A strain of Pseudomonas and its application in water toxicity monitoring

Abstract

The present invention belongs to the field of microbial application, and specifically relates to a strain of Pseudomonas and its application in water quality toxicity monitoring. The specific technical scheme is: a strain of Pseudomonas CIB‑66, which was deposited in the China Center for Type Culture Collection on May 23, 2022, with a deposit number of CCTCC NO: M2022638. The Pseudomonas is inoculated on the anode electrode, and the biofilm is started by forming a biofilm; the water body to be tested is injected into the anode, and the relationship between the pollutant concentration in the water body to be tested and the current inhibition rate is established according to the output total coulomb efficiency; the coulomb inhibition rate of the water body to be tested is calculated, and the concentration of the pollutant to be tested in the water body to be tested is obtained according to the coulomb inhibition rate. The present invention provides a new strain of Pseudomonas, which can monitor and detect toxic pollutants in water bodies in a pure bacterial system, has good application prospects for online early warning of water quality toxicity, has low cultivation costs, and is convenient for standard industrialization implementation.

Description

Technical Field

[0001] The invention belongs to the field of microbial application, and specifically relates to a strain of Pseudomonas and application thereof in water quality toxicity monitoring. Background Art

[0002] Traditional water quality testing methods, such as high performance liquid chromatography, mass spectrometry and atomic absorption spectrometry, are mainly based on physical or chemical techniques, with long testing times and high costs. In addition, the results obtained by these methods cannot reflect the biological toxicity of water bodies. To this end, the prior art has further developed biological methods to evaluate the comprehensive biological toxicity of water bodies, including the use of luminous bacteria, algae, fish and invertebrates for evaluation. However, existing biological toxicity evaluation methods generally have problems such as poor adaptability, long analysis cycle and low sensitivity.

[0003] Microorganisms have the characteristics of short reproduction time and rapid response to toxic environments, and may be suitable as sensing elements for biological methods of water quality monitoring. Among them, electrochemically active bacteria are a type of microorganism that can exchange electrons with electrodes and realize extracellular electron transfer. When electrochemically active bacteria are exposed to toxic pollutants, the activity of the electroactive biofilm (EAB) is inhibited and the current decreases accordingly. Based on this characteristic of electrochemically active bacteria, it is expected to detect the comprehensive toxicity of water quality by monitoring its current.

[0004] In the prior art, there are few studies on the use of electrochemically active bacteria to detect water toxicity, and mixed bacteria systems are generally used. However, in practice, the multi-community structure of mixed bacteria electroactive biofilms has problems such as being difficult to replicate.

[0005] Therefore, if a new pure bacterium with excellent electrochemical activity can be developed, it will have important research significance and practical value for water toxicity detection. Summary of the invention

[0006] The purpose of the invention is to provide a strain of Pseudomonas and application thereof in water quality toxicity monitoring.

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is: a strain of Pseudomonas ( Pseudomonas sp .) CIB-66, deposited in the China Center for Type Culture Collection on May 16, 2022, with the deposit number CCTCC NO: M2022638.

[0008] Preferably, the 16S rRNA gene sequence of Pseudomonas is shown as SEQ.ID.NO:1.

[0009] Accordingly, the Pseudomonas is used in water quality toxicity detection and / or monitoring.

[0010] Preferably, the Pseudomonas is used to detect and / or monitor whether water contains heavy metals, antibiotics, halogen elements or has biological toxicity, or to detect / monitor the concentration of heavy metals, antibiotics, and halogen elements in water.

[0011] Preferably, the application is performed using the electrochemical activity of the Pseudomonas.

[0012] Correspondingly, a water toxicity detection device inoculates the Pseudomonas onto an anode electrode.

[0013] Preferably, after inoculating the Pseudomonas, the electrode potential is adjusted to 0.2-0.3 V, and the biofilm is grown for 3-5 days to complete the biofilm initiation.

