A bioelectrochemical method for in situ monitoring of root exudates

Abstract

The application relates to a bioelectrochemical method for monitoring root exudates in situ, comprising placing a bioelectrochemical sensor in a water culture tank, wherein the bioelectrochemical sensor comprises an anode electrode, a cathode electrode and an external resistance connected in series through a wire, the anode electrode is arranged to be deep in the plant roots, and the cathode electrode is arranged in the water culture tank; plant rhizosphere microorganisms or microorganisms with extracellular electron transfer function are attached to the surface of the anode electrode to form a biofilm, the plant root exudates are sensed by the biofilm as an organic carbon source to generate a voltage signal, and the plant growth is stressed; the anode electrode pre-acclimated by the microorganisms generates protons and electrons, the protons are transmitted to the cathode electrode through mass transfer, and the electrons are transmitted to the cathode electrode through the wire; the external resistance voltage signal is measured and processed to judge the total amount change of the organic components in the root exudates.

Description

TECHNICAL FIELD

[0001] The present application relates to a bioelectrochemical method for in-situ monitoring of root exudates, and relates to the field of facility agriculture monitoring. BACKGROUND

[0002] Facility agriculture is a modern agricultural production mode that realizes intensive, efficient and sustainable development under relatively controllable environmental conditions by using industrialized means. Accurate monitoring of facility environmental parameters is very important for environmental regulation, and therefore, relevant designs for monitoring of environmental temperature, light, water, gas and fertilizer continue to emerge.

[0003] Plant roots can store organic matter synthesized by photosynthesis, and synthesize some endogenous auxins, cytokinins and other organic matter. During root growth, root exudates are released, including low molecular weight sugars, amino acids, organic acids and some phenolic substances. The components of root exudates of different nutrient genotypes of plants are significantly different. When nutrients and environmental stress exist, plants increase the secretion of mucilage, enzymes and some organic acids to adapt to the changing environment. Root exudates act on the surrounding environment to produce rhizosphere effects, affecting the ecological distribution and population composition of rhizosphere microorganisms, and are the main source of nutrients for rhizosphere microorganisms. At the same time, rhizosphere microorganisms also react to root exudates and affect the production and composition of exudates through various channels.

[0004] With in-depth research on plant physiology, it is gradually realized that root exudates have allelopathic effects and interaction mechanisms with microorganisms. Monitoring the content of root exudates is very important for reflecting the growth state of plants, and in-situ monitoring without disturbing root growth and destroying the rhizosphere environment state is more meaningful. Many existing monitoring root exudate collection devices have gradually changed from destructive sampling to in-situ collection, but the method of in-situ collection and subsequent separation and testing consumes a lot of time, and the composition and total amount of organic matter change over time, and cannot reflect the real-time dynamic situation of root exudates. SUMMARY

[0005] In view of the above problems, the purpose of the present application is to provide a bioelectrochemical method for in-situ monitoring of root exudates, which can monitor the organic components in the root exudates in-situ in real time.

[0006] In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is as follows: the bioelectrochemical method for in-situ monitoring of root exudates provided by the present application comprises:

[0007] The bioelectrochemical sensor is placed in the water culture tank, and the bioelectrochemical sensor comprises an anode electrode, a cathode electrode and an external resistance connected in series by wires, wherein the anode electrode is fixedly arranged at the root of the plant, and the cathode electrode is arranged in the water culture tank.

[0008] Plant rhizosphere microorganisms or microorganisms with extracellular electron transfer function adhere to the surface of the anode electrode to form a biofilm, and the plant root exudates are perceived by the biofilm as an organic carbon source to generate a voltage signal to stress the plant growth.

[0009] The anode electrode pre-acclimated by the microorganism generates protons and electrons, the protons are transmitted to the cathode electrode by mass transfer, and the electrons are transmitted to the cathode electrode by the wire;

[0010] The voltage signal of the external resistance is measured and processed to determine the total amount change of the organic components in the root exudates.

[0011] Further, the anode electrode is pre-acclimated by the microorganism, including: using EM bacterial agent or microbial fertilizer to acclimate in an independent electrochemical system.

[0012] Further, before testing, the bioelectrochemical sensor is calibrated, including: testing the standard curve of the organic components under different internal resistance conditions without concentration, for subsequent measurement concentration comparison calculation.

[0013] Further, a filter membrane or a filtering device is arranged at the water inlet of the anode electrode.

