CN113292169A

CN113292169A - Method for synchronously removing nitrobenzene and vanadate in water environment through electric stimulation and inoculum

Info

Publication number: CN113292169A
Application number: CN202110711707.3A
Authority: CN
Inventors: 张宝刚; 李宗岩
Original assignee: China University of Geosciences Beijing
Current assignee: China University of Geosciences Beijing
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2021-08-24
Anticipated expiration: 2041-06-25
Also published as: CN113292169B

Abstract

One or more embodiments of the present disclosure relate to the technical field of environmental management, and in particular, to a method for synchronously removing nitrobenzene and vanadate in an aqueous environment by electrical stimulation and an inoculum, wherein the method includes: inoculating an inoculum into a bioreactor, wherein the inoculum is a sediment of a vanadium-polluted site of the Panzhihua; acclimating the inoculum; injecting first synthetic underground water at least containing nitrobenzene, pentavalent vanadium and a carbon source into a bioreactor containing the domesticated inoculum, and performing synchronous removal reaction of the nitrobenzene and the pentavalent vanadium under the stimulation of 0.3V-1.2V voltage; wherein the simultaneous removal reaction is carried out for at least 48 hours; maintaining an anaerobic environment within the bioreactor during the simultaneous removal reaction. The scheme can stably and efficiently remove nitrobenzene and pentavalent vanadium in the water environment at the same time.

Description

Method for synchronously removing nitrobenzene and vanadate in water environment through electric stimulation and inoculum

Technical Field

One or more embodiments of the specification relate to the technical field of environmental management, and in particular relate to a method and an inoculum for synchronously removing nitrobenzene and vanadate in an aqueous environment through electrical stimulation.

Background

The rapid development of economy, the increasing demand of human beings, the increasing discharge of waste gas and waste water, untreated waste water is artificially discharged into the environment wantonly, so that the environment contains a large amount of organic matters and heavy metals, and the threat to human health and the steady state of an ecosystem is formed.

Nitroaromatics (NACs) are an important component of recalcitrant xenobiotics and have been widely used in dyes, pigments, pharmaceuticals, pesticides, rubber chemicals, engineering plastics, explosives and other fields. Nitrobenzene (NB), as one of the NACs, is widely produced as an organically synthesized explosive, pesticide, solvent and intermediate.

As is well known, NACs are used in various aspects of productive life, improper use of NACs causes its entry into surface water, ground water and soil, and NB, one of NACs, is a serious environmental pollution problem due to its toxicity and potential environmental impact, has a bitter almond taste, and at the same time, NB can enter the human body through skin and respiration, damaging human organs, and has been reported to affect the development of the male reproductive system, testis, so as to produce histological changes in testis, cell depletion, and change sperm motility and morphology. At the same time, absorption or inhalation of more than a certain amount of NB by the human body can lead to hemolysis, impairment of liver and kidney functions, and even canceration of vital organs of the body. NB capacity to be discharged into the environment is increasing and health risks are not considered.

Vanadium (V) is one of five major transition metal elements commonly found in the natural environment, is equal to zinc (Zn) in the earth crust, is about twice as high as copper (Cu) and is as much as ten times as high as lead (Pb), and is widely distributed in igneous and sedimentary rocks and minerals among minerals, crude oil, and coal. The source of V in the environment comes primarily from three areas: weathering of natural rock, combustion of fuels such as coal, petroleum and the like, mining and smelting of minerals containing V and the like, in modern industries, V is mainly applied to steel for improving strength and corrosion resistance, but also in many other industries including pigments, catalysts and pharmaceuticals. Due to the excellent performance of V, the demand of people for V is getting bigger and bigger, people start intensive mining and smelting activities, and therefore more and more V enters the environment, and environmental pollution is caused. Smelting activities volatilize at high temperature to release a large amount of toxic metals including V, Zn and Cu, and enter soil through an atmospheric sedimentation process to cause soil pollution. A large amount of waste gas and waste water can be generated in the vanadium ore smelting process, and the waste water directly enters underground water to cause underground water pollution. In addition, the exhaust gas enters the ground water through rain wash and leaching, causing pollution.

V is one of the essential trace elements for human body and animals and plants, is vital to human health and growth and development of animals and plants, and the human body can promote the hematopoietic function of human body, regulate the functions of heart blood vessels and kidneys, reduce blood sugar and the like by taking a proper amount of V, the effects of V and molybdenum (Mo) are similar, the nitrogen fixation effect of plants can be improved, the photosynthesis efficiency of plants can be improved, and the V plays an irreplaceable role in the aspects of improving yield and quality. Excessive V concentration can affect the growth of plants, when the content of the soil V is higher, soybean leaves can turn yellow and wither, and the yield of overground parts and root systems is obviously reduced. When excessive V is ingested by a human body, thyroid gland activity disorder and renal failure can be caused; when the daily intake of V exceeds 3ppm, it causes diarrhea, green tongue and hematological changes, threatening the health of the human body.

It is worth noting that the coexistence of NB and v (v) in the environment is a complex and serious problem due to the increasing human activities caused by the rapid development of economy. The composite pollution to NB and V (V) generally coexists in mine development, dye synthesis, smelting and processing fields and near farmlands. Meanwhile, due to the strong toxicity of NB, NB cannot be removed very effectively by using the conventional in situ bioremediation technology, and thus, an efficient method for treating NB and v (v) combined pollution in the environment is urgently needed.

Disclosure of Invention

The embodiment of the specification describes a method and an inoculum for synchronously removing nitrobenzene and vanadate in an aqueous environment by electrical stimulation, which can efficiently and stably remove nitrobenzene and pentavalent vanadium from water simultaneously.

In a first aspect, the embodiments of the present disclosure provide a method for synchronously removing nitrobenzene and vanadate in an aqueous environment by electrical stimulation, where the method includes: inoculating an inoculum into a bioreactor, wherein the inoculum is a sediment of a vanadium-polluted site of the Panzhihua; acclimating the inoculum; injecting first synthetic underground water at least containing nitrobenzene, pentavalent vanadium and a carbon source into a bioreactor containing the domesticated inoculum, and performing synchronous removal reaction of the nitrobenzene and the pentavalent vanadium under the stimulation of 0.3V-1.2V voltage; wherein the simultaneous removal reaction is carried out for at least 48 hours; maintaining an anaerobic environment within the bioreactor during the simultaneous removal reaction.

In one embodiment, the synchronous removal reaction of nitrobenzene and pentavalent vanadium under the stimulation of 0.3V-1.2V voltage is specifically as follows: under the stimulation of 0.6V voltage, the synchronous removal reaction of nitrobenzene and pentavalent vanadium is carried out.

In one embodiment, the initial concentration of pentavalent vanadium in the first synthetic groundwater is 10mg/L and the initial concentration of nitrobenzene is 10 mg/L.

In one embodiment, the ratio of inoculum volume of inoculum to bioreactor volume is 1: 5.

in one embodiment, the carbon source is ethanol and the initial concentration in the first synthetic groundwater is 100mg/L COD.

In one embodiment, said acclimating said inoculum comprises: injecting second synthetic groundwater into the bioreactor containing the inoculum to perform acclimatization reaction; the second synthetic groundwater contains pentavalent vanadium, nitrobenzene and a carbon source.

In one embodiment, the initial concentration of pentavalent vanadium in the second synthetic groundwater is 10mg/L, the initial concentration of nitrobenzene is 10mg/L, and the initial concentration of carbon source is 100mg/L COD.