[0014] Accordingly, a method for detecting water toxicity comprises the following steps:

[0015] (1) inoculating the Pseudomonas onto the anode electrode to form a biofilm to initiate the biofilm;

[0016] (2) Injecting the water to be tested into the anode, and establishing a relationship between the pollutant concentration in the water to be tested and the current suppression rate based on the output total coulomb efficiency;

[0017] (3) Calculate the coulomb suppression rate of the water body to be tested, and obtain the concentration of the pollutant to be tested in the water body to be tested based on the coulomb suppression rate.

[0018] The present invention has the following beneficial effects: The present invention provides a new strain of Pseudomonas CIB-66, which can monitor and detect toxic pollutants (heavy metals, antibiotics, halogen elements) in water bodies in a pure bacterial system. The anode biofilm constructed by strain CIB-66 has extremely high sensitivity for monitoring toxic pollution in water bodies, which can reach the ppb level, has good application prospects for online early warning of water quality toxicity, has low cultivation cost, and is convenient for standard industrialization implementation.

[0019] The Pseudomonas CIB-66 provided by the present invention has strong environmental adaptability and can survive well in high-salt and alkaline environments. When constructing a detection system, a high-salt and / or alkaline environment can be used to prevent contamination by miscellaneous bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the culture medium growth of Pseudomonas CIB-66;

[0021] Figure 2 is the growth curve of Pseudomonas CIB-66 at different pH;

[0022] Figure 3 is the growth curve of Pseudomonas CIB-66 under different salinities;

[0023] Figure 4 This is a photo of the biofilm formed by Pseudomonas CIB-66;

[0024] Figure 5 The sensor is sensitive to different concentrations of Cu 2+ Current-time response diagram of

[0025] Figure 6 Cu 2+ Concentration-current inhibition rate curve;

[0026] Figure 7 Sensor response to different Ni concentrations 2+ Current-time response diagram of

[0027] Figure 8 For you 2+ Concentration-current inhibition rate curve. DETAILED DESCRIPTION

[0028] The present invention provides a new strain of Pseudomonas ( Pseudomonas sp .) CIB-66. The Pseudomonas was deposited in the China Center for Type Culture Collection (Wuhan University, No. 299, Bayi Road, Wuhan City, Hubei Province) on May 16, 2022, with a deposit number of CCTCC NO: M 2022638. The 16S rRNA gene sequence of the strain is shown in SEQ.ID.NO: 1.

[0029] The present invention also provides a method for detecting heavy metals in water based on the Pseudomonas, specifically using the Pseudomonas to construct a Pseudomonas biofilm for detecting water pollutants with high sensitivity and rapid response, and then using the biofilm to detect water quality. The specific method is:

[0030] The Pseudomonas CIB-66 is inoculated on the anode electrode. The bioanode is preferably carbon felt (the size is determined according to actual needs, such as 2.0 cm×2.0 cm), and the cathode is preferably a platinum sheet (the size is determined according to actual needs, such as 1.0 cm×1.0 cm). The anode and cathode are connected to the negative electrode and the positive electrode respectively, and a reference electrode, such as Ag / AgCl, is set in the cathode chamber. The Pseudomonas inoculation ratio is 10% (v / v) of the working volume of each reactor, and the live bacteria concentration in the inoculated Pseudomonas culture solution is 10 12 CFU / L, adjust the electrode potential to 0.2-0.3 V, and allow the biofilm to grow for 3-5 days until three repeated baseline currents appear, completing the initiation of the biofilm.

[0031] The optional positive electrolyte composition is: 4.58g Na2HPO4, 2.45g NaH2PO4·2H2O, 1.2g CH3COONa, 0.31g NH4Cl, 0.13g KCl, 1mL vitamins, 1mL minerals, 1L deionized water. The pH is adjusted to 7.20±0.02.