[0014] Further, the electrode material of the anode electrode and the cathode electrode uses a material with high specific surface area, including carbon brush, carbon cloth or carbon felt.

[0015] Further, a catalyst is further arranged on the cathode electrode, the catalyst including ferricyanide, sulfurous acid and / or methyl viologen to improve the oxidation performance of the cathode electrode.

[0016] Further, a voltage signal acquisition device and a computer are further arranged, the voltage signal acquisition device is used to acquire the voltage signal of the external resistance, and the computer processes the acquired voltage signal to determine the total amount change of the organic components in the root exudates.

[0017] Further, an electrochemical workstation is further included, the internal resistance of the anode electrode is fitted by equivalent circuit through alternating current impedance test, the charge transfer internal resistance is fitted, and then the activity degree of the rhizosphere microorganism is determined.

[0018] Further, when the electrochemical workstation is used for testing, the detection is performed under open circuit condition, the anode electrode is the working electrode of the electrochemical workstation, the cathode electrode is the counter electrode of the electrochemical workstation, the Ag / AgCl electrode is the reference electrode of the electrochemical workstation, the scanning frequency and amplitude are set, the obtained Nyquist spectrum is fitted by equivalent circuit, and the charge transfer internal resistance is obtained.

[0019] Further, a constant potential instrument is also included, by inserting a reference electrode beside the anode electrode and connecting the constant potential instrument with the anode electrode and the cathode electrode through a three-electrode mode, the electronic transfer rate is controlled by adjusting the constant potential voltage and the electronic transfer rate of the microorganism is controlled.

[0020] The in-situ monitoring of the application has the advantages of real-time dynamics, and is faster than the traditional sampling and testing method, and can be widely applied to plant root monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0021] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of the preferred embodiments and are not intended to limit the scope of the application. Throughout the drawings, like reference numerals will be used to refer to like components. In the drawings:

[0022] Figure 1 The structure diagram of the bioelectrochemical sensor of the embodiment of the application.

[0023] Figure 2 The plant root secretion state diagram of the embodiment of the application.

[0024] Figure 3 The calibration curve diagram of the bioelectrochemical sensor of the embodiment of the application.

[0025] In the drawings, the reference numerals are: 1-anode electrode, 2-cathode electrode, 3-wire, 4-external resistance, 5-voltage signal acquisition device, 6-computer, 7-root microorganism, 8-constant potential instrument, 9-reference electrode. DETAILED DESCRIPTION

[0026] It is to be understood that the terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises", "comprising", "includes", "including" and "has" are inclusive and therefore specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order in which they are described, unless specifically identified as an order dependent step. It is also to be understood that additional or alternative steps can be employed.

[0027] Although the terms first, second, third, etc. can be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms can be only used to distinguish one element, component, region, layer or section from another region, layer or section. Unless the context clearly indicates otherwise, terms such as "first", "second" and the like used herein do not imply a sequence or an order, but rather are used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the example embodiments.

[0028] For ease of description, spatial relative terms can be used herein to describe the relationship of one element or feature to another element or feature as shown in the figures, such as "inner", "outer", "inside", "outside", "lower", "upper", etc. Such spatial relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures.

[0029] The method for in-situ collection and subsequent separation and testing consumes a lot of time, and the organic matter is prone to change in composition and total amount over time, which cannot reflect the real-time dynamic situation of root exudates. The bioelectrochemical method for monitoring root exudates in-situ provided by the application comprises: placing a bioelectrochemical sensor in a hydroponic tank, the bioelectrochemical sensor comprising an anode electrode, a cathode electrode and an external resistance connected in series through a wire, wherein the anode electrode is arranged to be deep in the plant roots, and the cathode electrode is arranged in the hydroponic tank; plant rhizosphere microorganisms or microorganisms with extracellular electron transfer function adhere to the surface of the anode electrode to form a biofilm, plant root exudates are sensed by the biofilm as an organic carbon source and generate a voltage signal, and the plant growth is stressed; the anode electrode pre-acclimated by the microorganisms generates protons and electrons, the protons are transferred to the cathode electrode by mass transfer, and the electrons are transferred to the cathode electrode by the wire; the external resistance voltage signal is measured and processed to judge the total amount change of the organic components in the root exudates. The application has the advantages of real-time dynamics and faster speed than the traditional sampling + testing method.