In one embodiment, the acclimatization reaction lasts for 20 cycles, each cycle having a duration of 48 hours; wherein the second synthetic groundwater in the bioreactor is replaced once per cycle.

In a second aspect, the present illustrative embodiments provide a method for acclimatization of an inoculum for simultaneous removal of nitrobenzene and vanadate in an aqueous environment by electrical stimulation, comprising: inoculating an inoculum into a bioreactor, wherein the inoculum is a sediment of a vanadium-polluted site of the Panzhihua; the ratio of the inoculation volume of the inoculum to the volume of the bioreactor was 1: 5; injecting second synthetic groundwater into the bioreactor containing the inoculum to perform acclimatization reaction; the second synthetic underground water contains pentavalent vanadium with initial concentration of 10mg/L, nitrobenzene with initial concentration of 10mg/L and ethanol with initial concentration of 100mg/L COD; the domestication reaction lasts for 20 periods, and the duration of each period is 48 hours; wherein the second synthetic groundwater in the bioreactor is replaced once per cycle.

In a third aspect, the present illustrative embodiments provide an inoculum acclimatized by the method of the second aspect.

The scheme provided by the embodiment of the specification can stably and efficiently remove nitrobenzene and pentavalent vanadium in the water environment at the same time.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 shows the results of a study of biodegrading nitrobenzene and pentavalent vanadium under different voltage stimuli; wherein a in fig. 1 shows the NB concentration change over the reactor 20 cycle at four different voltages; b in FIG. 1 shows the change in concentration of V (V) over the reactor 20 period at four different voltages; c in fig. 1 shows the variation of NB concentration in a single cycle at four different voltages; d in fig. 1 shows the pseudo first order kinetics of NB concentration change in a single cycle at four different voltages; in FIG. 1, e shows the variation of V (V) concentration in a single period at four different voltages; fig. 1, f, shows the pseudo first order kinetics of v (v) concentration change in a single cycle at four different voltages.

FIG. 2 shows the results of a study of NB with V (V) synchronous removal feature; wherein a in FIG. 2 shows the NB and V (V) concentration changes over the period of the experimental group reactor 20; b in FIG. 2 shows the change in V (V) concentration over the period of the experimental group reactor 20; c in fig. 2 shows NB removal efficiency in a single cycle; d in FIG. 2 shows a pseudo-first order kinetic model of NB for a single cycle; e in fig. 2 shows the v (v) removal efficiency in a single cycle; f in FIG. 2 shows a pseudo first order kinetic model of V (V) in a single cycle; g in FIG. 2 shows a graph of pH change over a single cycle; h in FIG. 2 shows the ORP variation in a single cycle.

FIG. 3 shows the identification of NB and V (V) reduced products; wherein (a) in fig. 3 shows TOC concentration; FIG. 3 (b) shows AN concentration and (c) NH₄ ⁺-N production; fig. 3 (d) shows a scanning electron microscope of the carbon felt biofilm structure; fig. 3 (e) shows the EDS results; fig. 3 (f) shows a time curve of the removal efficiency of dissolved total vanadium; fig. 3 (g) shows the XPS analysis result of the carbon felt surface layer biofilm.

FIG. 4 shows the effect of different influencing factors on the reduction of NB and V (V) in a typical cycle (48h) of E-NB-V; wherein a in FIG. 4 shows the effect of initial NB concentration on NB reduction in a typical cycle (48h) of E-NB-V; b in FIG. 4 shows the effect of initial NB concentration on V (V) reduction of E-NB-V in a typical cycle (48 h); c in FIG. 4 shows the effect of initial V (V) concentration on NB reduction in a typical cycle (48h) of E-NB-V; d in FIG. 4 shows the effect of the initial V (V) concentration on the V (V) reduction of E-NB-V in a typical cycle (48 h); FIG. 4, E, shows the effect of initial ethanol dose on NB reduction in a typical cycle (48h) of E-NB-V; the effect of the initial ethanol dose on the V (V) reduction of E-NB-V in a typical cycle (48h) is shown in fig. 4 at f.

FIG. 5 shows the kinetic fit of NB and V (V) removal effect under different influencing factors; wherein a in fig. 5 shows the effect of initial NB concentration on NB removal; b in fig. 5 shows the effect of initial NB concentration on v (v) removal; c in fig. 5 shows the sum of the effects of the initial v (v) concentration on NB removal; d in FIG. 5 shows the effect of the initial V (V) concentration on V (V) removal; e in fig. 5 shows the effect of the initial ethanol amount on NB removal; the effect of the initial ethanol amount on v (v) removal is shown in fig. 5 as e.

FIG. 6 shows a dilution curve of inoculated sludge with microbial communities in a bioreactor; wherein a in fig. 6 shows a dilutability curve of the microbial community at the anode; b in fig. 6 shows the dilutability curve of the microbial community at the cathode.

FIG. 7 shows the inoculum and the microbial structure and community in all bioreactors; wherein a in FIG. 7 shows a PCA anode plot based on OTU abundance data; b in fig. 7 shows a VENN anode plot based on OUT abundance data; c in fig. 7 shows a PCA cathode plot based on OTU abundance data; d in figure 7 shows the VENN cathode plot based on OTU abundance data; e in fig. 7 shows the relative abundance of the anodal gate microbial community; e in FIG. 7 shows the relative abundance of the microbial community of the genus anodic; g in fig. 7 shows the relative abundance of the cathodic phylum microbial communities; h in FIG. 7 shows the relative abundance of the cathodal microbial community.

Detailed Description

It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

A large amount of organic waste gas and waste water can be generated in a series of production and living processes such as development of mines, combustion of fuels, synthesis of pesticides and dyes and the like and enter the environment, so that organic pollution becomes one of water body pollution harming great harm. NB belongs to one of NACs and is widely applied to production of AN, dyes, pesticides, explosives and the like, the NB is widely used, so that the NB is inevitably released into the environment and causes serious pollution to soil and underground water, in the process of mining, waste gas and waste water containing heavy metals are generated along with the NB, organic matters and the heavy metals enter the soil through the disassembly process of electronic wastes, and enter a water body along with the action of rainwater and the like to cause pollution to the soil and the underground water.

When organic matters and heavy metals enter the environment at the same time, on one hand, the environment is threatened due to the toxicity of the organic matters and the heavy metals, and on the other hand, the organic matters and the heavy metals can generate larger toxicity to the environment due to the additive action or the synergistic action among the pollutants.

Abundant ferrovanadium ores are stored in the Panzhihua area in China, the use amount of explosives is increased along with more and more V resource exploitation required by production and life, in addition, a large amount of pesticides are used in the local agricultural production process, so that the explosives and NACs-NB ingredients in the pesticides enter soil, enter underground water through rainwater washing and leaching, in addition, in the exploitation process, V is enriched near smelting plants and farmlands through the modes of sedimentation, leaching and the like, and NB and V (V) exist in the underground water at the same time.

The valence and solubility of V influence the toxicity of V to human body and animals and plants. In nature, V, as a variable valence element, exists in several forms, mainly V (II), V (III), V (IV) and V (V), and among the valence states of vanadium, V (V) is the most stable in toxicity and easy to migrate. In contrast, V (IV) is relatively harmless, mostly in immobilized form, so that it is easily pre-incubated in the environment (near neutral pH), V (V) is reduced to V (IV), and V (V) is removed from water by neutral precipitation, which is an efficient and economical method for reducing V (V) pollution.