[0032] The optional cathode electrolyte composition is: 2.5g NaH2PO4·2H2O, 12.5g / L Na2HPO4·12H2O, 1L deionized water. The pH is adjusted to 7.20±0.02.

[0033] The vitamin composition is: 0.02g / L biotin, 0.02g / L folic acid, 0.1g / L pyridoxine hydrochloride, 0.05g / L riboflavin, 0.05g / L thiamine, 0.05g / L niacin, 0.05g / L pantothenic acid, 0.001g / L cobalamin, 0.05g / L p-aminobenzoic acid, 0.05g / L lipoic acid, and 1L deionized water.

[0034] The mineral composition is: 15g / L nitrosotriacetic acid, 60g / L MgSO4·7H2O, 5g / L MnSO4·H2O, 1g / L FeSO4·7H2O, 0.1g / L CuSO4·5H2O, 0.1g / L AlK(SO4)2·12H2O, 10g / L NaCl, 1g / L CaCl2·2H2O, 1g / L CoCl2·6H2O, 1.3g / L ZnCl2, 0.25g / L Na2MoO4, 0.25g / L NiCl2·6H2O, 1g / LH3BO3, 0.25g / L Na2WO4·2H2O, and 1L deionized water.

[0035] After the startup is completed, water with the pollutants to be detected (heavy metals as an example) is injected into the anode, and the relationship between the actual heavy metal concentration and the current suppression rate is established based on the output total coulomb efficiency. The total coulomb efficiency (Q) is obtained by integrating the current over time. The total coulomb efficiency calculation formula is as follows:

[0036]

[0037] Where I is the current in A, t is the time in seconds (s), and ti is the integral over time. This formula means: calculate the area of ​​the region with time as the horizontal axis and current as the vertical axis.

[0038] Current suppression rate calculation formula: IR(%)=(Cnon-Cn) / Cnon×100%

[0039] Where: Cnon is the total coulombic efficiency when there is no heavy metal, and Cn is the total coulombic efficiency when a certain heavy metal is added.

[0040] According to the above formula, the total coulomb amount of the water body to be tested and the simulated water body to be tested (without the corresponding heavy metal) are calculated respectively, and the coulomb amount suppression rate (IR) of the water body to be tested is calculated. The formula for calculating the coulomb amount suppression rate is as follows:

[0041]

[0042] Among them, Qnon refers to the total coulomb of the simulated water body to be tested, and Qn is the total coulomb of the water body to be tested.

[0043] Substituting the inhibition rate into the inhibition rate-concentration curve equation of the corresponding known heavy metal for calculation, the specific concentration of the heavy metal to be tested in the water body to be tested can be obtained. The method for obtaining the inhibition rate-concentration curve equation of the heavy metal is: adding different concentrations of the same heavy metal (such as Cu 2+ ), the total charge under different heavy metal concentrations was calculated by chronoamperometry, and the inhibition rate under each heavy metal concentration was calculated by taking only the anolyte (without adding heavy metals) as the blank control group. The concentration was taken as the independent variable and the inhibition rate as the variable to establish a correlation regression equation, that is, the inhibition rate-concentration curve equation.

[0044] In practice, water samples can be collected first, and the types of heavy metals in the water can be determined using existing ICP-MS and other methods, and then the present method can be used to determine the specific concentration of the heavy metals in the water to be tested.

[0045] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0046] Example 1: Screening and identification of electroactive microorganisms

[0047] 1. Select the long-term domestication of the inventor's research group at a high current density (current density > 0.25mA / cm 2 ) state as the inoculum source, which is a mixed system of autotrophic and heterotrophic bacteria with sodium acetate as the substrate.