[0030] Example embodiments of the present application will be described herein below with reference to the accompanying drawings. While example embodiments of the present application are shown in the drawings, it is understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present application will be thoroughly and completely understood, and so that the scope of the present application will be conveyed completely to those skilled in the art.

[0031] Example 1: As Figure 1 , Figure 2As shown, the in-situ monitoring root exudates bioelectrochemical sensor provided by the embodiment includes an anode electrode 1, a cathode electrode 2, a wire 3 and an external resistance 4.

[0032] The anode electrode 1 is arranged in the plant root, wherein the plant root includes taproot and lateral root, and a corresponding number of anode electrodes 1 can be arranged in the taproot and lateral root according to needs. The arrangement of multiple anode electrodes 1 can make the test more comprehensive. Further, the anode electrode 1 can be arranged in the solution according to the growth environment of the plant, and the anode electrode 1 can be arranged in the solution at different depths according to the growth of the plant root system. The cathode electrode 2 is arranged in the growth environment of the cultivated plant, and can be arranged in the nutrient solution. The number of the cathode electrode 2 is matched with the number of the anode electrode 1.

[0033] Each anode electrode 1 and each cathode electrode 2 are connected by the wire 3. The rhizosphere microorganism 7 can be enriched in the anode electrode 1. When the root exudates are more, the rhizosphere microorganism is more active, and the oxidation reaction occurs to decompose the organic matter to form protons and electrons. The protons are transmitted to the cathode electrode 1 by mass transfer, and the electrons are transmitted to the cathode electrode 1 by the wire 3 to form a complete closed loop. The reaction principle of forming the closed loop is that the organic matter in the plant root exudates is decomposed to generate carbon dioxide and water, the anode electrode 1 generates protons and electrons under the catalysis of the rhizosphere microorganism 7, the electrons are transmitted to the cathode electrode 2 by the wire, the protons are transmitted to the anode electrode 1 by mass transfer, and the protons and the electrons jointly generate water, so that the reaction can be continuously carried out. The electrons form an electric current through the external circuit and are recorded by the acquisition device as an output electric signal, and the protons must consume and transmit the electrons of the cathode electrode 2 through mass transfer, so that the loop is formed and the electric signal is continuously formed. It should be noted that the bioelectrochemical sensor provided by the embodiment takes the organic acid secreted by the plant root system as the substrate when monitoring the concentration of the organic acid. The biofilm is formed on the surface of the anode electrode 1 to consume the root organic acid and generate a voltage signal for the operation of the device itself, without external power supply. At the same time, the bioelectrochemical sensor can slow down the problem of microalgae caused by the high concentration of organic matter in the environment of the root system.

[0034] The anode electrode 1 and the cathode electrode 2 are connected in series with the external resistance 4. Since the anode electrode 1 and the cathode electrode 2 function as a battery, although the wire, the medium and the microorganism connected will generate a part of internal resistance, the external resistance 4 is arranged to avoid the short circuit of the circuit. The resistance value of the external resistance 4 can be in the range of 1-50000Ω, and the resistance value of the external resistance 4 is as small as possible to avoid the consumption of the external resistance 4 to the circuit electronics and further consume a large amount of organic acid to affect the acid-base balance of the soil or solution.

[0035] In a preferred embodiment, a voltage signal acquisition device (DAQ) 5 is further arranged, and the voltage signal acquisition device 5 is used to acquire the voltage of the external resistance 4.

[0036] In a preferred embodiment, the microorganism in the embodiment simultaneously assumes the functions of the sensitive element and the conversion element, and the embodiment is further provided with a computer 6 for processing the voltage signal collected by the external resistor 4 to determine the change in the total amount of organic components in the root exudates. For example, the computer 6 can eliminate gross errors by using Fourier transform, box plot, etc. When a significant stress peak value appears in the signal, it indicates that the total amount of root exudates has increased, and the plant is under environmental stress. The response of root exudates to the lack of nutrients in the environment of the root system is more intuitive than other indicators (such as pH and EC). Since the fluctuations in environmental factors are caused by the exchange of substances on the root surface, directly testing the differences in substance exchange is conducive to early warning of environmental stress and avoiding irreversible damage caused by large environmental stress on plants, and timely supplement of corresponding nutrients (such as Fe and P) can achieve precise regulation.