The method for removing nitrogen-containing organic matters in underground water mainly comprises a physical method, a chemical method and a biological method. The details are as follows.

1. Physical method

(1) Adsorption: the adsorption is mainly to remove organic matters from water by adsorbing the organic matters with low concentration in the water on the material by using the relevant material through adsorption. The adsorbing material mainly comprises activated carbon, sand gaps, zeolite, different types of clay minerals and the like.

(2) Microwave radiation: microwave radiation is mainly realized by directly transmitting microwave energy to a material through intermolecular interaction and an applied electromagnetic field, and organic pollutants in soil and underground water are pyrolyzed or volatilized in cooperation with a microwave absorbent, so that the concentration of organic matters in the soil and the underground water is reduced.

(3) Liquid-liquid extraction: the recyclable organic phase in the wastewater is extracted and reused by utilizing a similar compatibility principle, so that the effect of circular economy is achieved.

2. Chemical process

(1) Photocatalytic oxidation: the photocatalytic oxidation technology mainly utilizes ultraviolet light in a light source to irradiate a semiconductor which can be excited by light, conduction band electrons are generated, electron holes are left, the generated electron holes and organic matters adsorbed on a semiconductor catalytic material generate an oxidation-reduction reaction, and the organic matters are decomposed to generate carbon dioxide and water and are finally removed.

(2) Ozone oxidation: ozone has strong oxidizability, and is introduced into organic polluted wastewater, and organic matters are oxidized by the oxidizability of the ozone and finally degraded to generate micromolecules or even mineralized substances, so that the effect of removing organic pollutants is achieved.

(3) Wet oxidation: the method is mainly used for oxidizing organic matters into carbon dioxide, water and small molecular organic matters by using compressed air or high temperature and high pressure as an oxidant, and is suitable for treating high-concentration and relatively difficultly-degraded organic wastewater.

3. The biological method comprises the following steps: the biological method for degrading organic matters is mainly divided into aerobic degradation and anaerobic degradation, and the microorganisms degrade and mineralize the organic matters in the wastewater by creating an environment suitable for the growth of the microorganisms and using an aerobic or anaerobic means. Meanwhile, in the process of degrading organic matters by microorganisms, carbon sources such as glucose, sodium acetate, ethanol and the like which are beneficial to the degradation of the refractory organic matters by the microorganisms are artificially added to synergistically degrade the toxic organic matters into small molecular substances by the microorganisms, so that the harmfulness of the wastewater is reduced.

Aiming at the problem of increasingly serious groundwater heavy metal pollution, researchers in all countries of the world carry out sufficient investigation and research on the problem of how to repair groundwater heavy metal, and certain progress is made. Currently, ex-situ remediation and in-situ remediation are the main techniques for remediating heavy metal pollution in groundwater. The details are as follows.

1. Ectopic repair

The ex-situ remediation technology mainly utilizes an extraction technology to extract polluted underground water from an in-situ mode to the ground surface, and restores the polluted underground water to a standard level through related technologies or means, or transports the underground water polluted by heavy metals to an off-site mode for further treatment. The ectopic repair technology is convenient and easy to operate, the repair effect takes effect immediately, but the technology has the limitations that the energy consumption is high, the required maintenance and operation cost is high, and the like.

2. In situ remediation

(1) Permeable reactive barrier technology

In recent years, permeable reactive barrier technology, PRB for short, has developed rapidly as one of in-situ groundwater pollution technologies. The PRB system is a system filled with an active material grid body in the underground environment, when pollution in underground water diffuses and pollution plumes pass through the active material grid body, the pollution components in the pollution plumes are firstly adsorbed, precipitated and then degraded and removed by the active material, and therefore the underground water is purified. In PRB, active materials play a major role in removing groundwater pollutants in PRB, and in the remediation process, zeolite, activated carbon, zero-valent iron and microorganisms are mainly used as the active materials. The PRB has the advantages of good restoration capability, long-term stability, economy, convenience and the like.

(2) In-situ chemical repair technique

The in-situ chemical repairing technology is a technology that utilizes the redox property of chemical agents, injects or mixes the chemical agents with the oxidation or reduction property into polluted groundwater environment, and the chemical agents play a role in destroying and degrading pollutants into low-valence and non-toxic substances. This technique is commonly used to remediate groundwater contaminated with cr (vi), v (v). The in-situ chemical remediation technology has the advantages of high heavy metal removal efficiency, relatively low investment cost, short remediation period and the like, but when the types of heavy metals in the underground water environment are more, chemical agents in the in-situ chemical remediation process may generate secondary pollution, and meanwhile, a new button for heavy metal pollution can be triggered.

(3) In-situ electrical repair technique

The in-situ electro-remediation technology mainly utilizes electrokinetic effects such as electrodialysis, electromigration and electrophoresis to form an electric field gradient, pollutants are dispersed and converted in the formed electric field, then converted into charged ions, and then forcedly migrated and eliminated, so that the effect of remedying heavy metals is finally achieved. The in-situ electric remediation technology can be applied to heavy metal remediation and removal of organic matters in underground water, for example, by the electric remediation technology, pollution concentration of trichloroethylene in soil is reduced, and therefore threat to underground water is reduced.

(4) In situ bioremediation technique

The in-situ bioremediation technology is a technology for removing and degrading pollution components in underground water, including organic matters and heavy metals, and purifying the underground water by adding nutrient substances, such as organic carbon sources or other trace elements, or introducing air into the polluted underground water without changing the original environment of the underground water and under the action of inherent microorganisms or artificially selected functional strains in the underground water environment. If the microorganism can reduce V (V) and phenanthrene (Phe) in the underground water under the action of the additional carbon source methanol, the in-situ bioremediation technology has the advantages of instant treatment effect, no secondary pollution, economical operation cost and the like, and the technology can be combined with other technologies to attract attention.

In conclusion, the technology for repairing underground water organic matter and heavy metal combined pollution is evaluated, the physical method is simple and effective, the removal amount is large, but the process is complex, and the equipment maintenance cost is high. By chemical methods, new secondary pollution may be generated in the treatment process. The biological method has the advantages of environmental friendliness and cost effectiveness, and is a preferred method for treating double pollution of organic matters and heavy metals in underground water. The method is used for treating the underground water polluted by nitrogen-containing organic matters and V heavy metals by combining the complicated organic matter and heavy metal pollution conditions in the underground water, the operation cost and the secondary pollution problem, and the in-situ remediation technology is the best choice.

The in situ repair technique includes the following two techniques.

1 Bioprosthetic applications

Aiming at the pollution of organic matters and heavy metals in underground water, the bioremediation method is the preferable method for treating the double pollution of the organic matters and the heavy metals in the underground water from the aspects of environmental friendliness and treatment cost. Because of being environment-friendly, economical and effective, more and more scholars are devoted to the research on the aspect of repairing the organic matter and heavy metal composite pollution by using a biological method, and the application prospect is increasingly wide.