[0048] Scrape the biofilm, quantify to 1 mL with phosphate buffer, and press 10 -2 ~10 -5Dilution gradient, take 100µL of each gradient of bacterial solution and evenly spread it on the plate separation and purification medium. After the colonies appear, use the plate streak separation method to purify the strains, pick strains of different colors, shapes, and sizes and inoculate them on the separation and purification medium (plate), and culture them in a 26℃ constant temperature incubator for 24 hours. After the colonies grow, observe whether their characteristics are consistent. If there are foreign bacteria, separate and purify them repeatedly until pure bacteria are obtained, and record the morphology and characteristics of each bacterial group.

[0049] The separation and purification culture medium components are: Na2HPO4 4.58g / L, NaH2PO4‧2H2O 2.45g / L, CH3COONa 2g / L, NH4Cl 0.31g / L, KCl 0.13g / L, yeast extract 0.4g / L, vitamins 1ml / L, mineral elements 1ml / L, and deionized water 1L; the above components are mixed evenly, sterilized at 121°C and high temperature and high pressure for 20min, and then 15g of agar powder is added to prepare a solid culture medium.

[0050] The vitamin composition is: 0.02g / L biotin, 0.02g / L folic acid, 0.1g / L pyridoxine hydrochloride, 0.05g / L riboflavin, 0.05g / L thiamine, 0.05g / L niacin, 0.05g / L pantothenic acid, 0.001g / L cobalamin, 0.05g / L p-aminobenzoic acid, 0.05g / L lipoic acid, and 1L deionized water.

[0051] The mineral composition is: 15g / L nitrosotriacetic acid, 60g / L MgSO4·7H2O, 5g / L MnSO4·H2O, 1g / L FeSO4·7H2O, 0.1g / L CuSO4·5H2O, 0.1g / L AlK(SO4)2·12H2O, 10g / L NaCl, 1g / L CaCl2·2H2O, 1g / L CoCl2·6H2O, 1.3g / L ZnCl2, 0.25g / L Na2MoO4, 0.25g / L NiCl2·6H2O, 1g / LH3BO3, 0.25g / L Na2WO4·2H2O, and 1L deionized water.

[0052] The purified microorganisms obtained by screening were inoculated into the anode (carbon felt, 2.0cm×2.0cm, thickness of 0.3cm) of the sterilized bioelectric reactor at the same live bacterial concentration and 10% (v / v) of the working volume of the reactor. The cathode of the reactor was a platinum sheet (1.0cm×1.0cm). The anode and cathode were connected to the negative electrode and the positive electrode respectively, and a reference electrode Ag / AgCl was set in the cathode chamber. The electrode potential was adjusted to 0.3V, and the electrolyte was the same as the specific implementation method. The biofilm was grown at 26°C for 3 days. After the electrical signal was stable, the current stability and current power density values ​​between the strains were compared, and the colony CIB-66 with the highest current power density was selected. The specific selection method is: each colony is inoculated into different sensors for biofilm domestication and operation, and the output of the electrical signal is observed. Under the same conditions, the electrical signal output by CIB-66 exceeds 1mA.

[0053] 2. Identification of CIB-66. The polymerase chain reaction (PCR) product was sent to Chengdu Qingke Biology for sequencing. The sequencing result is shown in SEQ.ID.NO: 1. It was compared with the 16S rRNA gene sequence of the existing Pseudomonas standard strain, and physiological and biochemical analysis was performed, and it was identified as Pseudomonas sp. Pseudomonas CIB-66 was preserved in the China Center for Type Culture Collection with the collection number CCTCC NO: M 2022638. The growth diagram of Pseudomonas CIB-66 in culture medium is shown in Figure 1 shown.

[0054] The Pseudomonas CIB-66 can tolerate a pH of 6 to 10 and a high salt environment of 50 g / L NaCl. The growth curves of Pseudomonas CIB-66 at different pH values ​​are shown in FIG. Figure 2 The growth curves under different salinities are shown in Figure 3 shown.

