led out via a titanium wire 6. The reference electrode 5 and the counter electrode 4 are located in the lower chamber 2 and the upper chamber 1, respectively. The upper chamber 1 and the lower chamber 2 are respectively equipped with an inlet 7 and an outlet 8 for continuous flow operation. For ease of operation, the counter electrode 4 and the reference electrode 5 are fixed by hollow threads, and the upper chamber 1 and the lower chamber 2 are screwed together to complete the sensor installation and fixation of the working electrode 3.

[0046] The installation and reuse method of the above sensor is as follows: Cut 1 piece of 1 A 1cm carbon felt is used as the working electrode 3 and placed on the ring 9 inside the lower chamber 2. A titanium wire 6 is inserted into the carbon felt and led out from the titanium wire hole 10. A 4cm platinum wire is used as the counter electrode 4 and installed in the counter electrode hole of the upper chamber 1. An Ag / AgCl electrode is used as the reference electrode 5 and installed in the reference electrode hole of the lower chamber 2. The upper chamber 1 is rotated and screwed into the lower chamber 2. The carbon felt is fixed and sealed by the compression between the upper chamber 1 and the lower chamber 2. After one experiment, the upper chamber 1 and the lower chamber 2 are unscrewed, cleaned, and the carbon felt is replaced for reuse.

[0047] First, the culture was scaled up using LB medium. Acinetobacter baylyi When it grows to OD 600 When the value is approximately 1.0, it is used for subsequent experiments; then, the bacterial culture is centrifuged and resuspended in DM medium to obtain... Acinetobacter baylyi DM bacterial suspension; then, 300 mL of Acinetobacter baylyi The DM bacterial suspension was simultaneously introduced into 6 identical [cells / portions]. Figure 7The sensors shown are numbered 1-6 according to their sequence. The flow rate is 2 mL / min, and the bacterial suspension is self-circulated for 10 min to prepare an early biofilm. Next, 150 mL of DM medium with added toxic contaminants and 150 mL of DM medium without added toxic contaminants are introduced into sensors 1-3 (DM medium with added toxic contaminants) and sensors 4-6 (DM medium without added toxic contaminants), respectively. All DM media are supplemented with 10 mM tryptophan (Trp) or 10 mM sodium acetate as the sole carbon source. The flow rate is 2 mL / min, and the DM medium is self-circulated for 120 min to expose the bacteria to the toxic contaminants in the medium. Finally, the electrical signals of sensors 1-6 are analyzed by chronoamperometry, and the inhibitory effect of toxic contaminants on the electrical signals is analyzed by the formula Inhibition rate = |MN| / M × 100% (I), where M is the electrical signal of sensors 4-6 and N is the electrical signal of sensors 1-3.

[0048] By comparison Acinetobacter baylyi The sensitivity of biotoxicity determination was assessed by analyzing the difference in electrical signals under conditions of no toxicity exposure (Control) and exposure to heavy metal contaminants.

[0049] The results are as follows Figure 8-15 As shown, it can be seen that compared with common substrates, Acinetobacter baylyi Using the sensitizing substrate tryptophan provides higher sensitivity for determining the biotoxicity of heavy metal pollutants. Specifically, Acinetobacter baylyi When using sodium acetate, a common substrate, as the sole carbon source, 0.1 mg / L Hg resulted in only a 4.2% inhibition rate of the electrical signal. Figure 8 , Figure 11 ), while when using the sensitizing substrate tryptophan (Trp), the electro-signal inhibition rate reached 50.3% ( Figure 12 , Figure 15 This means that the electrical signal suppression rate was increased by 12.0 times. Similarly, when tryptophan was used as a substrate, 0.1 mg / L Pb was measured. 2+ The inhibition rate of biotoxic electrical signals was increased by 11.7 times (compared to...). Figure 9 , Figure 11 and Figure 13 , Figure 15 ), will determine 0.1 mg / L Cd 2+ The inhibition rate of biotoxic electrical signals was increased by 11.2 times (compared to...). Figure 10 , Figure 11 and Figure 14 , Figure 15 This study demonstrates that using tryptophan as a sensitizing substrate improves the sensitivity of Acinetobacter baylyi in determining the biotoxicity of heavy metal pollutants.

[0050] Example 3 Further verification showed that the sensitizing substrate also had a sensitizing effect on other electrochemically active bacteria and other types of toxic pollutants.

[0051] Using common EAB pattern strains Shewanella loihica PV-4 involved toxic exposure to two benzene derivatives (benzene and trichlorobenzene). The culture medium consisted of 10 g / L peptone, 10 g / L NaCl, and 5 g / L yeast extract; the culture temperature was 25°C, the shaking speed was 150 rpm, and the culture conditions were aerobic, with a culture time of 2 days. Three groups were established: Group A included culture medium with added trichlorobenzene (5 mg / L); Group B included culture medium with added benzene (5 mg / L); and Group C included culture medium without added pollutants. Each group had three replicates.