Aiming at the environment with composite pollution of persistent refractory organic matters and heavy metals, the bioremediation method is relied on in a single aspect, the detoxification of the persistent organic matters and the heavy metals cannot be rapidly and effectively carried out, and the dual toxicity of the composite pollution can cause the biological poisoning to reduce the degradation effect. And a proper amount of electrical stimulation is combined with bioremediation, so that the defects of multiple side reactions, high economic cost and the like of single electric-based remediation can be overcome, and the damage of double toxicants to microorganisms can be overcome. Especially in the complex pollution environment, the electrical stimulation bioremediation technology is widely applied.

In summary, bioremediation and electrical stimulation bioremediation are widely applied, and in combination with the complex organic matter and heavy metal pollution condition in underground water, the description uses a mixed microorganism remediation and electrical stimulation microorganism mode to treat underground water polluted by nitrogen-containing organic matter and V heavy metal, and explores a response mechanism for removing complex pollutants by microorganisms in a nitrogen-containing organic matter and V (V) complex pollution environment.

The research is to research the removal of nitrogenous organic matters and V (V) by respectively using a mixed microorganism repair technology and an electrical stimulation biological repair technology. Firstly, the feasibility of simultaneously removing the two is verified, the identification of a pollutant intermediate product and the research of central experiment influence factors are carried out, and finally, a 16S rRNA sequencing technology is combined to probe the microbial community structure and functional microorganisms to analyze and evaluate a possible reaction mechanism.

As NB is a refractory toxic organic matter capable of carrying out co-metabolism, the experiment adopts an electrical stimulation mixed microorganism method, ethanol is used as an electron donor, V (V) is used as an electron acceptor, and batch experimental research is carried out through appropriate amount of electrical stimulation. Through domestication, under the condition of analyzing and researching 48h in a period, the removal conditions of NB and V (V) are researched, the optimal voltage is determined, then a central experiment group is arranged under the optimal voltage, long-period domestication is carried out, the removal of the NB and V (V) in the period and the change of NB intermediate products and V (V) reduction products in the period are researched, then the research on the influence factors of the reactor under the condition of NB and V (V) combined pollution under the optimal voltage is carried out, and finally the change of the microbial community is analyzed, and the functional microorganisms which have the main functions of NB degradation and V (V) reduction under the electric stimulation are determined.

According to the scheme provided by the embodiment of the description, the underground water bioremediation process simulating NB and V (V) (potential of Hydrogen) combined pollution is researched through the condition of electrically stimulating microorganisms, the change of the optimal voltage and other main operation parameters to the reactor is researched, the functional microbial genera of the optimal voltage reactor are deeply excavated, and theoretical support is provided for the practical application of electrically stimulating microorganism remediation under the combined pollution of organic matters and heavy metals in the underground water.

Next, the embodiments provided in the present specification will be specifically described.

Example 1, test materials and chemical reagents

Experimental Material

In the experiment of degrading nitrobenzene and vanadate by microorganisms under electric stimulation, 4 250 mL-sized serum bottles wrapped by tinfoil are prepared as batch experiment bioreactors, 200mL of synthetic groundwater (shown in table 1) and 50mL of inoculated sludge are added into each serum bottle, 1cm multiplied by 0.5 cm-sized carbon felts are selected as cathodes and anodes, the carbon felts are fixed by titanium wires with the diameter of 1mm, the carbon felts are all below the groundwater page of the reactor, one end of a double-ended tiger clamp lead is clamped between a cathode pole and an anode pole, the other end of the double-ended tiger clamp lead is clamped between a constant power supply output port, and the reactor is operated at a position where the constant power supply is regulated to a specified voltage. All reactors were incubated at 25. + -. 2 ℃ in the absence of light, the experimental water being ultrapure water. The inoculated sludge is the sediment collected from the vanadium-polluted site of the Panzhihua.

TABLE 1 groundwater composition

Chemical reagent

NB was purchased from Sigma, USA, and the purity was 99.5%. NaVO is used in the experiment₃(analytical reagent, national chemical group Co., Ltd.) as a source of V (V),

in the experiment, ethanol was used as the experimental carbon source and four experimental groups (shown in Table 2) were set up, namely E-NB-V, E-NB, E-V and B-NB-V, since NB is insoluble in water. E-NB-V represents a bioreactor containing NB and V (V) at optimal voltages, E-NB and E-V represent only NB and V (V), respectively, at optimal voltages. B-NB-V represents no voltage but NB and V (V), both NB and V (V) initially at a concentration of 10 mg/L. The amount of ethanol added corresponds to a Chemical Oxygen Demand (COD) of 100 mg/L.

Table 2 four sets of reactors

Example 2

Voltage preference feasibility study

Synthetic groundwater containing ethanol at initial concentrations of 10mg/LNB and initial 10mg/L of V (V) and initial concentration of 100mg/L COD was acclimated to the bioreactor for 20 cycles of operation using different voltages, i.e., 0.3V, 0.6V, 0.9V and 1.2V, and water was changed every 48h to assess optimal voltage-synchronized NB biodegradation and V (V) bioreduction processes. The removal of NB and v (v) was monitored during the acclimation period. When the NB and V (V) removal effect was stable in all reactors, the next experiment was carried out with an operation period of 48 h.

After the reactor acclimation was completed, NB and v (v) removal performance was evaluated in three consecutive reaction cycles (48h per operating cycle). The concentration levels of NB and v (v) remained consistent with those of the previous acclimation stage. Meanwhile, other initial voltages are fixed, i.e., 0.3V, 0.6V, 0.9V, and 1.2V. And carrying out a pseudo-first-order kinetic model on the obtained data to obtain a removal rate constant.

The removal performance of NB and v (v) at four different voltages was evaluated, the optimum voltage was selected, and then four experimental reactors were run under the optimum carbon source system, and the reactors prepared were as shown in table 3. Consistent with the previous conditions, the acclimation is carried out for 20 periods, water is changed every 48h, and when the removal performance of NB and V (V) in the reactors of the four experimental groups tends to be stable, the next experiment is started. The removal performance of NB and v (v) was tested in three consecutive experimental cycles of operation (48h) and the data obtained was subjected to a pseudo-first order kinetic model to obtain the reactor removal target contaminant rate constant.

Measurement of physical and chemical Properties

In the sampling process, a small amount of nitrogen is injected by an injector to perform sampling detection by using air pressure difference, and a collected water sample is pretreated by using a 0.22 mu m filter.

The pH was measured by a pH meter (pH-201, Hanna, Italy). NB and AN are tested by using AN improved reduction azo method, the main principle is that in AN acidic solution containing copper sulfate, zinc powder reacts to generate reduced hydrogen, NB is reduced to AN, a diazo coupling reaction is utilized to generate a purple red dye, and the colorimetric determination is carried out on medicines required by NB and AN test and shown in table 3. Spectrophotometer (DR6000, HACH, USA) for monitoring NH₄ ⁺-N. The concentration of total vanadium dissolved was determined by inductively coupled plasma mass spectrometry (ICP-MS) (X series, Thermo Fisher, germany). Total organic carbon was measured using a total carbon analyzer (TOC-5000, Shimadzu, Japan). The precipitate was separated from the biomass and carbon felt by sonication and collected by centrifugation at 8000 rpm. The composition of the precipitate passes throughThe amount-dispersed X-ray was detected on a scanning electron microscope (JEOL JAX-840, Hitachi Limited, Japan). The valency of the resulting precipitate was investigated by X-ray photoelectron spectroscopy (XPS) (JEOL JAX-840, Hitachi Limited, Japan). All instruments in the experiment are shown in table 4.