[0037] In a preferred embodiment, an electrochemical workstation is further included, and the resistance of the anode electrode 1 is fitted by equivalent circuit through alternating current impedance test, and the charge transfer resistance can be fitted. The charge transfer resistance depends on the activation rate of the electrode surface, and the activation rate of the electrode surface depends on the redox activity of the rhizosphere microorganism, so the activity of the rhizosphere microorganism can be determined according to the alternating current impedance test, and the interaction between the root exudates and the rhizosphere microorganism in the growth process of the plant can be comprehensively evaluated. With the secretion of a large amount of root exudates, the activity of the rhizosphere microorganism is tested, and the plant growth is quickly diagnosed in combination with the plant physiological indicators and the soil environmental indicators, for example, alfalfa nodule bacteria can release volatile organic compounds to acidify the rhizosphere soil, and increase the activity of iron-reducing enzymes to promote the absorption of iron by alfalfa. During the test, the detection is carried out under open circuit condition, under two-electrode condition, the anode electrode 1 is used as the working electrode of the electrochemical workstation, the cathode electrode 2 is used as the counter electrode of the electrochemical workstation, the Ag / AgCl electrode is used as the reference electrode of the electrochemical workstation, the scanning frequency range is 100 kHz to 10 mHz, the amplitude is 5 mV, the Nyquist spectrum obtained is fitted by software to obtain the charge transfer resistance.

[0038] In a preferred embodiment, an external potentiostat 8 is further included, by inserting a reference electrode 9 (vs Ag / AgCl) beside the anode electrode 1 and connecting it to the potentiostat through a three-electrode mode (a three-electrode system contains two loops, one loop is composed of the working electrode and the reference electrode, which is used to test the electrochemical reaction process of the working electrode, and the other loop is composed of the working electrode and the auxiliary electrode, which plays the role of forming a loop to transfer electrons), the constant potential voltage is adjusted between 0.1-0.5V, and the potentiostat 8 is almost open circuit between the reference electrode 9 and the anode electrode 1, only adjusting the electromotive force of the anode electrode 1, without affecting the loop of the anode and cathode electrodes, the constant potential voltage instrument 8 can control the electron transfer rate of the external circuit, and further control the electron transfer rate of the microorganism (i.e. the nutrient solution environment), on the one hand, to avoid the problem of plant root hypoxia caused by more root exudates, and on the other hand, to avoid consuming necessary root exudates when the plant needs a small amount of root exudates to adjust the ion permeation concentration on the root surface.

[0039] In a preferred embodiment, the wire 3 can be made of titanium wire.

[0040] In a preferred embodiment, the electrode materials of the anode electrode 1 and the cathode electrode 2 can use high specific surface area materials such as carbon brush, carbon cloth, carbon felt, etc.

[0041] In a preferred embodiment, a catalyst is further provided on the cathode electrode 1 to improve the efficiency of the catalyst, which includes ferricyanide, sulfite and / or methyl viologen, etc.

[0042] In a preferred embodiment, a 0.45μm filter membrane or filter device is provided at the water inlet of the anode electrode 1 to ensure the independence of the anode electrode microbial environment and improve the sensitivity and stability of the bioelectrochemical sensor.

[0043] Embodiment two: the embodiment also provides a bioelectrochemical method for in-situ monitoring of root exudates, which comprises:

[0044] The above bioelectrochemical sensor is placed in a water culture tank;

[0045] The rhizosphere microorganism 7 of the plant or the microorganism with extracellular electron transfer function adheres to the surface of the anode electrode 1 to form a biofilm, and the plant root exudates are sensed by the biofilm as an organic carbon source and generate a voltage signal to stress the plant growth;

[0046] The anode electrode 1 pre-acclimated by the microorganism produces protons and electrons, the protons are transferred to the cathode electrode 2 through mass transfer, and the electrons are transferred to the cathode electrode 2 through the wire 3;

[0047] The voltage signal of the external resistance 4 is measured and processed to determine the total amount change of the organic components in the root exudates.

[0048] In a preferred embodiment, the anode electrode 1 is pre-acclimated by microorganisms, including: using EM microbial agents or microbial fertilizers, etc. in an independent electrochemical system for acclimation, the reason for choosing such microorganisms is to improve the concentration of anode biofilm, and such mixed microorganisms are harmless to plants and do not pose food safety problems. After the acclimation of the biofilm, the anode electrode is put into actual use.