[0052] from Figure 16 It can be seen from some parts that toxic pollutants have a significant impact. Shewanella loihica PV-4 metabolism. The three groups of samples were distributed in different regions. Furthermore, the cluster analysis of the samples was completely consistent with the sample grouping. Figure 17 This indicates that the impact of toxic pollutant exposure on metabolites is greater than the differences between groups. Compared with group C, the number of metabolic pathways with significantly downregulated metabolite enrichment in groups A and B were 19 and 2, respectively, with one common metabolic pathway being tyrosine metabolism (…). Figure 18-19 Therefore, tyrosine is... Shewanella loihica PV-4 is a sensitizing substrate for benzene series pollutants.

[0053] Example 4 Using tyrosine, a sensitizing substrate, and sodium lactate, a commonly used substrate, as the sole carbon source, respectively, and employing the same testing methods as in Example 2, the results were analyzed. Shewanella loihica PV-4 was used to determine the biotoxicity of trichlorobenzene at concentrations of 1 mg / L and 5 mg / L, with both substrates at a concentration of 10 mM.

[0054] The results are as follows Figure 20-25 As shown, it can be seen that when Shewanella loihica When PV-4 is used with sodium lactate, a common substrate, as the sole carbon source, 1 mg / L of trichlorobenzene does not significantly inhibit its activity. Shewanella loihica The electrical signal of PV-4 has a suppression rate of -6.9%. Figure 20 , Figure 22 And when Shewanella loihica When PV-4 uses the sensitizing substrate tyrosine as the sole carbon source, 1 mg / L TCB has an effect on... Shewanella loihica The electrical signal suppression rate of PV-4 reached 9.2% ( Figure 23 , Figure 25Therefore, the biotoxicity of 1 mg / L trichlorobenzene was successfully identified only when the sensitizing substrate tyrosine was used as the sole carbon source. Similarly, Shewanella loihica When PV-4 used tyrosine as the sole carbon source, the measured electro-signal inhibition rate of 5 mg / L TCB increased by 2.67 times (compared to...). Figure 21 , Figure 22 and Figure 24 , Figure 25 These results indicate that using substrates from pathways that downregulate metabolite enrichment as sensitizing substrates improves... Shewanella loihica Sensitivity of PV-4 in determining TCB biotoxicity.

[0055] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for screening sensitizing substrates to enhance the sensitivity of electrochemically active bacteria in determining biotoxicity, characterized in that, Includes the following steps: Metabolomics was used to screen for downregulated metabolites in electrochemically active bacteria after exposure to toxic pollutants, which reduced their sensitivity. The metabolic pathways enriched by the downregulated metabolites were analyzed, and the substrates of the downregulated metabolites enriched the metabolic pathways were used as sensitizing substrates to complete the screening of sensitizing substrates.

2. The method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in determining biotoxicity, as described in claim 1, is characterized in that... The electrochemically active bacteria include Acinetobacter bengal or Leuishwarbella.

3. The method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in determining biotoxicity, as described in claim 2, is characterized in that... When the electrochemically active bacteria is Acinetobacter bengal, the toxic pollutant is a heavy metal pollutant; or, When the electrochemically active bacteria is *L. reishi*, the toxic pollutant is a benzene series pollutant.

4. The method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in determining biotoxicity, as described in claim 1, is characterized in that... The sensitizing substrates include one or more of tyrosine, tryptophan, and phenylalanine.

5. The method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in determining biotoxicity, as described in claim 4, is characterized in that... The sensitizing substrate is tyrosine and / or tryptophan.

6. The method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in determining biotoxicity, as described in claim 1, is characterized in that... The metabolomics mentioned are targeted metabolomics, non-targeted metabolomics, or targeted metabolomics.

7. The method for screening sensitizing substrates to improve the sensitivity of electrochemically active bacteria in determining biotoxicity, as described in claim 1, is characterized in that... The method used to analyze the metabolic pathways that downregulate metabolite enrichment is KEGG enrichment analysis.

8. The application of a sensitizing substrate obtained by the method described in any one of claims 1-7 in improving the sensitivity of biotoxicity determination in electrochemically active bacteria.

9. A method for determining the biotoxicity of electrochemically active bacteria, characterized in that, Using the sensitizing substrate obtained by the method described in any one of claims 1-7, the biotoxicity of electrochemically active bacteria was determined under the condition that the sensitizing substrate was used as the sole carbon source for metabolism.

10. The method for determining the biotoxicity of electrochemically active bacteria according to claim 9, characterized in that, The amount of the sensitizing substrate added is 1-10 mM.

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