TABLE 3 Chemicals

TABLE 4 Experimental instruments

Study of influencing factors

The study of the influence experiments was carried out in an E-NB-V reactor using the single variable principle.

The effect of initial NB concentration was first studied, with the initial NB concentration gradient being 5mg/L, 10mg/L, 15mg/L, and 20mg/L in that order.

Next, the influence of the initial V (V) concentrations was investigated, wherein the initial V (V) concentrations were varied to 5mg/L, 10mg/L, 15mg/L, and 20mg/L, respectively.

Finally, the influence of the initial ethanol concentration is studied, and the concentration gradient of the initial ethanol concentration is 50mg/L COD, 100mg/L COD, 150mg/L COD and 200mg/L COD. The concentration of NB and V (V) was varied under different conditions of influence.

From the above-mentioned studies, it was found that the removal performance was the best when the initial NB concentration was 10mg/L, the initial V (V) concentration was 10mg/L, and the initial ethanol concentration was 100mg/L COD.

Microbial community evolution study

The samples and collections of the microorganisms from the cathodes and anodes of the experimental groups E-NB-V, E-NB, E-V and B-NB-V were subjected to an ultrasonic pretreatment. Use according to manufacturer's instructions

The SPIN kit (Qiagen, CA, usa) extracts and purifies genomic DNA from collected biomass samples. Then makePCR with primer pairs 515F and 806R: (A), (B), (C

9700, ABI, usa) amplified DNA. One previous study showed that the biomass in the bioreactor was predominantly dominated by bacteria, so only bacterial colonies were analyzed in this experiment. The amplification procedure for PCR was as follows: denaturation was first carried out at 95 ℃ for 2 minutes, followed by 25 cycles. Each cycle lasted 90s, including 30s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃. Finally, the extension was carried out at 72 ℃ for 5 minutes. The resulting amplicons were extracted from a 2% agarose gel and then purified and quantified using AxyPrep DNA GelExtraction Kit (Axygen Biosciences, Union City, Calif., USA) and QuantiFluor TM-ST (Promega, USA), respectively. After pooling at equimolar concentrations of purified amplicons according to standard methods, they were sent to the Shanghai Meiji Biotech company (Shanghai, China) for high-throughput sequencing on the Illumina MiSeq platform (USA). The sequenced data are subjected to low-quality sequence removal by using FLASH and Trimmomatic, OTU clustering is carried out by using UPARES, the similarity of the OTU clustering reaches 97%, and the alpha diversity is calculated by using Mothur.

Example 3 feasibility study of biodegradation of NB and V (V) reduction under different Voltage stimuli

After 20 periods of acclimatization experiments, most of NB, v (v), was removed almost completely as shown in a in fig. 1 and b) in fig. 1 among the four bioreactors to which different voltages were applied. Ethanol containing NB at an initial concentration of 10mg/L and NB at an initial concentration of 10mg/L V (V) and an initial concentration of 100mg/L COD was added to the bioreactor. All reactors were then run continuously for 3 cycles simultaneously, and at the end of the cycle 10mL of solution was withdrawn from each reactor to check for NB and V (V) concentrations. As shown in c of FIG. 1, the NB removal rate in 48h for the reactor with 0.6V applied was 94.3. + -. 1.32% higher than the removal rates with 0.3V (91.0. + -. 1.67%), 0.9V (89.6. + -. 1.15%) and 1.2V (91.6. + -. 1.37%). Under proper electrical stimulation, the degradation of NB can be promoted.

The removal tendency of v (v) is similar to NB (shown as e in fig. 1), and the microorganisms are stimulated by proper voltage to enhance the reduction of v (v). The highest V (V) removal efficiency was observed in the bioreactor with added glucose (87.2 ± 0.856%), followed by 85.7 ± 0.327%, 83.5 ± 1.70% and 85.5 ± 0.294% V (V) removal efficiency of the bioreactor at voltages 0.3V, 0.9V, 1.2V, respectively. Research has shown that electrical energy from microbial fuel cells can provide electrons to support microbial detoxification of contaminants such as vanadate. At the same time, the removal of NB and v (v) from the bioreactor under four different voltage stimuli during the course of the experiment in three consecutive operating cycles can be described with pseudo-first order kinetics as d in fig. 1 and f in fig. 1. Pseudo-first order kinetic models are widely used to describe the changes in the material in the bioreactor, using the pseudo-first order kinetic model of equation 3-1 to describe the degradation of NB and v (v).

C_t＝C₀·(1-e^-kt) (3-1)

Wherein, C_tIs the concentration of the contaminant at time t, C₀Is the concentration at the start time, k is the degradation rate constant, and t is the reaction time.

The pseudo first order kinetic equations of NB and V (V) of the bioreactors at four different voltages were obtained using equation 3-1 as shown in D of FIG. 1 and f of FIG. 1, and the pseudo first order kinetic rate constant of NB in the reactor at a voltage of 0.6V was 0.0534h as shown in Table 4-1^-1Higher than 0.3V (0.0445 h)^-1)，0.9V(0.0437h^-1) And 1.2V (0.0474 h)^-1) The pseudo first order kinetic rate constant of MEC-0.6V bioreactor reduction V (V) is 0.0438h^-1Also higher than MEC-0.3V (0.0404 h)^-1)，MEC-0.9V(0.0375h^-1) And MEC-1.2V (0.0419 h)^-1). Therefore, we chose 0.6V as the optimal voltage for constructing a bioelectrochemical system.

TABLE 4-1 pseudo first order kinetics equations and related parameters for contaminants in various bioreactors at different voltages

Example 4 synchronized NB and v (v) removal feature

After the bioelectrochemical reactor determined the optimal voltage to be 0.6V, the four central experimental group bioreactors were operated continuously as shown by a in fig. 2, B in fig. 2, c in fig. 2, d in fig. 2, E in fig. 2, f in fig. 2, g in fig. 2, and h in fig. 2, i.e., E-NB-V, E-NB, E-V, and B-NB-V, and all the central experimental groups were stable after 40 days (20 cycles) of acclimation experiments. As shown in a of the figure 2 and 2B) of the figure, NB of the bioreactor E-NB-V, E-NB and NB-V is stabilized within the ranges of 57.5 + -3.15%, 72.5 + -5.13% and 35.3 + -5.64%, respectively, and V (V) removal rates of E-NB-V, E-V and B-NB-V are stabilized within the ranges of 85.4 + -4.32%, 95.2 + -3.42% and 57.2 + -1.10%, respectively. After 20 cycles of operation, all bioreactors achieved stable performance in degrading NB and v (v) reduction.

In a typical 48h operation, NB is gradually removed in the NB-containing bioreactor (shown as c in fig. 2). The NB removal rate in E-NB reaches 96.2 +/-2.10%, and the average removal rate is 0.200 +/-0.004 mg/L.h. The NB removal kinetics (d in FIG. 2) can be well simulated by simulating a first order kinetic equation with a kinetic rate constant of 0.0649h^-1(Table 4-2). The result shows that under the electrical stimulation with the optimal voltage of 0.6V, the reduction of NB by microorganisms can be better stimulated, and more researches indicate that compared with other carbon sources, the addition of ethanol can better synchronously promote the reduction of NB by microorganisms.