[0055] Example 2: Application effect demonstration of CIB-66 in heavy metal monitoring of water quality

[0056] 1. Preparation of CIB-66 biosensor: Inoculate CIB-66 bacteria on the bioanode electrode. The bioanode is carbon felt (2.0cm×2.0cm), and the cathode is a platinum sheet (1.0cm×1.0cm). Connect the negative and positive electrodes of the electrochemical workstation respectively, and place it in the cathode chamber with Ag / AgCl as the reference electrode. The CIB-66 inoculation ratio is 10% (v / v) of the working volume of the reactor. Adjust the electrode potential to 0.3V, and let the biofilm grow for 3 days to complete the start-up of the biofilm. Set up multiple identical biosensors. After the biofilm is started, the photo of the formed biofilm is as follows: Figure 4 shown.

[0057] 2. Inject 50mL of organic-loaded water containing different concentrations of copper ions and nickel ions into the anode of each sensor to monitor heavy metals. The copper ion concentration is set to: 0μg / L, 20μg / L, 40μg / L and 60μg / L; the nickel ion concentration is set to: 0μg / L, 20μg / L, 40μg / L and 60μg / L. The group with 0 copper ion and nickel ion concentration is the blank control group, and the same group can be used.

[0058] The direct relationship between the actual metal concentration and the current suppression rate is established based on the output total coulomb efficiency. The total coulomb efficiency is obtained by integrating the current over time.

[0059] The current suppression rate is calculated by the following formula: IR (%) = (Cnon-Cn) / Cnon × 100%

[0060] Where: Cnon is the total coulomb efficiency when there is no heavy metal (blank control group), and Cn is the total coulomb efficiency when a certain heavy metal is added. Figures 5 to 8 shown.

[0061] according to Figures 5 to 8 CIB-66 bacteria were used as biofilm to monitor heavy metals in water bodies. There was a high correlation between different heavy metal concentrations and toxicity inhibition rates. The correlation (R2) between copper ion and nickel ion concentration and toxicity inhibition rate were 0.947 and 0.902, respectively.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Pseudomonas ( Pseudomonas sp.) CIB-66, characterized in that: It was deposited in the China Center for Type Culture Collection on May 16, 2022, with the deposit number CCTCC NO: M 2022638.

2. The use of Pseudomonas CIB-66 according to claim 1 in water toxicity detection and / or monitoring, characterized in that: The Pseudomonas CIB-66 is used in detecting and / or monitoring whether water contains heavy metals, wherein the heavy metals are nickel or copper.

3. The use of Pseudomonas CIB-66 according to claim 1 in water toxicity detection and / or monitoring, characterized in that: The Pseudomonas CIB-66 is used for detecting and / or monitoring the concentration of heavy metals in water, wherein the heavy metals are nickel or copper.

4. The use according to claim 2 or 3, characterized in that: The application is carried out by utilizing the electrochemical activity of the Pseudomonas CIB-66.

5. A water toxicity detection device, characterized in that: The Pseudomonas CIB-66 of claim 1 is inoculated onto the anode electrode. After the Pseudomonas CIB-66 is inoculated, the electrode potential is adjusted to 0.2-0.3V, and the biofilm is allowed to form for 3-5 days to complete the biofilm initiation.

6. A method for detecting water toxicity, characterized in that: The method comprises the following steps: (1) Inoculating the Pseudomonas CIB-66 described in claim 1 onto the anode electrode to form a biofilm to initiate the biofilm formation; (2) Injecting the water to be tested into the anode, and establishing a relationship between the pollutant concentration in the water to be tested and the current suppression rate based on the output total coulomb efficiency; Current suppression rate calculation formula: IR (%) = (Cnon-Cn) / Cnon × 100%: Cnon is the total coulomb efficiency without heavy metals, and Cn is the total coulomb efficiency when a certain heavy metal is added; (3) Calculating the coulomb suppression rate of the water body to be tested, and obtaining the concentration of the pollutant to be tested in the water body to be tested according to the coulomb suppression rate; the pollutant is nickel or copper.

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