[0049] The embodiment mainly tests the total amount of organic acid. Unless in a precise control environment such as a plant factory, the microorganisms on the surface of the anode electrode 1 can be acclimated to be more sensitive to a certain organic acid. For example, when using an anode electrode 1 that is more sensitive to citric acid, as the secretion of citric acid increases, the voltage signal of the bioelectrochemical sensor produces an increasing peak value based on the baseline, which can determine that the secretion of citric acid increases, and further judge that the plant growth releases symptoms such as P or Fe deficiency.

[0050] In a preferred embodiment, before testing, the bioelectrochemical sensor is calibrated, including: testing the standard curve of organic components at different concentrations under different internal resistance conditions, which is used for subsequent concentration comparison and calculation. After the bioelectrochemical sensor is acclimated by microorganisms, the standard curve under different raw material concentrations and different alternating current impedances is tested, as shown in Figure 3 When the bioelectrochemical sensor is actually used, the alternating current impedance of the bioelectrochemical sensor anode electrode 1 is tested after a period of time, and then it can be known which dimension of the standard curve is selected. This process does not need to disassemble the bioelectrochemical sensor, nor does it damage the internal conditions of the bioelectrochemical sensor.

[0051] Each embodiment in the specification is described in a progressive manner, and the same and similar parts between each embodiment can be referred to each other. Each embodiment focuses on the difference from other embodiments. In the description of the specification, the description of the terms "one embodiment", "some implementations" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the specification. In the description of the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the characteristics of the different embodiments or examples without contradiction.

[0052] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A bioelectrochemical method for in-situ monitoring of root exudates, characterized in that, include: A bioelectrochemical sensor is placed in a hydroponic tank. The bioelectrochemical sensor includes an anode electrode, a cathode electrode, and an external resistor connected in series by wires. The anode electrode is placed at a fixed depth at the plant roots, and the cathode electrode is placed in the hydroponic tank. Plant rhizosphere microorganisms or microorganisms with extracellular electron transfer function attach to the surface of the anode electrode to form a biofilm. Plant root secretions, as an organic carbon source, are sensed by the biofilm and generate voltage signals that have a stress effect on plant growth. The anode electrode, which has been pre-domesticated by microorganisms, produces protons and electrons. The protons are transferred to the cathode electrode via mass transfer, and the electrons are transferred to the cathode electrode via wires. The external resistance voltage signal is measured and processed to determine the change in the total amount of organic components in root exudates; Before testing, the bioelectrochemical sensor is calibrated, including by using a standard curve of the organic component under different internal resistance conditions at different concentrations, which is used for concentration comparison calculations in subsequent measurements. It is also equipped with a voltage signal acquisition device and a computer. The voltage signal acquisition device is used to acquire the voltage signal of the external resistor, and the computer processes the acquired voltage signal to determine the total change of organic components in the root exudate. It also includes an electrochemical workstation, which performs equivalent circuit fitting on the internal resistance of the anode electrode through AC impedance testing, fits the charge migration internal resistance, and then determines the activity level of rhizosphere microorganisms. When the electrochemical workstation is used for testing, the detection is carried out under open circuit conditions. The anode electrode is the working electrode of the electrochemical workstation, the cathode electrode is the counter electrode of the electrochemical workstation, and the Ag / AgCl electrode is the reference electrode of the electrochemical workstation. The scanning frequency and amplitude are set, and the obtained Nyquist spectrum is fitted with an equivalent circuit to obtain the charge migration internal resistance. It also includes an external potentiostat. By inserting a reference electrode next to the anode electrode and connecting it to the potentiostat in a three-electrode mode with the anode and cathode electrodes, the anode electromotive force can be adjusted by adjusting the constant potential voltage, thereby controlling the electron transfer rate and thus controlling the electron transfer rate of microorganisms.

2. The bioelectrochemical method for in-situ monitoring of root exudates according to claim 1, characterized in that, The anode electrode is pre-acclimated to microorganisms, including acclimation in a stand-alone electrochemical system using EM microbial agents.

3. The bioelectrochemical method for in-situ monitoring of root exudates according to claim 1, characterized in that, A filter device is installed at the water inlet of the anode electrode.

4. The bioelectrochemical method for in-situ monitoring of root exudates according to claim 1, characterized in that, The electrode materials for the anode and cathode electrodes are materials with high specific surface area, including carbon brushes, carbon cloth, or carbon felt.

5. The bioelectrochemical method for in-situ monitoring of root exudates according to claim 1, characterized in that, The cathode electrode is also provided with a catalyst, which includes ferricyanide or thionine to improve the reduction performance of the cathode electrode.

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