In a single cycle, the test group reactor pH tended to fall under stimulation of an appropriate voltage (0.6V), but the overall pH stabilized substantially between 7 and 8, while the oxidation-reduction potential (ORP) of the overall test group increased, the ORP in E-NB-V increased from-100 + -1.35 mV to-71.6 + -1.28 mV and 28.4mV increased, the ORP in E-NB increased from-97.9 + -0.75 mV to-65.1 + -0.87 mV and 32.8mV increased, the ORP in E-V increased from-99.9 + -1.32 mV to-72.9 + -0.86 mV and 27mV increased, and the ORP in B-NB-V increased from-102.2 + -1.3 mV to-76.2 + -1.23 mV and 26mV increased. The pH in the whole reactor is in a neutral environment, which is beneficial to the removal of V (V) reduction products V (IV) in the reactor, and the anaerobic reduction of NACs such as NB (NB) -like NACs is reported to be beneficial under a lower oxidation environment, although the oxidation-reduction potential of the whole experimental reactor is increased, which indicates that NB and V (V) are subjected to certain biological reduction under the electric stimulation, which indicates the high-efficiency performance of the bioelectrochemical system.

TABLE 4-2 pseudo first order kinetics equations and related parameters for contaminants in different bioreactors

Example 5 identification of reaction product

The total organic carbon concentration in E-NB-V, E-NB, B-NB-V, showed a downward trend (shown as a in FIG. 3), indicating partial NB mineralization. In a typical operation cycle, the total organic carbon removal rates of E-NB-V, E-NB and B-NB-V reach 61.1 + -0.26%, 65.7 + -0.75% and 37.1 + -0.92%, respectively, probably due to the common degradation of NB and ethanol. Previous studies have reported the biochemical reduction pathway of NB, where degradation of AN organic carbon source and degradation of a portion of the reduction products, NB, is reduced to AN. Researches indicate that after the nitroaromatic organic matters are degraded into aniline organic matters, the toxicity of the nitroaromatic organic matters is reduced, which shows that the toxicity of NB can be better reduced under an electrical stimulation microbial system. Within 48h of one operating cycle, AN accumulation of AN production of NB-reduced products was also observed during NB reduction in the studies of the examples of this specification. In a typical cycle, AN accumulates in E-NB at a maximum concentration of 3.36. + -. 0.15mg/L (b in FIG. 3). The AN amount generated in the E-NB-V and E-NB reactor system is increased firstly and then reduced, which indicates that the AN generated by NB reduction is further cracked and mineralized, and other organic matters such as catechol and the like are probably generated, and NH is detected in the system₄ ⁺N, NH at a maximum concentration of 1.14. + -. 0.064mg/L₄ ⁺Accumulation of-N in E-NB (c in FIG. 3) indicates partial degradation of AN and mineralization in the system. This result essentially follows the previously reported NB reduction pathway, where-NO on NB₂Reduction to-NH₂Generates AN, then degrades into short-chain olefin formic acid to enter TCA circulation, and generates a small amount of NH₄ ⁺-N. The toxicity of NB is strong, so that the TOC removal rate and AN production amount in B-NB-V are low.

The trend of total vanadium was similar to that of v (v) in the vanadium-containing bioreactor (f in fig. 3). In the experimental process, the removal rates of the total vanadium in the E-NB-V, E-V, B-NB-V are 71.7 +/-3.49%, 83.3 +/-1.24% and 50.5 +/-2.61%, respectively. This phenomenon indicates that soluble vanadium (V) has been reduced to V (IV) and converted to precipitate which is removed from the reaction system. When the reaction was complete, we collected the cathode carbon felt and showed the microbial attachment of the carbon felt by scanning electron microscopy images (d in fig. 3). Simultaneous spectroscopy (EDS) analysis indicated that the carbon felt contained element V (e in fig. 3). After the carbon felt attached with the microbial film is pretreated, the collected sample is subjected to XPS analysis, and a peak (g in figure 3) at 515.9eV is identified in a V2 p high-resolution spectrum corresponding to V (IV), which shows that V (IV) is a main reduction product converted by the microbe V (V), and V (V) is mainly reduced and degraded at a microbe cathode, so that the auxiliary effect of electric stimulation in a bioelectrochemical system is highlighted.

Example 6 investigation of influencing factors

NB removal efficiency decreased significantly with increasing initial NB concentration (a in FIG. 4). When the initial NB concentration was 5mg/L, the degradation rate of NB was slightly higher within 48h than the other NB concentrations and almost completely reduced degradation (a in FIG. 4). When the initial NB concentration was 20mg/L, the NB biodegradation efficiency decreased from 94.3. + -. 1.32% to 63.4. + -. 1.57% and the removal rate decreased from 0.196. + -. 0.003 mg/L.h to 0.132. + -. 0.003 mg/L.h. At the same time, the initial NB concentration affected the reductive degradation of V (V) by the microorganisms (b in FIG. 4), and when the initial NB concentration was increased from a concentration of 10mg/L to 20mg/L, the removal efficiency of V (V) was decreased from 87.2 + -0.239% to 63.9 + -0.789% and the removal rate was decreased from 0.182 + -0.0005 mg/L.h to 0.133 + -0.002 mg/L.h. The initial NB concentration increased, and the reduction rate of NB decreased greatly, indicating that when the initial NB concentration increased, the microorganism was caused to preferentially reduce V (V) due to the higher toxicity of NB than V (V). Previous reports indicate that when V (V) and Cr (VI) are reductively degraded by microorganisms while coexisting, the initial concentration of contaminants has a similar effect on the removal of the target contaminants.

The degradation rate of NB with V (V) decreases with increasing initial V (V) concentration (c in FIG. 4), probably due to V (V) vs NB toxicity of degradation products. Previous studies have shown that NO is present in the reaction system₃ ^-The rate of microbial degradation of p-chloronitrobenzene decreases with increasing concentration. An increase in the initial v (v) concentration exacerbates the competition of the microorganism for NB reduction, resulting in a decrease in NB reduction rate. At higher initial v (v) concentrations (d in fig. 4), an increase in v (v) concentration also has some negative impact on v (v) reduction, since v (v) reduction requires sufficient support from the carbon source. Even under the condition of high V (V) concentration, the microorganism can well perform biological NB reduction, and the high efficiency of the constructed system for degrading NB is reflected.

The bioreduction rates of NB and v (v) increased with increasing ethanol usage (e in fig. 4). When the initial ethanol dosage increased from 50mg/L COD to 200mg/L COD, the reduction efficiency of NB increased from 84.3 + -2.35% to 96.7 + -1.18%, while the reduction efficiency of V (V) also increased from 84.6 + -0.43% to 98.2 + -0.14%. Under this operating condition, V (V) is reduced faster than NB. The average removal rates for NB and V (V) and NB increased by 0.026. + -. 0.002 mg/L.multidot.h and 0.028. + -. 0.001 mg/L.multidot.h, respectively. Compared to NB, since NB is more toxic than v (v) and is not easily degraded by microorganisms, microorganisms are more prone to reduce v (v). When the carbon source is less, the microorganism reduces V (V) preferentially, and when the carbon source is sufficient, the microorganism degrades NB in a better reduction mode. In previous reports it was indicated that when the amount of carbon source is insufficient for microbial degradation of V (V) and PCP, the microorganisms preferentially degrade V (V), and that PCP is reduced by the microorganisms when the carbon source is sufficient.

Meanwhile, pseudo first-order kinetic simulation equations (a-f in FIG. 5) under different operation factor conditions in the reduction process of NB and V (V) are explored, and the pseudo first-order kinetic equations and related parameters under different influence factors are shown in the table 4-3. From the degradation rate parameters of the pseudo-first order kinetic modeling, the rate in the E-NB-V reactor gradually decreased as the NB and V (V) concentrations increased, respectively, and in particular, the NB degradation rate index increased from 0.0641h as the NB concentration increased from 5mg/L to 20mg/L^-1Reduced to 0.0205h^-1The degradation rate of NB is reduced by 0.0436h^-1And V (V) degradation rate index from 0.0597h^-1Reduced to 0.0196h^-1V (V) degradation rate decreasesAmplitude of 0.0401h^-1And conversely, when the V (V) concentration is increased from 5mg/L to 20mg/L, the NB degradation rate index is from 0.0736h^-1Reduced to 0.0363h^-1The degradation rate of NB is reduced by 0.0373h^-1And V (V) degradation rate index from 0.0485h^-1Reduced to 0.0163h^-1V (V) degradation rate is reduced by 0.0322h^-1. From the degradation rate, NB is more toxic than v (v), and v (v) is reduced preferentially to NB.

TABLE 4-3 pseudo-first order kinetic equations and related parameters for E-NB-V bioreactor influential factor studies

TABLE 4-3 continuation of the table

Example 7 evolution of microbial communities

TABLE 4-4 richness and diversity index of the anode microbial communities of the experimental inoculated sludge and the four experimental reactors

TABLE 4-5 abundance and diversity index of the experimental inoculated sludge and cathode microbial communities of four experimental reactors

It was found by the microbial community analysis that the microbial community had changed significantly, with the abundance of the microbes decreasing with increasing NB and v (v), as reflected by the sparsity curve (a in fig. 6 and b in fig. 6). From the Ace and Chao index changes, the abundance of the microbial community is reduced compared to the inoculum due to stress with NB and v (v) dual toxicity. From the Shannon and Simpson indices, the microbial diversity of the experimental groups E-NB-V, E-V, E-NB and B-NB-V decreased compared to the inoculum. This indicates that under the stimulation of a certain voltage, NB and V (V) double-complex contamination, a specific species in the microbial community is selected.

We studied the beta diversity of the major reactor cathode and anode microbial communities. Principal Component Analysis (PCA) based on microbiology levels showed that the microbial community at the cathode and anode of the E-NB-V reactor was significantly distant from other samples and the inoculum (a in FIG. 7 and c in FIG. 7), indicating that dual toxicity had a large impact on the structure of the microbial community in the co-existing system of NB and V (V). The single toxicity of NB or V (V) is also reflected by the distance of E-NB and E-V from the inoculum. However, from the anode (in FIG. 7 a), the distance between E-NB-V and the inoculum was further than that of the other groups, while from the cathode (c in FIG. 7), the distance between E-NB-V and the inoculum was closer than that of the other groups, indicating that NB was predominantly at the cathode and that the microorganisms were better at reducing NB degradation, whereas the anode was stressed by NB toxicity, which had a large impact on microbial community structure.

The VENN plot shows the differences and identities of the microbial community cathodes and anodes in the four bioreactors (b in fig. 7 and d in fig. 7). The anode and cathode had 164 and 305 OTUs, respectively, indicating the stable presence of bacteria having the ability to remove NB and V (V) in the anode and cathode of the bioreactor.

The relative abundance of bacteria at the gate level for the primary reactor anode and cathode is shown in fig. 7 e and fig. 7 g. From a bipolar microbial community, Proteobacteria and Firmicutes predominate in E-NB-V, accounting for 37.7% and 41.6% of the total germ line, respectively, significantly higher than the inoculum (21.1% and 4.12%), probably due to the stress of the two species to the toxic substances, i.e. NB or V (V). Proteobacteria and bacteroidata were significantly enriched in the anodic microbial community in E-NB and E-V with relative abundances of 50.5% and 31.2%, 12.0% and 7.09%, respectively, while the same was also found in the cathodic microbial community, indicating that certain genera of these two phyla may be involved in NB and V (V) reductive degradation. In B-NB-V, Chloroflexi and Halobacteta were significantly enriched compared to the inoculum, accounting for 23.7% and 24.2% of the total germ line, respectively, unlike other reaction systems, possibly due to the possible enrichment of the genera involved in NB and V (V) reduction under the effect of electrical stimulation.

Possible functional species of the microbial anode and cathode were identified at the genus level (f in fig. 7 and h in fig. 7). When NB and V (V) were added simultaneously to the reaction system, in the anodic microbial community, d, Sphingobium, Clostridium _ sensu _ stricoto _12, Longilinea, pseudooxonanthomonas, acetobacter in fig. 7 were enriched in the E-NB-V system, wherein Clostridium _ sensu _ stricoto _12 and acetobacter were the most abundant in E-NB-V, respectively 2.66 and 3.29%, and their proportions in the inoculum were only 0.012% and 0. Among them, Clostridium sense stricoto 12 is one of Clostridium species, which is itself an electroactive bacterium, enriched at the anode, and having the ability to transport electrons and thus promote the degradation of organic substances. The Clostridium sense strain 12 has a high abundance in MFC, and Clostridium sense strain 12 is one of the V-resistant bacteria, so the bacteria exhibit a good reducing power and a certain V resistance to V (V). Therefore, on the bioanode, Clostridium _ sensu _ stricoto _12 not only can be used as an electroactive bacterium to transfer electrons, but also has the function of reducing v (v) in a system to a certain extent. Furthermore, at the anode, the relative abundance of Pseudooxanthomonas in the inoculum was low (0.047%), but was abundant in E-NB-V (1.41%) and E-V. It has been reported that pseudooxanthomonas is enriched under the current of AN Electrical Microbial System (EMS) and has the ability to degrade lignin, it is presumed that pseudooxanthomonas as AN electroactive bacterium is enriched at the anode, on the one hand, ethanol, which is AN organic carbon source, is utilized to promote extracellular electron transfer and promote NB degradation, and on the other hand, it is reported that pseudooxanthomonas has the ability to degrade AN organic synthetic compound, it is presumed that partial degradation mineralization of the reduction product AN at the anode NB is caused by pseudooxanthomonas at the anode, which may be responsible for the decrease in TOC and the partial reduction in the amount of AN produced in AN E-NB-V system. Meanwhile, Pseudoxanthomonas was enriched at the anode of the E-V system, and it is presumed that the bacterium utilizes ethanol as an electron donor to promote the transfer of extracellular electrons, reducing V (V) in the solution. From the perspective of the cathodic microbial community, Clostridium _ sensu _ stricoto _12, Acetobacter and Longilinea are enriched in the E-NB-V and E-NB systems, and related reports indicate that Acetobacter has the effect of reducing trichloromethane dechlorination, so that the bacteria are presumed to be enriched at the cathode and possibly have a certain promoting effect on the reductive degradation of NB. Acetobacter can tend to utilize organic to donate electrons for biological reduction of V (V), so simultaneous reduction of NB and V (V) can be attributed in part to enrichment of Acetobacter. The genera Methanosarcina, which are related to NB degradation, are enriched in E-NB (4.89 and 4.90%) and B-NB-V (15.8 and 15.9%), which are important degrading bacteria that can degrade NACs, tetrachloroethylene (PCE) and Trichloroethylene (TCE). Methanosarcina is a V-resistant bacterium and can perform V (V) reduction, and Longilinea is a bacterium for anaerobically degrading V (V), and the bacterium is enriched in an E-NB-V (5.71%) system and an E-V (19.8%) system, can promote the degradation of V (V) in the system and can reduce the biotoxicity of V (V). Meanwhile, Sphingobium and Terrimonas are slightly higher than the inoculum (0.012% and 0.11%) in E-NB-V (0.97% and 0.17%), and studies have shown that Sphingobium can oxidize organic substances and convert the organic substances into small molecular organic substances, and Sphingobium is supposed to provide electrons for the reduction of NB by using ethanol organic substances and promote the reduction of NB and V (V). Meanwhile, relevant research indicates that Terrimonas can degrade polybrominated diphenyl ethers (PBDEs), and the bacteria are supposed to play a certain role in promoting the reduction of NB and the mineralization of product AN.

In summary, a certain current stimulation (0.6V) in the simulated aquifer can promote NB and V (V) bioreduction processes. Two electroactive bacteria, namely Clostridium _ sensu _ stricoto _12 and pseudomonas in the anode community can be enriched, electron transfer can be enhanced, and reduction of NB and V (V) can be promoted, and bioreduction of NB and V (V) can be realized by Methanosarcosina in the cathode community. Sphingobium is capable of reducing NB and V (V) using ethanol as a co-metabolizing substrate. NB partially mineralizes and produces AN, while v (v) is converted to insoluble v (iv) mainly by microbial reduction.

The research verifies that under a bioelectrochemical system, the microbes are promoted to perform NB and V (V) reduction, the method for electrically stimulating the microbes is applied to the NB and V (V) combined polluted groundwater environment for the first time, and the method is more effective compared with in-situ bioremediation. Meanwhile, functional microorganism species (Methanosarcina and Sphingobium) capable of simultaneously performing NB and V (V) reduction are discovered in the research. In production life, substances for promoting electron shuttling, such as biochar, lignite, nano zero-valent iron, elemental sulfur, natural minerals and the like, can be added to promote the process of reduction of NB and V (V).

In summary, the present illustrative examples investigated the feasibility of promoting bioreduction of microorganisms NB and v (v) under long-term conditions, preferably under four different voltage electrical stimuli, the feasibility of bioreduction of NB and v (v) at optimal voltage, the analysis of degradation products, the analysis of influential conditions, the changes in kinetics, and the investigation of microbial community changes, and concluded the following:

(1) the optimal voltage experiment shows that under the stimulation of 0.6V voltage, ethanol is an external carbon source, the removal rate of NB reaches 94.3 +/-1.32% within 48h, the removal rate of V (V) reaches 87.2 +/-0.856%, and the pseudo first order kinetic rate constant of NB is 0.0534h^-1The pseudo first order kinetic rate constant of V (V) is 0.0438h^-1All higher than the other voltage reactors. Therefore, 0.6V is the optimum voltage.

(2) After determining that the optimal voltage is 0.6V, four central experimental group bioreactors are operated until the electric-nitrobenzene-vanadium, the electric-nitrobenzene, the electric-vanadium, the nitrobenzene-vanadium, namely E-NB-V, E-NB, E-V and B-NB-V are stable, in a typical 48h operation, the NB removal rate in the E-NB reaches 96.2 +/-2.10%, and the average removal rate is 0.200 +/-0.004 mg/L.h. The NB quasi-first order kinetic rate constant is 0.0649h^-1. Under the electric stimulation with the optimal voltage of 0.6V, the microorganisms can well reduce NB.

(3) The reaction product after the end of the experiment was determined, NB was well reduced to AN, XPS, EDS and total vanadium analysis V (V) was reduced to V (IV).

(4) In the influential study of NB and V (V), V (V) was found to be reduced in preference to NB, indicating that NB is more toxic than V (V). The biodegradation rates of NB and v (v) increase with increasing ethanol usage. When the carbon source is less, the microorganism preferentially reduces V (V), and when the carbon source is sufficient, the reduction rate of NB and V (V) is increased under the electric stimulation.

(5) The 16S rRNA technology reveals that the microbial community changes, two electroactive bacteria, namely Clostridium _ sensu _ stricoto _12 and Pseudomonas are enriched in the anode community, the electron transfer can be enhanced, the reduction of NB and V (V) can be promoted, and the bioreduction of NB and V (V) can be realized by Methanosarcina and Sphingobium in the cathode community.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A method for synchronously removing nitrobenzene and vanadate in a water environment by electric stimulation is characterized by comprising the following steps:

inoculating an inoculum into a bioreactor, wherein the inoculum is a sediment of a vanadium-polluted site of the Panzhihua;

acclimating the inoculum;

injecting first synthetic underground water at least containing nitrobenzene, pentavalent vanadium and a carbon source into a bioreactor containing the domesticated inoculum, and performing synchronous removal reaction of the nitrobenzene and the pentavalent vanadium under the stimulation of 0.3V-1.2V voltage;

wherein the simultaneous removal reaction is carried out for at least 48 hours; maintaining an anaerobic environment within the bioreactor during the simultaneous removal reaction.

2. The method according to claim 1, wherein the synchronous removal reaction of nitrobenzene and pentavalent vanadium under the stimulation of 0.3V-1.2V voltage is specifically as follows: under the stimulation of 0.6V voltage, the synchronous removal reaction of nitrobenzene and pentavalent vanadium is carried out.

3. The method of claim 1 or 2, wherein the initial concentration of pentavalent vanadium in the first synthetic groundwater is 10mg/L and the initial concentration of nitrobenzene is 10 mg/L.

4. A method according to claim 1 or 2, wherein the ratio of inoculum volume of inoculum to bioreactor volume is 1: 5.

5. the method of claim 1 or 2, wherein the carbon source is ethanol and the initial concentration in the first synthetic groundwater is 100mg/L COD.

6. The method of claim 1 or 2, wherein said acclimating the inoculum comprises:

injecting second synthetic groundwater into the bioreactor containing the inoculum to perform acclimatization reaction; the second synthetic groundwater contains pentavalent vanadium, nitrobenzene and a carbon source.

7. The method of claim 6, wherein in the second synthetic groundwater the initial concentration of pentavalent vanadium is 10mg/L, the initial concentration of nitrobenzene is 10mg/L, and the initial concentration of carbon source is 100mg/L COD.

8. The method according to claim 6, wherein the acclimatization reaction lasts for 20 cycles, each cycle having a duration of 48 hours; wherein the second synthetic groundwater in the bioreactor is replaced once per cycle.

9. A domestication method of an inoculum for synchronously removing nitrobenzene and vanadate in a water environment by electric stimulation is characterized in that,

inoculating an inoculum into a bioreactor, wherein the inoculum is a sediment of a vanadium-polluted site of the Panzhihua; the ratio of the inoculation volume of the inoculum to the volume of the bioreactor was 1: 5;

injecting second synthetic groundwater into the bioreactor containing the inoculum to perform acclimatization reaction; the second synthetic underground water contains pentavalent vanadium with initial concentration of 10mg/L, nitrobenzene with initial concentration of 10mg/L and ethanol with initial concentration of 100mg/L COD;

the domestication reaction lasts for 20 periods, and the duration of each period is 48 hours; wherein the second synthetic groundwater in the bioreactor is replaced once per cycle.

10. An inoculum acclimatized by the method of claim 9.